@article {68011, title = {BRD2 compartmentalizes the accessible genome.}, journal = {Nature Genetics}, volume = {54}, year = {2022}, month = {2022 Apr 11}, pages = {481-491}, abstract = {

Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

}, keywords = {Animals, Cell Cycle Proteins, Chromatin, Chromosomes, Mammals, Nuclear Proteins, Protein Binding, Protein Domains, Transcription Factors}, issn = {1546-1718}, doi = {10.1038/s41588-022-01044-9}, author = {Xie, Liangqi and Dong, Peng and Qi, Yifeng and Hsieh, Tsung-Han S and English, Brian P and Jung, Seolkyoung and Chen, Xingqi and De Marzio, Margherita and Casellas, Rafael and Chang, Howard Y and Zhang, Bin and Tjian, Robert and Liu, Zhe} } @article {67806, title = {ecDNA hubs drive cooperative intermolecular oncogene expression.}, journal = {Nature}, volume = {600}, year = {2021}, month = {2021 Dec 01}, pages = {731-6}, abstract = {

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-04116-8}, author = {Hung, King L and Yost, Kathryn E and Xie, Liangqi and Shi, Quanming and Helmsauer, Konstantin and Luebeck, Jens and Sch{\"o}pflin, Robert and Lange, Joshua T and Chamorro Gonz{\'a}lez, Roc{\'\i}o and Weiser, Natasha E and Chen, Celine and Valieva, Maria E and Wong, Ivy Tsz-Lo and Wu, Sihan and Dehkordi, Siavash R and Duffy, Connor V and Kraft, Katerina and Tang, Jun and Belk, Julia A and Rose, John C and Corces, M Ryan and Granja, Jeffrey M and Li, Rui and Rajkumar, Utkrisht and Friedlein, Jordan and Bagchi, Anindya and Satpathy, Ansuman T and Tjian, Robert and Mundlos, Stefan and Bafna, Vineet and Henssen, Anton G and Mischel, Paul S and Liu, Zhe and Chang, Howard Y} } @article {65368, title = {3D ATAC-PALM: super-resolution imaging of the accessible genome.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Apr 01}, pages = {430-6}, abstract = {

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA{\textendash}fluorescence in situ hybridization (FISH), RNA{\textendash}FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

}, issn = {1548-7091}, doi = {10.1038/s41592-020-0775-2}, url = {http://www.nature.com/articles/s41592-020-0775-2}, author = {Xie, Liangqi and Dong, Peng and Chen, Xingqi and Hsieh, Tsung-Han S. and Banala, Sambashiva and De Marzio, Margherita and English, Brian P. and Qi, Yifeng and Jung, Seol Kyoung and Kieffer-Kwon, Kyong-Rim and Legant, Wesley R. and Hansen, Anders S. and Schulmann, Anton and Casellas, Rafael and Zhang, Bin and Betzig, Eric and Lavis, Luke D. and Chang, Howard Y. and Tjian, Robert and Liu, Zhe} } @article {65257, title = {MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions.}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Dec 23}, abstract = {

Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.

}, issn = {2050-084X}, doi = {10.7554/eLife.51449}, author = {Piccolo, Francesco M and Liu, Zhe and Dong, Peng and Hsu, Ching-Lung and Stoyanova, Elitsa I and Rao, Anjana and Tjian, Robert and Heintz, Nathaniel} } @article {49293, title = {Imaging dynamic and selective low-complexity domain interactions that control gene transcription.}, journal = {Science (New York, N.Y.)}, volume = {361}, year = {2018}, month = {2018 Jun 21}, pages = {eaar2555}, abstract = {

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs), but how they drive transactivation remains unclear. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for novel drugs targeting gene regulatory interactions implicated in disease.

}, issn = {1095-9203}, doi = {10.1126/science.aar2555}, author = {Chong, Shasha and Dugast-Darzacq, Claire and Liu, Zhe and Dong, Peng and Dailey, Gina M and Cattoglio, Claudia and Heckert, Alec and Banala, Sambashiva and Lavis, Luke and Darzacq, Xavier and Tjian, Robert} } @article {48997, title = {Robust model-based analysis of single-particle tracking experiments with Spot-On.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Jan 04}, abstract = {

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce {\textquoteright}Spot-On{\textquoteright}, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

}, issn = {2050-084X}, doi = {10.7554/eLife.33125}, author = {Hansen, Anders S and Woringer, Maxime and Grimm, Jonathan B and Lavis, Luke D and Tjian, Robert and Darzacq, Xavier} } @article {49022, title = {Visualizing transcription factor dynamics in living cells.}, journal = {The Journal of Cell Biology}, volume = {217}, year = {2018}, month = {2018 Jan 29}, pages = {1181-91}, abstract = {

The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level. The ability to observe the remarkably dynamic behavior of individual TFs in live cells at high spatiotemporal resolution has begun to provide novel mechanistic insights and promises new advances in deciphering causal-functional relationships of TF targeting, genome organization, and gene activation. In this review, we review current transcription imaging techniques and summarize converging results from various lines of research that may instigate a revision of models to describe key features of eukaryotic gene regulation.

}, issn = {1540-8140}, doi = {10.1083/jcb.201710038}, author = {Liu, Zhe and Tjian, Robert} } @article {48859, title = {A dynamic interplay of enhancer elements regulates Klf4 expression in na{\"\i}ve pluripotency.}, journal = {Genes \& Development}, volume = {31}, year = {2017}, month = {2017 Sep 01}, pages = {1795-1808}, abstract = {

Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Kr{\"u}ppel-like factor 4 (Klf4) expression in na{\"\i}ve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two na{\"\i}ve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying na{\"\i}ve pluripotency.

}, issn = {1549-5477}, doi = {10.1101/gad.303321.117}, author = {Xie, Liangqi and Torigoe, Sharon E and Xiao, Jifang and Mai, Daniel H and Li, Li and Davis, Fred P and Dong, Peng and Marie-Nelly, Herve and Grimm, Jonathan and Lavis, Luke and Darzacq, Xavier and Cattoglio, Claudia and Liu, Zhe and Tjian, Robert} } @article {47938, title = {Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.}, journal = {Genes and Development}, volume = {30}, year = {2016}, month = {2016 Sep 15}, pages = {2106-18}, abstract = {

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A {\textquotedblleft}step-wise{\textquotedblright} preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB{\textendash}promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II{\textendash}TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

}, doi = {10.1101/gad.285395.116}, author = {Zhang, Zhengjian and English, Brian P and Grimm, Jonathan B and Kazane, Stephanie A and Hu, Wenxin and Tsai, Albert and Inouye, Carla and You, Changjiang and Piehler, Jacob and Schultz, Peter G and Lavis, Luke D and Revyakin, Andrey and Tjian, Robert} } @article {47741, title = {Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Aug 03}, abstract = {

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form {\textquoteright}sticky{\textquoteright} decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

}, issn = {2050-084X}, doi = {10.7554/eLife.17056}, author = {Li, Li and Liu, Hui and Dong, Peng and Li, Dong and Legant, Wesley R and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert and Liu, Zhe} } @article {47032, title = {CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Aug 31}, pages = {11870-5}, abstract = {

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

}, issn = {1091-6490}, doi = {10.1073/pnas.1515692112}, author = {Deng, Wulan and Shi, Xinghua and Tjian, Robert and Lionnet, Timoth{\'e}e and Singer, Robert H} } @article {47034, title = {Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Aug 28}, abstract = {

Intrinsically disordered protein regions (IDRs) are peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. Here we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

}, issn = {2050-084X}, doi = {10.7554/eLife.07777}, author = {Zhang, Zhengjian and Boskovic, Zarko and Hussain, Mahmud M and Hu, Wenxin and Inouye, Carla and Kim, Han-Je and Abole, A Katherine and Doud, Mary K and Lewis, Timothy A and Koehler, Angela N and Schreiber, Stuart L and Tjian, Robert} } @article {47160, title = {Dynamics of CRISPR-Cas9 genome interrogation in living cells.}, journal = {Science (New York, N.Y.)}, volume = {350}, year = {2015}, month = {2015 Nov 13}, pages = {823-6}, abstract = {

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (\<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

}, issn = {1095-9203}, doi = {10.1126/science.aac6572}, author = {Knight, Spencer C and Xie, Liangqi and Deng, Wulan and Guglielmi, Benjamin and Witkowsky, Lea B and Bosanac, Lana and Zhang, Elisa T and El Beheiry, Mohamed and Masson, Jean-Baptiste and Dahan, Maxime and Liu, Zhe and Doudna, Jennifer A and Tjian, Robert} } @article {47734, title = {Imaging transcription: past, present, and future.}, journal = {Cold Spring Harbor Symposia on Quantitative Biology}, volume = {80}, year = {2015}, month = {2015}, pages = {1-8}, abstract = {

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

}, issn = {1943-4456}, doi = {10.1101/sqb.2015.80.027201}, author = {Coleman, Robert A and Liu, Zhe and Darzacq, Xavier and Tjian, Robert and Singer, Robert H and Lionnet, Timoth{\'e}e} } @article {47085, title = {A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015}, abstract = {

TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

}, issn = {2050-084X}, doi = {10.7554/eLife.08536}, author = {Li, Li and Martinez, Silvia Sanchez and Hu, Wenxin and Liu, Zhe and Tjian, Robert} } @article {45886, title = {3D imaging of Sox2 enhancer clusters in embryonic stem cells.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Dec 24}, abstract = {

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

}, issn = {2050-084X}, doi = {10.7554/eLife.04236}, author = {Liu, Zhe and Legant, Wesley R and Chen, Bi-Chang and Li, Li and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert} } @article {6894, title = {Charting Brachyury-mediated developmental pathways during early mouse embryogenesis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, year = {2014}, month = {2014 Mar 10}, pages = {4478-83}, abstract = {

To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator-Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

}, issn = {1091-6490}, doi = {10.1073/pnas.1402612111}, author = {Lolas, Macarena and Valenzuela, Pablo D T and Tjian, Robert and Liu, Zhe} } @article {6919, title = {Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.}, journal = {Cell}, volume = {156}, year = {2014}, month = {2014 Mar 13}, pages = {1274-85}, abstract = {

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in\ vivo and in\ vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to\ determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.01.062}, author = {Chen, Jiji and Zhang, Zhengjian and Li Li and Chen, Bi-Chang and Revyakin, Andrey and Hajj, Bassam and Legant, Wesley and Dahan, Maxime and Lionnet, Timoth{\'e}e and Betzig, Eric and Tjian, Robert and Liu, Zhe} } @article {6845, title = {Single-molecule tracking of the transcription cycle by sub-second RNA detection.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jan 28}, pages = {e01775}, abstract = {

Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (\~{}10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01775}, author = {Zhang, Zhengjian and Revyakin, Andrey and Grimm, Jonathan B and Lavis, Luke D and Tjian, Robert} } @article {6846, title = {Dual functions of TAF7L in adipocyte differentiation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Jan 8}, pages = {e00170}, abstract = {

The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.00170}, author = {Zhou, Haiying and Kaplan, Tommy and Li, Yan and Grubisic, Ivan and Zhang, Zhengjian and Wang, P Jeremy and Eisen, Michael B and Tjian, Robert} } @article {6518, title = {Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Aug 1}, pages = {1691-702}, abstract = {

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

}, keywords = {Fluorescence, Genetic, Humans, Microscopy, Molecular Imaging, Mutation, Promoter Regions, RNA Polymerase II, Sp1 Transcription Factor, Transcription, Transcription Factor TFIID, Video}, issn = {1549-5477}, doi = {10.1101/gad.194936.112}, author = {Revyakin, Andrey and Zhang, Zhengjian and Coleman, Robert A and Li, Yan and Inouye, Carla and Lucas, Julian K and Park, Sang-Ryul and Chu, Steven and Tjian, Robert} } @article {5411, title = {Control of embryonic stem cell lineage commitment by core promoter factor, TAF3.}, journal = {Cell}, volume = {146}, year = {2011}, month = {2011 Sep 2}, pages = {720-31}, abstract = {

Deciphering the molecular basis of pluripotency is fundamental to our understanding of development and embryonic stem cell function. Here, we report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In this context, TAF3 is required for endoderm lineage differentiation and prevents premature specification of neuroectoderm and mesoderm. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to a subset of chromosomal regions bound by CTCF/cohesin that are selectively associated with genes upregulated by TAF3. Notably, CTCF directly recruits TAF3 to promoter distal sites and TAF3-dependent DNA looping is observed between the promoter distal sites and core promoters occupied by TAF3/CTCF/cohesin. Together, our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions that safeguard the finely-balanced transcriptional programs underlying pluripotency.

}, keywords = {Animals, Cell Cycle Proteins, Cell Proliferation, Chromosomal Proteins, Developmental, Embryonic Stem Cells, Endoderm, Gene Expression Regulation, Genetic, Homeodomain Proteins, Humans, Mice, Non-Histone, Promoter Regions, Repressor Proteins, Teratoma, Transcription, Transcription Factor TFIID}, issn = {1097-4172}, doi = {10.1016/j.cell.2011.08.005}, author = {Liu, Zhe and Scannell, Devin R and Eisen, Michael B and Tjian, Robert} } @article {2384, title = {Core promoter recognition complex changes accompany liver development.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, year = {2011}, month = {2011 Mar 8}, pages = {3906-11}, abstract = {

Recent studies of several key developmental transitions have brought into question the long held view of the basal transcriptional apparatus as ubiquitous and invariant. In an effort to better understand the role of core promoter recognition and coactivator complex switching in cellular differentiation, we have examined changes in transcription factor IID (TFIID) and cofactor required for Sp1 activation/Mediator during mouse liver development. Here we show that the differentiation of fetal liver progenitors to adult hepatocytes involves a wholesale depletion of canonical cofactor required for Sp1 activation/Mediator and TFIID complexes at both the RNA and protein level, and that this alteration likely involves silencing of transcription factor promoters as well as protein degradation. It will be intriguing for future studies to determine if a novel and as yet unknown core promoter recognition complex takes the place of TFIID in adult hepatocytes and to uncover the mechanisms that down-regulate TFIID during this critical developmental transition.

}, issn = {1091-6490}, doi = {10.1073/pnas.1100640108}, author = {D{\textquoteright}Alessio, Joseph A and Ng, Raymond and Willenbring, Holger and Tjian, Robert} } @article {5307, title = {Sub-nuclear compartmentalization of core promoter factors and target genes.}, journal = {Cell Cycle}, volume = {10}, year = {2011}, month = {2011 Aug 1}, pages = {2405-6}, issn = {1551-4005}, author = {Yao, Jie and Tjian, Robert} } @article {2385, title = {Subnuclear segregation of genes and core promoter factors in myogenesis. (With commentary)}, journal = {Genes \& Development}, volume = {25}, year = {2011}, month = {2011 Mar 15}, pages = {569-80}, abstract = {

Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. Here we report a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD (plant homeodomain) finger appears to be required for the sequestration of TAF3 to the nuclear interior. We suggest that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation.

Commentary: Jie Yao in Bob Tijan{\textquoteright}s lab used a combination of confocal microscopy and dual label PALM in thin sections cut from resin-embedded cells to show that certain core transcription components and their target genes are spatially segregated in myoblasts, but not in differentiated myotubes, suggesting that such spatial segregation may play a role in guiding cellular differentiation.

}, issn = {1549-5477}, doi = {10.1073/pnas.1100640108}, author = {Yao, Jie and Fetter, Richard D and Hu, Ping and Betzig, Eric and Tjian, Robert} } @article {2386, title = {A new focus on plant sciences.}, journal = {Science}, volume = {330}, year = {2010}, month = {2010 Nov 19}, pages = {1021}, keywords = {Agriculture, Botany, Financing, Government, Plants, Research Support as Topic, United States}, issn = {1095-9203}, doi = {10.1073/pnas.1100640108}, author = {McCormick, Steven J and Tjian, Robert} } @article {2387, title = {Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation.}, journal = {Nature Reviews. Genetics}, volume = {11}, year = {2010}, month = {2010 Aug}, pages = {549-58}, abstract = {

The eukaryotic core promoter recognition complex was generally thought to play an essential but passive role in the regulation of gene expression. However, recent evidence now indicates that core promoter recognition complexes together with {\textquoteright}non-prototypical{\textquoteright} subunits may have a vital regulatory function in driving cell-specific programmes of transcription during development. Furthermore, new roles for components of these complexes have been identified beyond development; for example, in mediating interactions with chromatin and in maintaining active gene expression across cell divisions.

}, keywords = {Animals, Cell Differentiation, DNA, Embryonic Development, Female, Genetic, Germ Cells, Humans, Male, Promoter Regions, RNA Polymerase II, Transcription, Transcription Factors, Transcriptional Activation}, issn = {1471-0064}, doi = {10.1073/pnas.1100640108}, author = {Goodrich, James A and Tjian, Robert} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} } @article {2388, title = {Shifting players and paradigms in cell-specific transcription.}, journal = {Molecular Cell}, volume = {36}, year = {2009}, month = {2009 Dec 25}, pages = {924-31}, abstract = {

Historically, developmental-stage- and tissue-specific patterns of gene expression were assumed to be determined primarily by DNA regulatory sequences and their associated activators, while the general transcription machinery including core promoter recognition complexes, coactivators, and chromatin modifiers was held to be invariant. New evidence suggests that significant changes in these general transcription factors including TFIID, BAF, and Mediator may facilitate global changes in cell-type-specific transcription.

}, keywords = {Animals, Developmental, Gene Expression Regulation, Genetic, Mediator Complex, Models, Molecular, Promoter Regions, Protein Isoforms, TATA Box Binding Protein-Like Proteins, Tissue Distribution, Transcription, Transcription Factor TFIID}, issn = {1097-4164}, doi = {10.1073/pnas.1100640108}, author = {D{\textquoteright}Alessio, Joseph A and Wright, Kevin J and Tjian, Robert} } @article {2389, title = {Structures of three distinct activator-TFIID complexes.}, journal = {Genes \& Development}, volume = {23}, year = {2009}, month = {2009 Jul 1}, pages = {1510-21}, abstract = {

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, we determined the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their structures as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, our results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation.

}, keywords = {Hela Cells, Humans, Imaging, Immunoglobulins, Models, Molecular, Protein Binding, Protein Multimerization, Protein Structure, Proto-Oncogene Proteins c-jun, Quaternary, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIID, Tumor Suppressor Protein p53}, issn = {1549-5477}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Ma, Elizabeth and Grob, Patricia and Yang, Joyce L and Zhang, Yixi and Dailey, Gina and Nogales, Eva and Tjian, Robert} } @article {2391, title = {Wnt signaling targets ETO coactivation domain of TAF4/TFIID in vivo.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {106}, year = {2009}, month = {2009 Jan 6}, pages = {55-60}, abstract = {

Understanding the diverse activities of the multisubunit core promoter recognition complex TFIID in vivo requires knowledge of how individual subunits contribute to overall functions of this TATA box-binding protein (TBP)/TBP-associated factor (TAF) complex. By generating altered holo-TFIID complexes in Drosophila we identify the ETO domain of TAF4 as a coactivator domain likely targeted by Pygopus, a protein that is required for Wingless-induced transcription of naked cuticle. These results establish a coactivator function of TAF4 and provide a strategy to dissect mechanisms of TFIID function in vivo.

}, keywords = {Animals, Binding Sites, Cell Line, Drosophila, Drosophila Proteins, Humans, Intracellular Signaling Peptides and Proteins, Larva, Multiprotein Complexes, Proto-Oncogene Proteins, Signal Transduction, TATA-Binding Protein Associated Factors, Transcription Factor TFIID, Transcription Factors, Tumor, Wnt Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1100640108}, author = {Wright, Kevin J and Tjian, Robert} } @article {2392, title = {Codependent activators direct myoblast-specific MyoD transcription.}, journal = {Developmental Cell}, volume = {15}, year = {2008}, month = {2008 Oct}, pages = {534-46}, abstract = {

Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. Here, we identify MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Our cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinately recruit RNA polymerase II and form a preinitiation complex (PIC) to activate MyoD transcription in myoblasts. The role of FoxO3 in regulating muscle differentiation is confirmed in vivo by observed defects in muscle regeneration caused by MyoD downregulation in FoxO3 null mice. These data establish a mutual interdependence and functional link between two families of transcription activators serving as potential signaling sensors and regulators of cell fate commitment in directing tissue specific MyoD transcription.

}, keywords = {Animals, Cell Line, Forkhead Transcription Factors, Genetic, Immunohistochemistry, Knockout, Mice, Myoblasts, MyoD Protein, Paired Box Transcription Factors, RNA, RNA Interference, Small Interfering, Trans-Activators, Transcription, Transfection}, issn = {1878-1551}, doi = {10.1073/pnas.1100640108}, author = {Hu, Ping and Geles, Kenneth G and Paik, Ji-Hye and DePinho, Ronald A and Tjian, Robert} } @article {2396, title = {ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {105}, year = {2008}, month = {2008 May 6}, pages = {6656-61}, abstract = {

ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectoderm-derived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.

}, keywords = {Adipocytes, Animals, Cardiac, Cell Differentiation, Cell Line, Cell Lineage, Cell Proliferation, Chromatin Assembly and Disassembly, Chromosomal Proteins, Developmental, DNA-Binding Proteins, Embryo, Embryonic Development, Embryonic Stem Cells, Female, Genes, Germ Layers, Knockout, Male, Mammalian, Mice, Muscle, Myocytes, Non-Histone, Nuclear Proteins, Pluripotent Stem Cells, Skeletal, Transcription Factors}, issn = {1091-6490}, doi = {10.1073/pnas.1100640108}, author = {Gao, Xiaolin and Tate, Peri and Hu, Ping and Tjian, Robert and Skarnes, William C and Wang, Zhong} } @article {2394, title = {The future for Howard Hughes. Interview by Erika Check Hayden.}, journal = {Nature}, volume = {455}, year = {2008}, month = {2008 Oct 9}, pages = {718}, keywords = {Academies and Institutes, Creativeness, Disease, Humans, Internationality, Maryland, Molecular Biology, Research, Research Personnel}, issn = {1476-4687}, doi = {10.1073/pnas.1100640108}, author = {Tjian, Robert} } @article {2393, title = {MyoD targets TAF3/TRF3 to activate myogenin transcription.}, journal = {Molecular Cell}, volume = {32}, year = {2008}, month = {2008 Oct 10}, pages = {96-105}, abstract = {

Skeletal muscle differentiation requires a cascade of transcriptional events to control the spatial and temporal expression of muscle-specific genes. Until recently, muscle-specific transcription was primarily attributed to prototypic enhancer-binding factors, while the role of core promoter recognition complexes in directing myogenesis remained unknown. Here, we report the development of a purified reconstituted system to analyze the properties of a TAF3/TRF3 complex in directing transcription initiation at the Myogenin promoter. Importantly, this new complex is required to replace the canonical TFIID to recapitulate MyoD-dependent activation of Myogenin. In vitro and cell-based assays identify a domain of TAF3 that mediates coactivator functions targeted by MyoD. Our findings also suggest changes to CRSP/Mediator in terminally differentiated myotubes. This switching of the core promoter recognition complex during myogenesis allows a more balanced division of labor between activators and TAF coactivators, thus providing another strategy to accommodate cell-specific regulation during metazoan development.

}, keywords = {Animals, Cell Line, Genetic, Homeodomain Proteins, Mice, Multiprotein Complexes, Muscle Fibers, MyoD Protein, Myogenin, Promoter Regions, Protein Structure, Protein Subunits, Recombinant Proteins, Skeletal, TATA Box Binding Protein-Like Proteins, Tertiary, Trans-Activators, Transcription Factor TFIID, Transcription Initiation Site, Transcriptional Activation}, issn = {1097-4164}, doi = {10.1073/pnas.1100640108}, author = {Deato, Maria Divina E and Marr, Michael T and Sottero, Theo and Inouye, Carla and Hu, Ping and Tjian, Robert} } @article {2397, title = {The nucleosome remodeling factor (NURF) regulates genes involved in Drosophila innate immunity.}, journal = {Developmental Biology}, volume = {316}, year = {2008}, month = {2008 Apr 15}, pages = {538-47}, abstract = {

The Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF has the ability to alter chromatin structure and regulate transcription. Previous studies have shown that mutation of Drosophila NURF induces melanotic tumors, implicating NURF in innate immune function. Here, we show that NURF mutants exhibit identical innate immune responses to gain-of-function mutants in the Drosophila JAK/STAT pathway. Using microarrays, we identify a common set of target genes that are activated in both mutants. In silico analysis of promoter sequences of these defines a consensus regulatory element comprising a STAT-binding sequence overlapped by a binding-site for the transcriptional repressor Ken. NURF interacts physically and genetically with Ken. Chromatin immunoprecipitation (ChIP) localizes NURF to Ken-binding sites in hemocytes, suggesting that Ken recruits NURF to repress STAT responders. Loss of NURF leads to precocious activation of STAT target genes.

}, keywords = {Animals, Chromosomal Proteins, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Gene Expression Profiling, Gene Expression Regulation, Genetic, Genome, Immunity, Innate, Non-Histone, Oligonucleotide Array Sequence Analysis, Phenotype, Recombinant Proteins, Reverse Transcriptase Polymerase Chain Reaction, RNA, Signal Transduction, Transcription}, issn = {1095-564X}, doi = {10.1073/pnas.1100640108}, author = {Kwon, So Yeon and Xiao, Hua and Glover, Bradley P and Tjian, Robert and Wu, Carl and Badenhorst, Paul} } @article {2399, title = {Structural changes in TAF4b-TFIID correlate with promoter selectivity.}, journal = {Molecular Cell}, volume = {29}, year = {2008}, month = {2008 Jan 18}, pages = {81-91}, abstract = {

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.

}, keywords = {B-Lymphocytes, Cell Line, Computer-Assisted, Electron, Genetic, Hela Cells, Humans, Image Processing, Imaging, Microscopy, Models, Molecular, Organ Specificity, Promoter Regions, Protein Conformation, Protein Interaction Mapping, Proto-Oncogene Proteins c-jun, Sp1 Transcription Factor, Structure-Activity Relationship, TATA-Binding Protein Associated Factors, Three-Dimensional, Transcription Factor TFIIA, Transcription Factor TFIID, Transcriptional Activation, Tumor}, issn = {1097-2765}, doi = {10.1073/pnas.1100640108}, author = {Liu, Wei-Li and Coleman, Robert A and Grob, Patricia and King, David S and Florens, Laurence and Washburn, Michael P and Geles, Kenneth G and Yang, Joyce L and Ramey, Vincent and Nogales, Eva and Tjian, Robert} } @article {2395, title = {TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription.}, journal = {Genes \& Development}, volume = {22}, year = {2008}, month = {2008 Sep 1}, pages = {2353-8}, abstract = {

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways.

}, keywords = {Animals, Cells, Cultured, Drosophila, Drosophila Proteins, Enhancer Elements, Gene Expression Regulation, Genetic, Promoter Regions, RNA Polymerase II, TATA Box, TATA-Box Binding Protein, Transcription, Transcription Factors}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Hsu, Jer-Yuan and Juven-Gershon, Tamar and Marr, Michael T and Wright, Kevin J and Tjian, Robert and Kadonaga, James T} } @article {2401, title = {Daniel E. Koshland, Jr. 1920-2007.}, journal = {Cell}, volume = {130}, year = {2007}, month = {2007 Aug 24}, pages = {579-80}, keywords = {20th Century, 21st Century, Biochemistry, History, United States}, issn = {0092-8674}, doi = {10.1073/pnas.1100640108}, author = {Tjian, Robert} } @article {2404, title = {IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback.}, journal = {Genes \& Development}, volume = {21}, year = {2007}, month = {2007 Jan 15}, pages = {175-83}, abstract = {

It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Here, we report evidence for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. Our studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions.

}, keywords = {5{\textquoteright} Untranslated Regions, Animals, Biological, Drosophila melanogaster, Drosophila Proteins, Eukaryotic Initiation Factor-4E, Feedback, Forkhead Transcription Factors, Gene Expression Regulation, Genetic, Insulin, Models, Phosphorylation, Physiological, Protein Biosynthesis, Receptor, Ribosomes, Signal Transduction, Transcription, Transfection}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Marr, Michael T and D{\textquoteright}Alessio, Joseph A and Puig, Oscar and Tjian, Robert} } @article {2405, title = {Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription.}, journal = {The EMBO Journal}, volume = {26}, year = {2007}, month = {2007 Jan 10}, pages = {79-89}, abstract = {

Metazoans have evolved multiple paralogues of the TATA binding protein (TBP), adding another tunable level of gene control at core promoters. While TBP-related factor 1 (TRF1) shares extensive homology with TBP and can direct both Pol II and Pol III transcription in vitro, TRF1 target sites in vivo have remained elusive. Here, we report the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. We found 354 TRF1-binding sites genome-wide with approximately 78\% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol III-dependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. We provide direct evidence that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). Our studies suggest that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila and possibly other insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription.

}, keywords = {Animals, Base Sequence, Binding Sites, Chromatin Immunoprecipitation, Deoxyribonuclease I, DNA Polymerase III, Drosophila melanogaster, Genetic, Genome, Molecular Sequence Data, Oligonucleotide Probes, Protein Binding, RNA, Small Nucleolar, Telomeric Repeat Binding Protein 1, Transcription, Transcription Factor TFIIIB}, issn = {0261-4189}, doi = {10.1073/pnas.1100640108}, author = {Isogai, Yoh and Takada, Shinako and Tjian, Robert and Kele\c s, S{\"u}nd{\"u}z} } @article {2403, title = {Switching of the core transcription machinery during myogenesis.}, journal = {Genes \& Development}, volume = {21}, year = {2007}, month = {2007 Sep 1}, pages = {2137-49}, abstract = {

Transcriptional mechanisms that govern cellular differentiation typically include sequence-specific DNA-binding proteins and chromatin-modifying activities. These regulatory factors are assumed necessary and sufficient to drive both divergent programs of proliferation and terminal differentiation. By contrast, potential contributions of the basal transcriptional apparatus to orchestrate cell-specific gene expression have been poorly explored. In order to probe alternative mechanisms that control differentiation, we have assessed the fate of the core promoter recognition complex, TFIID, during skeletal myogenesis. Here we report that differentiation of myoblast to myotubes involves the disruption of the canonical holo-TFIID and replacement by a novel TRF3/TAF3 (TBP-related factor 3/TATA-binding protein-associated factor 3) complex. This required switching of core promoter complexes provides organisms a simple yet effective means to selectively turn on one transcriptional program while silencing many others. Although this drastic but parsimonious transcriptional switch had previously escaped our attention, it may represent a more general mechanism for regulating cell type-specific terminal differentiation.

}, keywords = {Animals, Cell Differentiation, Cell Line, Developmental, Gene Expression Regulation, Genetic, Humans, Mice, Models, Muscle, Muscle Development, Muscle Fibers, Myofibrils, Nuclear Proteins, RNA Interference, Skeletal, TATA Box Binding Protein-Like Proteins, Transcription, Transcription Factor TFIID}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Deato, Maria Divina E and Tjian, Robert} } @article {2400, title = {Transcription of histone gene cluster by differential core-promoter factors.}, journal = {Genes \& Development}, volume = {21}, year = {2007}, month = {2007 Nov 15}, pages = {2936-49}, abstract = {

The 100 copies of tandemly arrayed Drosophila linker (H1) and core (H2A/B and H3/H4) histone gene cluster are coordinately regulated during the cell cycle. However, the molecular mechanisms that must allow differential transcription of linker versus core histones prevalent during development remain elusive. Here, we used fluorescence imaging, biochemistry, and genetics to show that TBP (TATA-box-binding protein)-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription. Importantly, TRF2-depleted polytene chromosomes display severe chromosomal structural defects. This selective usage of TRF2 and TBP provides a novel mechanism to differentially direct transcription within the histone cluster. Moreover, genome-wide chromatin immunoprecipitation (ChIP)-on-chip analyses coupled with RNA interference (RNAi)-mediated functional studies revealed that TRF2 targets several classes of TATA-less promoters of \>1000 genes including those driving transcription of essential chromatin organization and protein synthesis genes. Our studies establish that TRF2 promoter recognition complexes play a significantly more central role in governing metazoan transcription than previously appreciated.

}, keywords = {Animals, Base Sequence, Biological, Cells, Chromatin Immunoprecipitation, Chromosomes, Cluster Analysis, Cultured, Drosophila, Firefly, Fluorescence, Genes, Genetic, Histones, Immunohistochemistry, In Situ Hybridization, Insect, Luciferases, Models, Molecular Sequence Data, Multigene Family, Nucleic Acid, Promoter Regions, Reporter, RNA Interference, Sequence Homology, TATA Box Binding Protein-Like Proteins, TATA-Box Binding Protein, Transcription}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Isogai, Yoh and Keles, S{\"u}nd{\"u}z and Prestel, Matthias and Hochheimer, Andreas and Tjian, Robert} } @article {2407, title = {An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis.}, journal = {Nature}, volume = {442}, year = {2006}, month = {2006 Aug 10}, pages = {700-4}, abstract = {

The sterol regulatory element binding protein (SREBP) family of transcription activators are critical regulators of cholesterol and fatty acid homeostasis. We previously demonstrated that human SREBPs bind the CREB-binding protein (CBP)/p300 acetyltransferase KIX domain and recruit activator-recruited co-factor (ARC)/Mediator co-activator complexes through unknown mechanisms. Here we show that SREBPs use the evolutionarily conserved ARC105 (also called MED15) subunit to activate target genes. Structural analysis of the SREBP-binding domain in ARC105 by NMR revealed a three-helix bundle with marked similarity to the CBP/p300 KIX domain. In contrast to SREBPs, the CREB and c-Myb activators do not bind the ARC105 KIX domain, although they interact with the CBP KIX domain, revealing a surprising specificity among structurally related activator-binding domains. The Caenorhabditis elegans SREBP homologue SBP-1 promotes fatty acid homeostasis by regulating the expression of lipogenic enzymes. We found that, like SBP-1, the C. elegans ARC105 homologue MDT-15 is required for fatty acid homeostasis, and show that both SBP-1 and MDT-15 control transcription of genes governing desaturation of stearic acid to oleic acid. Notably, dietary addition of oleic acid significantly rescued various defects of nematodes targeted with RNA interference against sbp-1 and mdt-15, including impaired intestinal fat storage, infertility, decreased size and slow locomotion, suggesting that regulation of oleic acid levels represents a physiologically critical function of SBP-1 and MDT-15. Taken together, our findings demonstrate that ARC105 is a key effector of SREBP-dependent gene regulation and control of lipid homeostasis in metazoans.

}, keywords = {Amino Acid Sequence, Animals, Biomolecular, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Cholesterol, Homeostasis, Humans, Lipid Metabolism, Mediator Complex, Mice, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Protein Binding, Protein Structure, Sterol Regulatory Element Binding Proteins, Tertiary, Transcription Factors, Transcriptional Activation}, issn = {1476-4687}, doi = {10.1073/pnas.1100640108}, author = {Yang, Fajun and Vought, Bryan W and Satterlee, John S and Walker, Amy K and Jim Sun, Z-Y and Watts, Jennifer L and DeBeaumont, Rosalie and Saito, R Mako and Hyberts, Sven G and Yang, Shaosong and Macol, Christine and Iyer, Lakshmanan and Tjian, Robert and van den Heuvel, Sander and Hart, Anne C and Wagner, Gerhard and N{\"a}{\"a}r, Anders M} } @article {2411, title = {Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {103}, year = {2006}, month = {2006 Feb 21}, pages = {2594-9}, abstract = {

Cell-type-selective expression of the TFIID subunit TAF(II)105 (renamed TAF4b) in the ovary is essential for proper follicle development. Although a multitude of signaling pathways required for folliculogenesis have been identified, downstream transcriptional integrators of these signals remain largely unknown. Here, we show that TAF4b controls the granulosa-cell-specific expression of the proto-oncogene c-jun, and together they regulate transcription of ovary-selective promoters. Instead of using cell-type-specific activators, our findings suggest that the coactivator TAF4b regulates the expression of tissue-specific genes, at least in part, through the cell-type-specific induction of c-jun, a ubiquitous activator. Importantly, the loss of TAF4b in ovarian granulosa cells disrupts cellular morphologies and interactions during follicle growth that likely contribute to the infertility observed in TAF4b-null female mice. These data highlight a mechanism for potentiating tissue-selective functions of the basal transcription machinery and reveal intricate networks of gene expression that orchestrate ovarian-specific functions and cell morphology.

}, keywords = {Animals, Developmental, Female, Gene Expression Regulation, Genetic, Gonadotropins, Granulosa Cells, Horses, Messenger, Mice, Mutant Strains, Ovarian Follicle, Ovary, Promoter Regions, Proto-Oncogene Proteins c-jun, Rats, RNA, TATA-Binding Protein Associated Factors, Transcription, Transcription Factor TFIID, Up-Regulation}, issn = {0027-8424}, doi = {10.1073/pnas.1100640108}, author = {Geles, Kenneth G and Freiman, Richard N and Liu, Wei-Li and Zheng, Shuang and Voronina, Ekaterina and Tjian, Robert} } @article {2408, title = {Coactivator cross-talk specifies transcriptional output.}, journal = {Genes \& Development}, volume = {20}, year = {2006}, month = {2006 Jun 1}, pages = {1458-69}, abstract = {

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. Here we have used RNA interference (RNAi) in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. We exploited the robust response of the metallothionein genes to heavy metal as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, we find that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a "checkpoint complex" for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, we find that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, we find that the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation.

}, keywords = {Animals, Cells, Chromatin Immunoprecipitation, Cultured, Drosophila, Gene Expression, Genetic, Metallothionein, Models, Polymerase Chain Reaction, Promoter Regions, RNA Interference, Transcription}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Marr, Michael T and Isogai, Yoh and Wright, Kevin J and Tjian, Robert} } @article {2410, title = {Cryo-electron microscopy studies of human TFIID: conformational breathing in the integration of gene regulatory cues.}, journal = {Structure}, volume = {14}, year = {2006}, month = {2006 Mar}, pages = {511-20}, abstract = {

The multisubunit transcription factor TFIID is essential for directing eukaryotic promoter recognition and mediating interactions with activators/cofactors during assembly of the preinitiation complex. Despite its central role in transcription initiation and regulation, structural knowledge of the TFIID complex has so far been largely limited to electron microscopy studies of negatively stained samples. Here, we present a cryo-electron microscopy 3D reconstruction of the large endogenous human TFIID complex. The improved cryopreservation has allowed for a more detailed definition of the structural elements in the complex and for the detection, by an extensive statistical analysis of the data, of a conformational opening and closing of the cavity central to the TFIID architecture. We propose that these density rearrangements in the structure are a likely reflection of the plasticity of the interactions between TFIID and its many partner proteins.

}, keywords = {Cryoelectron Microscopy, Gene Expression Regulation, Genetic, Humans, Protein Binding, Protein Structure, Tertiary, Transcription, Transcription Factor TFIID}, issn = {0969-2126}, doi = {10.1073/pnas.1100640108}, author = {Grob, Patricia and Cruse, Michael J and Inouye, Carla and Peris, Marian and Penczek, Pawel A and Tjian, Robert and Nogales, Eva} } @article {2409, title = {Nutrient availability and growth: regulation of insulin signaling by dFOXO/FOXO1.}, journal = {Cell Cycle}, volume = {5}, year = {2006}, month = {2006 Mar}, pages = {503-5}, abstract = {

Organisms adjust their rate of growth depending on the availability of nutrients. Thus, when environmental conditions limit nutrients, growth is slowed and is only restored after food again becomes abundant. Many aspects of the molecular mechanisms that govern this complex control system remain unknown. However, it has been shown that the insulin/IGF-1 (insulin-like growth factor 1) receptor pathway, together with the FOXO family of transcription factors, play an important role in this process. Recent studies with the fruit fly Drosophila melanogaster have provided new insights into the regulatory circuitry that controls both growth and gene expression in response to nutrient availability.

}, keywords = {Animal Nutritional Physiological Phenomena, Animals, Drosophila melanogaster, Drosophila Proteins, Forkhead Transcription Factors, Insulin, Receptor, Signal Transduction}, issn = {1551-4005}, doi = {10.1073/pnas.1100640108}, author = {Puig, Oscar and Tjian, Robert} } @article {2406, title = {TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {103}, year = {2006}, month = {2006 Aug 15}, pages = {12347-52}, abstract = {

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, we find that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. Our analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on our results, we propose that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID.

}, keywords = {Animals, Cell Line, Drosophila melanogaster, Drosophila Proteins, Enzyme Stability, Gene Expression Regulation, Genetic, Humans, Macromolecular Substances, Models, Molecular, Protein Subunits, RNA Interference, TATA Box, TATA-Binding Protein Associated Factors, Transcription, Transcription Factor TFIID}, issn = {0027-8424}, doi = {10.1073/pnas.1100640108}, author = {Wright, Kevin J and Marr, Michael T and Tjian, Robert} } @article {2412, title = {In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.}, journal = {Cell}, volume = {123}, year = {2005}, month = {2005 Dec 29}, pages = {1241-53}, abstract = {

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington{\textquoteright}s disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. Here, we report the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. We demonstrate that both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. Our results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus.

}, keywords = {Animals, Biological Assay, Cell-Free System, Cells, Cercopithecus aethiops, COS Cells, Cultured, Dopamine D2, Down-Regulation, Genetic, Mice, Mutation, Nerve Tissue Proteins, Neurons, Nuclear Proteins, Protein Binding, Protein Subunits, Rats, Receptors, Sp1 Transcription Factor, TFII, Transcription, Transcription Factor TFIID, Transcription Factors}, issn = {0092-8674}, doi = {10.1073/pnas.1100640108}, author = {Zhai, Weiguo and Jeong, Hyunkyung and Cui, Libin and Krainc, Dimitri and Tjian, Robert} } @article {2414, title = {Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID.}, journal = {Genes \& Development}, volume = {19}, year = {2005}, month = {2005 Apr 1}, pages = {794-803}, abstract = {

The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF(II)105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult.

}, keywords = {Aging, Animals, Cell Division, Infertility, Male, Mice, Spermatids, Spermatogenesis, Spermatogonia, TATA-Binding Protein Associated Factors, Testis, Transcription Factor TFIID}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Falender, Allison E and Freiman, Richard N and Geles, Kenneth G and Lo, Kirk C and Hwang, KeumSil and Lamb, Dolores J and Morris, Patricia L and Tjian, Robert and Richards, JoAnne S} } @article {2415, title = {Structural studies of the human PBAF chromatin-remodeling complex.}, journal = {Structure}, volume = {13}, year = {2005}, month = {2005 Feb}, pages = {267-75}, abstract = {

ATP-dependent chromatin remodeling is one of the central processes responsible for imparting fluidity to chromatin and thus regulating DNA transactions. Although knowledge on this process is accumulating rapidly, the basic mechanism (or mechanisms) by which the remodeling complexes alter the structure of a nucleosome is not yet understood. Structural information on these macromolecular machines should aid in interpreting the biochemical and genetic data; to this end, we have determined the structure of the human PBAF ATP-dependent chromatin-remodeling complex preserved in negative stain by electron microscopy and have mapped the nucleosome binding site using two-dimensional (2D) image analysis. PBAF has an overall C-shaped architecture{\textendash}with a larger density to which two smaller knobs are attached{\textendash}surrounding a central cavity; one of these knobs appears to be flexible and occupies different positions in each of the structures determined. The 2D analysis of PBAF:nucleosome complexes indicates that the nucleosome binds in the central cavity.

}, keywords = {Binding Sites, Chromosomal Proteins, Humans, Imaging, Molecular Conformation, Non-Histone, Nucleosomes, Three-Dimensional, Transcription Factors}, issn = {0969-2126}, doi = {10.1073/pnas.1100640108}, author = {Leschziner, Andres E and Lemon, Bryan and Tjian, Robert and Nogales, Eva} } @article {2413, title = {Transcriptional feedback control of insulin receptor by dFOXO/FOXO1.}, journal = {Genes \& Development}, volume = {19}, year = {2005}, month = {2005 Oct 15}, pages = {2435-46}, abstract = {

The insulin signaling pathway, which is conserved in evolution from flies to humans, evolved to allow a fast response to changes in nutrient availability while keeping glucose concentration constant in serum. Here we show that, both in Drosophila and mammals, insulin receptor (InR) represses its own synthesis by a feedback mechanism directed by the transcription factor dFOXO/FOXO1. In Drosophila, dFOXO is responsible for activating transcription of dInR, and nutritional conditions can modulate this effect. Starvation up-regulates mRNA of dInR in wild-type but not dFOXO-deficient flies. Importantly, FOXO1 acts in mammalian cells like its Drosophila counterpart, up-regulating the InR mRNA level upon fasting. Mammalian cells up-regulate the InR mRNA in the absence of serum, conditions that induce the dephosphorylation and activation of FOXO1. Interestingly, insulin is able to reverse this effect. Therefore, dFOXO/FOXO1 acts as an insulin sensor to activate insulin signaling, allowing a fast response to the hormone after each meal. Our results reveal a key feedback control mechanism for dFOXO/FOXO1 in regulating metabolism and insulin signaling.

}, keywords = {Animals, Cell Line, Drosophila, Drosophila Proteins, Evolution, Food Deprivation, Forkhead Transcription Factors, Genetic, Humans, Insulin, Messenger, Mice, Molecular, Receptor Protein-Tyrosine Kinases, RNA, Signal Transduction, Transcription, Up-Regulation}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Puig, Oscar and Tjian, Robert} } @article {2417, title = {Distinct conformational states of nuclear receptor-bound CRSP-Med complexes.}, journal = {Nature Structural \& Molecular Biology}, volume = {11}, year = {2004}, month = {2004 Jul}, pages = {664-71}, abstract = {

The human CRSP-Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-Med complex. To test this, we conducted a structural analysis of CRSP-Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP-Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- Med structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of Med220 within the complex.

}, keywords = {Bacterial Proteins, Binding Sites, Calcitriol, Genetic, Hela Cells, Humans, Protein Binding, Protein Conformation, Receptors, Thyroid Hormone, Trans-Activators, Transcription}, issn = {1545-9993}, doi = {10.1073/pnas.1100640108}, author = {Taatjes, Dylan J and Schneider-Poetsch, Tilman and Tjian, Robert} } @article {2420, title = {Myc-interacting protein 1 target gene profile: a link to microtubules, extracellular signal-regulated kinase, and cell growth.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {101}, year = {2004}, month = {2004 Jan 13}, pages = {458-63}, abstract = {

To study the role of the transcription factor Myc-interacting protein 1 (MIZ-1) in activating various target genes after induction with the microtubule disrupting agent T113242, we have used small interfering RNA duplexes (siRNAs) to knockdown the expression of MIZ-1. As expected, depletion of MIZ-1 resulted in the inhibition of T113242-dependent activation of the low-density lipoprotein receptor (LDLR) gene in hepatocytes. Cells transfected with MIZ-1 siRNAs also exhibited growth arrest. In addition, inhibition of the extracellular signal-regulated kinase (ERK) pathway inhibited T113242-induced nuclear accumulation of MIZ-1 and activation of LDLR. Gene expression microarray analysis under various induction conditions identified other T113242-activated genes affected by a decrease in MIZ-1 and inhibition of the ERK pathway. We also found that the accumulation of MIZ-1 in the nucleus is influenced by cell-cell contact and/or growth. Taken together, our studies suggest that MIZ-1 regulates a specific set of genes that includes LDLR and that the ERK pathway plays a role in the activation of target promoters by MIZ-1.

}, keywords = {Cell Division, Cell Line, DNA-Binding Proteins, Flow Cytometry, Fluorescent Antibody Technique, Gene Expression Profiling, Humans, Indirect, Kruppel-Like Transcription Factors, Mitogen-Activated Protein Kinases, RNA Interference, Transcription Factors}, issn = {0027-8424}, doi = {10.1073/pnas.1100640108}, author = {Ziegelbauer, Joseph and Wei, Joyce and Tjian, Robert} } @article {2416, title = {Polybromo protein BAF180 functions in mammalian cardiac chamber maturation.}, journal = {Genes \& Development}, volume = {18}, year = {2004}, month = {2004 Dec 15}, pages = {3106-16}, abstract = {

BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRalpha, VDR, and PPARgamma. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRalpha and PPARgamma. Embryonic aggregation analyses reveal that in contrast to PPARgamma-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARbeta2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARbeta2. These studies reveal unique functions of PBAF in cardiac chamber maturation.

}, keywords = {Animals, Cell Aggregation, Chromatin Assembly and Disassembly, Congenital, Developmental, DNA Primers, Gene Expression Regulation, Heart Defects, Immunoprecipitation, In Situ Hybridization, In Situ Nick-End Labeling, Knockout, Mice, Nuclear Proteins, Oligonucleotide Array Sequence Analysis, Placenta, Receptors, Retinoic Acid, Reverse Transcriptase Polymerase Chain Reaction, S100 Proteins, Transcription Factors}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Wang, Zhong and Zhai, Weiguo and Richardson, James A and Olson, Eric N and Meneses, Juanito J and Firpo, Meri T and Kang, Chulho and Skarnes, William C and Tjian, Robert} } @article {2419, title = {Regulatory diversity among metazoan co-activator complexes.}, journal = {Nature Reviews. Molecular Cell Biology}, volume = {5}, year = {2004}, month = {2004 May}, pages = {403-10}, abstract = {

Transcription is a stepwise process that involves many specialized proteins and protein complexes, all of which must work together to express a given gene in a spatially and temporally regulated manner. An integral step in this regulatory process is carried out by large, multisubunit co-activator complexes, which have diverse roles in transcriptional control. Their diversity and large size allows for many potential regulatory inputs, but how is the versatility and specificity of these co-activator complexes determined?

}, keywords = {Animals, DNA-Binding Proteins, Evolution, Gene Expression Regulation, Genetic, Genetic Variation, Humans, Molecular, Promoter Regions, Repressor Proteins, Species Specificity, Transcription, Transcription Factors}, issn = {1471-0072}, doi = {10.1073/pnas.1100640108}, author = {Taatjes, Dylan J and Marr, Michael T and Tjian, Robert} } @article {2418, title = {Structure and function of CRSP/Med2; a promoter-selective transcriptional coactivator complex.}, journal = {Molecular Cell}, volume = {14}, year = {2004}, month = {2004 Jun 4}, pages = {675-83}, abstract = {

The multi-subunit, human CRSP coactivator-also known as Mediator (Med)-regulates transcription by mediating signals between enhancer-bound factors (activators) and the core transcriptional machinery. Interestingly, different activators are known to bind distinct subunits within the CRSP/Med complex. We have isolated a stable, endogenous CRSP/Med complex (CRSP/Med2) that specifically lacks both the Med220 and the Med70 subunits. The three-dimensional structure of CRSP/Med2 was determined to 31 A resolution using electron microscopy and single-particle reconstruction techniques. Despite lacking both Med220 and Med70, CRSP/Med2 displays potent, activator-dependent transcriptional coactivator function in response to VP16, Sp1, and Sp1/SREBP-1a in vitro using chromatin templates. However, CRSP/Med2 is unable to potentiate activated transcription from a vitamin D receptor-responsive promoter, which requires interaction with Med220 for coactivator recruitment, whereas VDR-directed activation by CRSP/Med occurs normally. Thus, it appears that CRSP/Med may be regulated by a combinatorial assembly mechanism that allows promoter-selective function upon exchange of specific coactivator targets.

}, keywords = {CCAAT-Enhancer-Binding Proteins, DNA-Binding Proteins, Genes, Genetic, Hela Cells, Herpes Simplex Virus Protein Vmw65, Humans, Macromolecular Substances, Promoter Regions, Protein Structure, Protein Subunits, Regulator, Sp1 Transcription Factor, Sterol Regulatory Element Binding Protein 1, Tertiary, Trans-Activators, Transcription Factors, Transcriptional Activation, Vitamin D Response Element}, issn = {1097-2765}, doi = {10.1073/pnas.1100640108}, author = {Taatjes, Dylan J and Tjian, Robert} } @article {2425, title = {Bromodomains mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries.}, journal = {Molecular Cell}, volume = {11}, year = {2003}, month = {2003 Feb}, pages = {365-76}, abstract = {

Bromodomains bind acetylated histone H4 peptides in vitro. Since many chromatin remodeling complexes and the general transcription factor TFIID contain bromodomains, they may link histone acetylation to increased transcription. Here we show that yeast Bdf1 bromodomains recognize endogenous acetyl-histone H3/H4 as a mechanism for chromatin association in vivo. Surprisingly, deletion of BDF1 or a Bdf1 mutation that abolishes histone binding leads to transcriptional downregulation of genes located at heterochromatin-euchromatin boundaries. Wild-type Bdf1 protein imposes a physical barrier to the spreading of telomere- and mating-locus-proximal SIR proteins. Biochemical experiments indicate that Bdf1 competes with the Sir2 deacetylase for binding to acetylated histone H4. These data suggest an active role for Bdf1 in euchromatin maintenance and antisilencing through a histone tail-encoded boundary function.

}, keywords = {Acetylation, Biological, Down-Regulation, Fungal, Genes, Heterochromatin, Histones, Hydrogen Bonding, Models, Mutation, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Silent Information Regulator Proteins, Transcription Factor TFIID, Transcription Factors}, issn = {1097-2765}, doi = {10.1073/pnas.1100640108}, author = {Ladurner, Andreas G and Inouye, Carla and Jain, Rajan and Tjian, Robert} } @article {2421, title = {Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway.}, journal = {Genes \& Development}, volume = {17}, year = {2003}, month = {2003 Aug 15}, pages = {2006-20}, abstract = {

The Drosophila insulin receptor (dInR) regulates cell growth and proliferation through the dPI3K/dAkt pathway, which is conserved in metazoan organisms. Here we report the identification and functional characterization of the Drosophila forkhead-related transcription factor dFOXO, a key component of the insulin signaling cascade. dFOXO is phosphorylated by dAkt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant dFOXO lacking dAkt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. dFOXO activation in S2 cells induces growth arrest and activates two key players of the dInR/dPI3K/dAkt pathway: the translational regulator d4EBP and the dInR itself. Induction of d4EBP likely leads to growth inhibition by dFOXO, whereas activation of dInR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of dFOXO in fly tissues regulates organ size by specifying cell number with no effect on cell size. Our results establish dFOXO as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation.

}, keywords = {Amino Acid, Amino Acid Sequence, Animals, Biological, Cell Count, Cell Division, Cell Nucleus, Chromones, Drosophila, Drosophila Proteins, Enzyme Inhibitors, Feedback, Forkhead Transcription Factors, Gene Expression Regulation, Insect Proteins, Insulin, Models, Molecular Sequence Data, Morpholines, Physiological, Post-Translational, Protein Processing, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Receptor, Recombinant Proteins, Sequence Homology, Signal Transduction, Trans-Activators, Transcription Factors}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Puig, Oscar and Marr, Michael T and Ruhf, M Laure and Tjian, Robert} } @article {2424, title = {Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression.}, journal = {Genes \& Development}, volume = {17}, year = {2003}, month = {2003 Jun 1}, pages = {1309-20}, keywords = {Animals, Developmental, Drosophila, Drosophila Proteins, Gene Expression Regulation, Genetic, Male, Messenger, Organ Specificity, Promoter Regions, RNA, RNA Polymerase III, Spermatogenesis, TATA Box Binding Protein-Like Proteins, Telomeric Repeat Binding Protein 2, Transcription}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {Hochheimer, Andreas and Tjian, Robert} } @article {2426, title = {Regulating the regulators: lysine modifications make their mark.}, journal = {Cell}, volume = {112}, year = {2003}, month = {2003 Jan 10}, pages = {11-7}, abstract = {

Decades of research have uncovered much of the molecular machinery responsible for establishing and maintaining proper gene transcription patterns in eukaryotes. Although the composition of this machinery is largely known, mechanisms regulating its activity by covalent modification are just coming into focus. Here, we review several cases of ubiquitination, sumoylation, and acetylation that link specific covalent modification of the transcriptional apparatus to their regulatory function. We propose that potential cascades of modifications serve as molecular rheostats that fine-tune the control of transcription in diverse organisms.

}, keywords = {Acetylation, Basic-Leucine Zipper Transcription Factors, Cell Nucleus, DNA-Binding Proteins, Gene Expression Regulation, Genetic, Humans, Lysine, Messenger, Models, Promoter Regions, RNA, RNA Polymerase II, Saccharomyces cerevisiae Proteins, Small Ubiquitin-Related Modifier Proteins, Trans-Activators, Transcription, Transcription Factors, Transcriptional Activation, Ubiquitins}, issn = {0092-8674}, doi = {10.1073/pnas.1100640108}, author = {Freiman, Richard N and Tjian, Robert} } @article {2423, title = {Targeting genes and transcription factors to segregated nuclear compartments.}, journal = {Current Opinion in Cell Biology}, volume = {15}, year = {2003}, month = {2003 Jun}, pages = {296-303}, abstract = {

With increasingly detailed images of nuclear structures revealed by advanced microscopy, a remarkably compartmentalized cell nucleus has come into focus. Although this complex nuclear organization remains largely unexplored, some progress has been made in deciphering the functional aspects of various subnuclear structures, revealing how this elaborate framework can influence gene activation. Several recent studies have helped illustrate how cells might utilize the nuclear architecture as an additional level of transcriptional control, perhaps by targeting genes and regulatory factors to specific sites within the nucleus that are designated for active RNA synthesis.

}, keywords = {Animals, Cell Nucleus Structures, Gene Expression, Humans, Protein Transport, Transcription Factors}, issn = {0955-0674}, doi = {10.1073/pnas.1100640108}, author = {Isogai, Yoh and Tjian, Robert} } @article {2422, title = {Transcription regulation and animal diversity.}, journal = {Nature}, volume = {424}, year = {2003}, month = {2003 Jul 10}, pages = {147-51}, abstract = {

Whole-genome sequence assemblies are now available for seven different animals, including nematode worms, mice and humans. Comparative genome analyses reveal a surprising constancy in genetic content: vertebrate genomes have only about twice the number of genes that invertebrate genomes have, and the increase is primarily due to the duplication of existing genes rather than the invention of new ones. How, then, has evolutionary diversity arisen? Emerging evidence suggests that organismal complexity arises from progressively more elaborate regulation of gene expression.

}, keywords = {Animals, Chromatin, DNA, Ecosystem, Gene Expression Regulation, Genetic, Humans, Nucleic Acid, Regulatory Sequences, Transcription, Transcription Factors}, issn = {1476-4687}, doi = {10.1073/pnas.1100640108}, author = {Levine, Michael and Tjian, Robert} } @article {2430, title = {Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation.}, journal = {Genes \& Development}, volume = {16}, year = {2002}, month = {2002 Jun 1}, pages = {1339-44}, abstract = {

Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

}, keywords = {Binding Sites, Chromatin, Electron, Electrophoresis, Genetic, Glutathione Transferase, Hela Cells, Humans, Immunoblotting, Ligands, Microscopy, Models, Molecular, Polyacrylamide Gel, Protein Binding, Protein Conformation, Protein Structure, Recombinant Fusion Proteins, RNA Polymerase II, Tertiary, Trans-Activators, Transcription}, issn = {0890-9369}, doi = {10.1073/pnas.1100640108}, author = {N{\"a}{\"a}r, Anders M and Taatjes, Dylan J and Zhai, Weiguo and Nogales, Eva and Tjian, Robert} } @article {2429, title = {Neurodegeneration. A glutamine-rich trail leads to transcription factors.}, journal = {Science }, volume = {296}, year = {2002}, month = {2002 Jun 21}, pages = {2149-50}, keywords = {Animals, Brain, CREB-Binding Protein, DNA-Binding Proteins, Dopamine D2, Gene Expression Regulation, Genetic, Humans, Huntington Disease, Mice, Models, Nerve Tissue Proteins, Neurodegenerative Diseases, Neurons, Nuclear Proteins, Peptides, Promoter Regions, Protein Binding, Protein Structure, Receptors, Sp1 Transcription Factor, TATA-Binding Protein Associated Factors, Tertiary, Trans-Activators, Transcription, Transcription Factor TFIID, Transcription Factors, Trinucleotide Repeat Expansion}, issn = {1095-9203}, doi = {10.1073/pnas.1100640108}, author = {Freiman, Richard N and Tjian, Robert} } @article {2428, title = {Redundant role of tissue-selective TAF(II)105 in B lymphocytes.}, journal = {Molecular and Cellular Biology}, volume = {22}, year = {2002}, month = {2002 Sep}, pages = {6564-72}, abstract = {

Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAF(II)s). The recent discovery of multiple TBP-related factors and tissue-specific TAF(II)s suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAF(II)105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAF(II)105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAF(II)105, we have generated TAF(II)105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAF(II)105. TAF(II)105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAF(II)105 in B cells is likely redundant with the function of other TAF(II)105-related cellular proteins.

}, keywords = {Animals, B-Lymphocytes, Blotting, Cell Division, Cell Separation, DNA-Binding Proteins, Dose-Response Relationship, Drug, Erythrocytes, Flow Cytometry, Genetic, Immunoglobulin A, Immunoglobulin G, Immunoglobulin M, Lipopolysaccharides, Messenger, Mice, Precipitin Tests, RNA, Spleen, TATA-Binding Protein Associated Factors, Time Factors, Transcription, Transcription Factor TFIID, Transcription Factors, Transcriptional Activation, Western}, issn = {0270-7306}, doi = {10.1073/pnas.1100640108}, author = {Freiman, Richard N and Albright, Shane R and Chu, Leslie E and Zheng, Shuang and Liang, Hong-Erh and Sha, William C and Tjian, Robert} } @article {2431, title = {Structure, function, and activator-induced conformations of the CRSP coactivator.}, journal = {Science}, volume = {295}, year = {2002}, month = {2002 Feb 8}, pages = {1058-62}, abstract = {

The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.

}, keywords = {CCAAT-Enhancer-Binding Proteins, Chromatin, Computer-Assisted, DNA-Binding Proteins, Electron, Genetic, Hela Cells, Herpes Simplex Virus Protein Vmw65, Humans, Image Processing, Imaging, Macromolecular Substances, Microscopy, Models, Precipitin Tests, Protein Binding, Protein Structure, Protein Subunits, Quaternary, Recombinant Fusion Proteins, Recombinant Proteins, Sterol Regulatory Element Binding Protein 1, Tertiary, Three-Dimensional, Trans-Activators, Transcription, Transcription Factors, Transcriptional Activation}, issn = {1095-9203}, doi = {10.1073/pnas.1100640108}, author = {Taatjes, Dylan J and N{\"a}{\"a}r, Anders M and Andel, Frank and Nogales, Eva and Tjian, Robert} } @article {2427, title = {TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila.}, journal = {Nature}, volume = {420}, year = {2002}, month = {2002 Nov 28}, pages = {439-45}, abstract = {

Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. Here we report the antibody affinity purification of a Drosophila TRF2-containing complex that contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding led us to potential target genes containing TRF2-responsive promoters. We have used a combination of in vitro and in vivo assays to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. We also identified additional TRF2-responsive target genes involved in DNA replication and cell proliferation. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila.

}, keywords = {Animals, Cell Line, Chromatin, Drosophila, Drosophila Proteins, Gene Expression Regulation, Genes, Genetic, Insect, Macromolecular Substances, Nuclease Protection Assays, Oligonucleotide Array Sequence Analysis, Precipitin Tests, Proliferating Cell Nuclear Antigen, Promoter Regions, Protein Binding, RNA Interference, Substrate Specificity, Telomeric Repeat Binding Protein 2, Transcription Factors, Transcription Initiation Site}, issn = {0028-0836}, doi = {10.1073/pnas.1100640108}, author = {Hochheimer, Andreas and Zhou, Sharleen and Zheng, Shuang and Holmes, Michael C and Tjian, Robert} }