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.

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

The molecular and cellular architecture of the organs in a whole mouse is revealed through optical clearing.

A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms governing learning and memory. We developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) insertion in vivo by pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach allows for single-synapse resolution maps of plasticity in genetically targeted neurons during memory formation. We investigated the relationship between synapse-level and cell-level memory encodings by mapping synaptic plasticity and cFos expression in hippocampal CA1 pyramidal cells upon contextual fear conditioning (CFC). We observed a strong correlation between synaptic plasticity and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins.

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems. Expected final online publication date for the , Volume 45 is July 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems. Expected final online publication date for the , Volume 45 is July 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems.

Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable 'confined target search' mechanism. PTFs such as FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-PTF MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domains, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested that transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.