@article {68226, title = {Kinetic principles underlying pioneer function of GAGA transcription factor in live cells.}, journal = {Nature Structural and Molecular Biology}, volume = {29}, year = {2022}, month = {2022 Jul 01}, pages = {665-676}, abstract = {

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF{\textquoteright}s DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF{\textquoteright}s neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

}, keywords = {Animals, Chromatin, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Kinetics, Transcription Factors}, issn = {1545-9985}, doi = {10.1038/s41594-022-00800-z}, author = {Tang, Xiaona and Li, Taibo and Liu, Sheng and Wisniewski, Jan and Zheng, Qinsi and Rong, Yikang and Lavis, Luke D and Wu, Carl} } @article {67647, title = {Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin.}, journal = {eLife}, volume = {10}, year = {2021}, month = {2021 Jul 27}, abstract = {

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending {\textquoteright}tug-of-war{\textquoteright} that controls a temporally shifting window of accessibility for the transcription initiation machinery.

}, issn = {2050-084X}, doi = {10.7554/eLife.69387}, author = {Kim, Jee Min and Visanpattanasin, Pat and Jou, Vivian and Liu, Sheng and Tang, Xiaona and Zheng, Qinsi and Li, Kai Yu and Snedeker, Jonathan and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {67671, title = {Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.}, journal = {Molecular Cell}, volume = {81}, year = {2021}, month = {2021 Sep 02}, pages = {3560-3575}, abstract = {

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S.\ cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator{\textquoteright}s key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2021.07.022}, author = {Nguyen, Vu Q and Ranjan, Anand and Liu, Sheng and Tang, Xiaona and Ling, Yick Hin and Wisniewski, Jan and Mizuguchi, Gaku and Li, Kai Yu and Jou, Vivian and Zheng, Qinsi and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {65581, title = {Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 Apr 27}, abstract = {

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

}, issn = {2050-084X}, doi = {10.7554/eLife.55667}, author = {Ranjan, Anand and Nguyen, Vu Q and Liu, Sheng and Wisniewski, Jan and Kim, Jee Min and Tang, Xiaona and Mizuguchi, Gaku and Elalaoui, Ejlal and Nickels, Timothy J and Jou, Vivian and English, Brian P and Zheng, Qinsi and Luk, Ed and Lavis, Luke D and Lionnet, Timoth{\'e}e and Wu, Carl} } @article {65176, title = {Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.}, journal = {ACS Central Science}, volume = {5}, year = {2019}, month = {2019 Sep 25}, pages = {1602-1613}, abstract = {

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.9b00676}, author = {Zheng, Qinsi and Ayala, Anthony X and Chung, Inhee and Weigel, Aubrey V and Ranjan, Anand and Falco, Natalie and Grimm, Jonathan B and Tkachuk, Ariana N and Wu, Carl and Lippincott-Schwartz, Jennifer and Singer, Robert H and Lavis, Luke D} } @article {48908, title = {Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.}, journal = {Genes \& Development}, volume = {31}, year = {2017}, month = {2017 Oct 01}, pages = {1958-1972}, abstract = {

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

}, issn = {1549-5477}, doi = {10.1101/gad.304782.117}, author = {Xiao, Hua and Wang, Feng and Wisniewski, Jan and Shaytan, Alexey K and Ghirlando, Rodolfo and Fitzgerald, Peter C and Huang, Yingzi and Wei, Debbie and Li, Shipeng and Landsman, David and Panchenko, Anna R and Wu, Carl} } @article {65034, title = {Comment on "A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme".}, journal = {Science}, volume = {353}, year = {2016}, month = {2016 Jul 22}, pages = {358}, abstract = {

Watanabe et al (Reports, 12 April 2013, p. 195) study the yeast SWR1/SWR-C complex responsible for depositing the histone variant H2A.Z by replacing nucleosomal H2A with H2A.Z. They report that reversal of H2A.Z replacement is mediated by SWR1 and related INO80 on an H2A.Z nucleosome carrying H3K56Q. Using multiple assays and reaction conditions, we find no evidence of such reversal of H2A.Z exchange.

}, keywords = {Adenosine Triphosphatases, Chromatin Assembly and Disassembly, Histones, Multienzyme Complexes, Nucleosomes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1095-9203}, doi = {10.1126/science.aad5921}, author = {Wang, Feng and Ranjan, Anand and Wei, Debbie and Wu, Carl} } @article {46973, title = {H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Jun 27}, abstract = {

The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

}, issn = {2050-084X}, doi = {10.7554/eLife.06845}, author = {Ranjan, Anand and Wang, Feng and Mizuguchi, Gaku and Wei, Debbie and Huang, Yingzi and Wu, Carl} } @article {6910, title = {The catalytic subunit of the SWR1 remodeler is a histone chaperone for the H2A.Z-H2B dimer.}, journal = {Molecular Cell}, volume = {53}, year = {2014}, month = {2014 Feb 6}, pages = {498-505}, abstract = {

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 {\r A} resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2014.01.010}, author = {Hong, Jingjun and Feng, Hanqiao and Wang, Feng and Ranjan, Anand and Chen, Jianhong and Jiang, Jiansheng and Ghirlando, Rodolfo and Xiao, T Sam and Wu, Carl and Bai, Yawen} } @article {45166, title = {Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 May 20}, pages = {e02203}, abstract = {

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.02203}, author = {Wisniewski, Jan and Hajj, Bassam and Chen, Jiji and Mizuguchi, Gaku and Xiao, Hua and Wei, Debbie and Dahan, Maxime and Wu, Carl} } @article {45778, title = {Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, year = {2014}, month = {2014 Dec 9}, pages = {17480-5}, abstract = {

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 {\textmu}m. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an \~{}4-{\textmu}m-deep volume, with lateral and axial localization precisions of \~{}20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

}, issn = {1091-6490}, doi = {10.1073/pnas.1412396111}, author = {Hajj, Bassam and Wisniewski, Jan and El Beheiry, Mohamed and Chen, Jiji and Revyakin, Andrey and Wu, Carl and Dahan, Maxime} } @article {5522, title = {Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013}, pages = {60-3}, abstract = {

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2277}, author = {Abrahamsson, Sara and Chen, Jiji and Hajj, Bassam and Stallinga, Sjoerd and Katsov, Alexander Y and Wisniewski, Jan and Mizuguchi, Gaku and Soule, Pierre and Mueller, Florian and Darzacq, Claire Dugast and Darzacq, Xavier and Wu, Carl and Bargmann, Cornelia I and Agard, David A and Dahan, Maxime and Gustafsson, Mats G L} } @article {6413, title = {Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.}, journal = {Cell}, volume = {154}, year = {2013}, month = {2013 Sep 12}, pages = {1220-31}, abstract = {

The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer\ for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.08.018}, author = {Nguyen, Vu Q and Ranjan, Anand and Stengel, Florian and Wei, Debbie and Aebersold, Ruedi and Wu, Carl and Leschziner, Andres E} } @article {6412, title = {Nucleosome-free region dominates histone acetylation in targeting SWR1 to promoters for H2A.Z replacement.}, journal = {Cell}, volume = {154}, year = {2013}, month = {2013 Sep 12}, pages = {1232-45}, abstract = {

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S.\ cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2013.08.005}, author = {Ranjan, Anand and Mizuguchi, Gaku and Fitzgerald, Peter C and Wei, Debbie and Wang, Feng and Huang, Yingzi and Luk, Ed and Woodcock, Christopher L and Wu, Carl} } @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} }