Managing the Steady State Chromatin Landscape by Nucleosome Dynamics.

Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.

Slow Down to Catch Up.

In this issue of Cell, Cassidy et al. (2019) show that, in Drosophila melanogaster, developmental abnormalities resulting from loss of repressors such as microRNAs can be suppressed by slow metabolism. They additionally provide insight into the underlying mechanism that connects metabolic state with developmental outcomes.

An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II.

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Transcriptional Regulators Compete with Nucleosomes Post-replication.

Every nucleosome across the genome must be disrupted and reformed when the replication fork passes, but how chromatin organization is re-established following replication is unknown. To address this problem, we have developed Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq) to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. We find that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. The most conspicuous changes are at promoters that have high levels of RNA polymerase II (RNAPII) stalling and DNA accessibility and show specific enrichment for the BRM remodeler. Enhancer chromatin is also disrupted during replication, suggesting a role for transcription factor (TF) competition in nucleosome re-establishment. Thus, the characteristic nucleosome landscape emerges from a uniformly packaged genome by the action of TFs, RNAPII, and remodelers minutes after replication fork passage.

The pausing zone and control of RNA polymerase II elongation by Spt5: Implications for the pause-release model.

The pause-release model of transcription proposes that 40-100 bases from the start site RNA Pol II pauses, followed by release into productive elongation. Pause release is facilitated by the PTEFb phosphorylation of the RNA Pol II elongation factor, Spt5. We mapped paused polymerases by eNET-seq and found frequent pausing in zones that extend ∼0.3-3 kb into genes even when PTEFb is inhibited. The fraction of paused polymerases or pausing propensity declines gradually over several kb and not abruptly as predicted for a discrete pause-release event. Spt5 depletion extends pausing zones, suggesting that it promotes the maturation of elongation complexes to a low-pausing state. The expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the CTR1 domain diminished pausing throughout genes. By contrast, mutants that prevent the phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus, distinct Spt5 phospho-isoforms set the balance between pausing and elongation.

Cooperative binding between distant transcription factors is a hallmark of active enhancers.

Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.

Pioneers Invade the Nucleosomal Landscape.

Two papers in Molecular Cell (Kubik et al., 2018; Yan et al., 2018) explore the mechanisms by which transcription factors bind their sites in chromatin, providing fresh insights into the much-debated question of how transcription factors can be "pioneers."

Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates.

Nucleosomes are disrupted during transcription and other active processes, but the structural intermediates during nucleosome disruption in vivo are unknown. To identify intermediates, we mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by sequencing. At the first nucleosome position downstream of the transcription start site, we identified unwrapped intermediates, including hexasomes that lack either proximal or distal contacts. Inhibiting topoisomerases or depleting histone chaperones increased unwrapping, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased unwrapping. Our results indicate that positive torsion generated by elongating RNAPII causes transient loss of histone-DNA contacts. Using this mapping approach, we found that nucleosomes flanking human CTCF insulation sites are similarly disrupted. We also identified diagnostic subnucleosomal particle remnants in cell-free human DNA data as a relic of transcribed genes from apoptosing cells. Thus identification of subnucleosomal fragments from nuclease protection data represents a general strategy for structural epigenomics.

Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID.

Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We re-examined the role of TFIID by rapid depletion of S. cerevisiae TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Taf1, Taf2, Taf7, Taf11, and Taf13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing, and TATA-less gene classes. The magnitude of TFIID dependence varies with growth conditions, although this variation is similar genome-wide. Many studies have suggested differences in gene-regulatory mechanisms between TATA and TATA-less genes, and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID participates in expression of nearly all yeast mRNAs and that differences in regulation between these two gene categories is due to other properties.

JMJD5 couples with CDK9 to release the paused RNA polymerase II.

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase.

Nucleosomes are barriers to transcription in vitro; however, their effects on RNA polymerase in vivo are unknown. Here we describe a simple and general strategy to comprehensively map the positions of elongating and arrested RNA polymerase II (RNAPII) at nucleotide resolution. We find that the entry site of the first (+1) nucleosome is a barrier to RNAPII for essentially all genes, including those undergoing regulated pausing farther upstream. In contrast to the +1 nucleosome, gene body nucleosomes are low barriers and cause RNAPII stalling both at the entry site and near the dyad axis. The extent of the +1 nucleosome barrier correlates with nucleosome occupancy but anticorrelates with enrichment of histone variant H2A.Z. Importantly, depletion of H2A.Z from a nucleosome position results in a higher barrier to RNAPII. Our results suggest that nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z.

Capitalizing on disaster: Establishing chromatin specificity behind the replication fork.

Eukaryotic genomes are packaged into nucleosomal chromatin, and genomic activity requires the precise localization of transcription factors, histone modifications and nucleosomes. Classic work described the progressive reassembly and maturation of bulk chromatin behind replication forks. More recent proteomics has detailed the molecular machines that accompany the replicative polymerase to promote rapid histone deposition onto the newly replicated DNA. However, localized chromatin features are transiently obliterated by DNA replication every S phase of the cell cycle. Genomic strategies now observe the rebuilding of locus-specific chromatin features, and reveal surprising delays in transcription factor binding behind replication forks. This implies that transient chromatin disorganization during replication is a central juncture for targeted transcription factor binding within genomes. We propose that transient occlusion of regulatory elements by disorganized nucleosomes during chromatin maturation enforces specificity of factor binding.

Asymmetric nucleosomes flank promoters in the budding yeast genome.

Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.

Pore dynamics and conductance of RyR1 transmembrane domain.

Ryanodine receptors (RyR) are calcium release channels, playing a major role in the regulation of muscular contraction. Mutations in skeletal muscle RyR (RyR1) are associated with congenital diseases such as malignant hyperthermia and central core disease (CCD). The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Previously, we have reported a hypothetical structure of the RyR1 pore-forming region, obtained by homology modeling and supported by mutational scans, electrophysiological measurements, and cryo-electron microscopy. Here, we utilize the expanded model encompassing six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the proposed pore structure can sustain ion currents measured in single-channel experiments. We observe a stable pore structure on timescales of 0.2 µs, with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. We further suggest that stability of the selectivity filter critically depends on the interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of polar substitution I4897T results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca(2+) conductance.

Structural determinants of skeletal muscle ryanodine receptor gating.

Ryanodine receptor type 1 (RyR1) releases Ca(2+) from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca(2+). However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830-Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.

Nucleation and spreading maintain Polycomb domains every cell cycle.

Gene repression by the Polycomb pathway is essential for metazoan development. Polycomb domains, characterized by trimethylation of histone H3 lysine 27 (H3K27me3), carry the memory of repression and hence need to be maintained to counter the dilution of parental H3K27me3 with unmodified H3 during replication. Yet, how locus-specific H3K27me3 is maintained through replication is unclear. To understand H3K27me3 recovery post-replication, we first define nucleation sites within each Polycomb domain in mouse embryonic stem cells. To map dynamics of H3K27me3 domains across the cell cycle, we develop CUT&Flow (coupling cleavage under target and tagmentation with flow cytometry). We show that post-replication recovery of Polycomb domains occurs by nucleation and spreading, using the same nucleation sites used during de novo domain formation. By using Polycomb repressive complex 2 (PRC2) subunit-specific inhibitors, we find that PRC2 targets nucleation sites post-replication independent of pre-existing H3K27me3. Thus, competition between H3K27me3 deposition and nucleosome turnover drives both de novo domain formation and maintenance during every cell cycle.

Increased EZH2 function in regulatory T cells promotes their capacity to suppress autoimmunity by driving effector differentiation prior to activation.

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here we assessed whether increased EZH2 activity in Treg cells would improve Treg cell function. Using an Ezh2 gain-of-function mutation, Ezh2 Y641F , we found that Treg cells expressing Ezh2 Y641F displayed an increased effector Treg phenotype and were poised for improved homing to organ tissues. Expression of Ezh2 Y641F in Treg cells led to more rapid remission from autoimmunity. H3K27me3 profiling and transcriptomic analysis revealed a redistribution of H3K27me3, which prompted a gene expression profile in naïve Ezh2 Y641F Treg cells that recapitulated aspects of CD28-activated Ezh2 WT Treg cells. Altogether, increased EZH2 activity promotes the differentiation of effector Treg cells that can better suppress autoimmunity. Highlights: EZH2 function promotes effector differentiation of Treg cells.EZH2 function promotes Treg cell migration to organ tissues.EZH2 function in Treg cells improves remission from autoimmunity.EZH2 function poises naïve Treg cells to adopt a CD28-activated phenotype.

The Histone Chaperone Spn1 Preserves Chromatin Protections at Promoters and Nucleosome Positioning in Open Reading Frames.

Spn1 is a multifunctional histone chaperone that associates with RNA polymerase II during elongation and is essential for life in eukaryotes. While previous work has elucidated regions of the protein important for its many interactions, it is unknown how these domains contribute to the maintenance of chromatin structure. Here, we employ digestion by micrococcal nuclease followed by single-stranded library preparation and sequencing (MNase-SSP) to characterize chromatin structure in Saccharomyces cerevisiae expressing wild-type or mutants of Spn1. We mapped protections of all sizes genome-wide, and surprisingly, we observed a widespread loss of short fragments over nucleosome-depleted regions (NDRs) at promoters in the Spn1-K192N-containing strain, indicating critical functions of Spn1 in maintaining normal chromatin architecture outside open reading frames. Additionally, there are shifts in DNA protections in the Spn1 mutant expressing strains over open reading frames, which indicate changes in nucleosome and subnucleosome positioning. This was observed in markedly different mutant Spn1 strains, demonstrating that multiple functions of Spn1 are required to maintain proper chromatin structure in open reading frames. Taken together, our results reveal a previously unknown role of Spn1 in the maintenance of NDR architecture and deepen our understanding of Spn1-dependent chromatin maintenance over transcribed regions.

Human cytomegalovirus induces neuronal gene expression for viral maturation.

Viral invasion of the host cell causes some of the most dramatic changes in biology. Human cytomegalovirus (HCMV) extensively remodels host cells, altering nuclear shape and generating a cytoplasmic viral-induced assembly compartment (vIAC). How these striking morphology changes take place in the context of host gene regulation is still emerging. Here, we discovered that histone variant macroH2A1 is essential for producing infectious progeny. Because virion maturation and cellular remodeling are closely linked processes, we investigated structural changes in the host cell upon HCMV infection. We discovered that macroH2A1 is necessary for HCMV-induced reorganization of the host nucleus, cytoskeleton, and endoplasmic reticulum. Furthermore, using RNA-seq we found that while all viral genes were highly expressed in the absence of macroH2A1, many HCMV-induced host genes were not. Remarkably, hundreds of these HCMV-induced macroH2A1-dependent host genes are associated with neuronal synapse formation and vesicle trafficking. Knock-down of these HCMV-induced neuronal genes during infection resulted in malformed vIACs and smaller plaques, establishing their importance to HCMV infection. Together, our findings demonstrate that HCMV manipulates host gene expression by hijacking a dormant neuronal secretory pathway for efficient virion maturation.

Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes.

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation. Furthermore, we show that LLPS of CBX2 is required for the demarcation and deposition of H3K27me3 and is essential for cellular differentiation. Our findings uncover new functional roles of LLPS in the formation of facultative heterochromatin and unravel a new mechanism by which low-abundant proteins nucleate many other proteins to form compartments that enable them to execute their functions.