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Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase-separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation in living animals. Here, we show in vivo evidence of a critical nonenzymatic repressive function of cPRC1 component CBX2 in the male germline. CBX2 is up-regulated as spermatogonial stem cells differentiate and is required to repress genes that were active in stem cells. CBX2 forms condensates (similar to previously described Polycomb bodies) that colocalize with target genes bound by CBX2 in differentiating spermatogonia. Single-cell analyses of mosaic Cbx2 mutant testes show that CBX2 is specifically required to produce differentiating A1 spermatogonia. Furthermore, the region of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical, which distinguishes this from a mechanism that is reliant on histone modification., (© 2023 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities., (© 2019 Plys et al.; Published by Cold Spring Harbor Laboratory Press.)

Development requires the expression of master regulatory genes necessary to specify a cell lineage. Equally significant is the stable and heritable silencing of master regulators that would specify alternative lineages. This regulated gene silencing is carried out by Polycomb group (PcG) proteins, which must be correctly recruited only to the subset of their target loci that requires lineage-specific silencing. A recent study by Erceg and colleagues (pp. 590-602) expands on a key aspect of that targeting: The same DNA elements that recruit PcG complexes to a repressed locus also encode transcriptional enhancers that function in different lineages where that locus must be expressed. Thus, PcG targeting elements overlap with enhancers., (© 2017 Jaensch et al.; Published by Cold Spring Harbor Laboratory Press.)

Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR., (© 2017 Mueller et al.; Published by Cold Spring Harbor Laboratory Press.)

The trithorax group of genes (trxG) was identified in mutational screens that examined developmental phenotypes and suppression of Polycomb mutant phenotypes. The protein products of these genes are primarily involved in gene activation, although some can also have repressive effects. There is no central function for these proteins. Some move nucleosomes about on the genome in an ATP-dependent manner, some covalently modify histones such as methylating lysine 4 of histone H3, and some directly interact with the transcription machinery or are a part of that machinery. It is interesting to consider why these specific members of large families of functionally related proteins have strong developmental phenotypes., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

Nucleosome occupancy plays a key role in regulating access to eukaryotic genomes. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of DNA sequence in this regulation remains unclear, particularly in mammals. To address this problem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV). We show that nucleosome redistribution peaks at 24 h post-KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro-assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 h post-KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans.

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.

Silencing of homeotic genes requires the Polycomb repressive complex 1 (PRC1) family of protein complexes, which are composed of Polycomb-group (PcG) proteins and frequently include other subunits. We discuss here two aspects of PRC1 that might contribute to this activity. Inhibiting the action of remodeling factors via chromatin compaction is believed to be one mechanism by which PRC1 represses genes. We show that PRC1s from fly and mouse have conserved this activity as complexes. Additionally, we provide evidence that a different subunit in the mouse complex retains the conserved repression activity and that activity appears to be mediated by charge interactions. We show that Zeste interacts specifically with the Ph subunit of PRC1 and discuss the possibility of these factors contributing to spreading of PRC1 complexes. Our results suggest that one aspect of PRC1 repression is likely to be mediated by charge-charge interactions.

Eukaryotic DNA is wrapped around a histone protein core to constitute the fundamental repeating units of chromatin, the nucleosomes. The affinity of the histone core for DNA depends on the nucleotide sequence; however, it is unclear to what extent DNA sequence determines nucleosome positioning in vivo, and if the same rules of sequence-directed positioning apply to genomes of varying complexity. Using the data generated by high-throughput DNA sequencing combined with chromatin immunoprecipitation, we have identified positions of nucleosomes containing the H2A.Z histone variant and histone H3 trimethylated at lysine 4 in human CD4(+) T-cells. We find that the 10-bp periodicity observed in nucleosomal sequences in yeast and other organisms is not pronounced in human nucleosomal sequences. This result was confirmed for a broader set of mononucleosomal fragments that were not selected for any specific histone variant or modification. We also find that human H2A.Z nucleosomes protect only approximately 120 bp of DNA from MNase digestion and exhibit specific sequence preferences, suggesting a novel mechanism of nucleosome organization for the H2A.Z variant.

The distribution of nucleosomes along the genome is a significant aspect of chromatin structure and is thought to influence gene regulation through modulation of DNA accessibility. However, properties of nucleosome organization remain poorly understood, particularly in mammalian genomes. Toward this goal we used tiled microarrays to identify stable nucleosome positions along the HOX gene clusters in human cell lines. We show that nucleosome positions exhibit sequence properties and long-range organization that are different from those characterized in other organisms. Despite overall variability of internucleosome distances, specific loci contain regular nucleosomal arrays with 195-bp periodicity. Moreover, such arrays tend to occur preferentially toward the 3' ends of genes. Through comparison of different cell lines, we find that active transcription is correlated with increased positioning of nucleosomes, suggesting an unexpected role for transcription in the establishment of well-positioned nucleosomes.

p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a beta-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.

The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner.

Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. We have used purified proteins and complexes reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1.

To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA.

Alteration of nucleosomes by ATP-dependent remodeling complexes represents a critical step in the regulation of transcription. The human SWI/SNF (hSWI/SNF) family is composed of complexes that contain either Brg1 or hBrm as the central ATPase; however, these separate complexes have not been compared functionally. Here we describe the establishment of cell lines that express epitope-tagged Brg1 and hBrm and a characterization of the complexes associated with these two ATPases. We show that Brg1 fractionates into two complexes that differ in activity and subunit composition, whereas hBrm is found in one complex with lower activity than the Brg1 complexes. These three complexes can remodel nucleosomal arrays, increase restriction enzyme accessibility, and hydrolyze ATP in a DNA-dependent manner. The three complexes differ markedly in their ability to remodel mononucleosomal core particles. We also show that the hBrm complex and one of the Brg1 complexes contain components of the mammalian Sin3 (mSin3) complex. In addition, we have found that Brg1, hBrm, and BAF155 can interact specifically with mSin3A in vitro, showing a direct association of hSWI/SNF complexes with proteins involved in gene repression. These unexpected functional characteristics indicate that these hSWI/SNF complexes play diverse regulatory roles.

The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood. We show that mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro. The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled beta-globin promoter. Interestingly, we find that two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain. In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB. We conclude that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling.

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited, processes that might be opposed by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, which are components of human SWI/SNF (hSWI/SNF) complexes, were recently shown to be phosphorylated during mitosis. This suggested that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, we have purified hSWI/SNF complexes at different stages of the cell cycle, and found that hSWI/SNF was inactive in cells blocked in G2-M. Mitotic hSWI/SNF contained Brg1 but not hBrm, and was phosphorylated on at least two subunits, hSWI3 and Brg1. In vitro, active hSWI/SNF from asynchronous cells can be phosphorylated and inactivated by ERK1, and reactivated by dephosphorylation. hSWI/SNF isolated as cells traversed mitosis regained activity when its subunits were dephosphorylated either in vitro or in vivo. We propose that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis.

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation.

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse genes, including human c-myc and c-fos, some HIV genes, and the Drosophila heat shock loci. To characterize the mechanisms that regulate pausing, we have established an in vitro system using the human hsp7O gene. We demonstrate that nucleosome formation increases by >100-fold the duration of a transcriptional pause on the human hsp7O gene in vitro at the same location as pausing is observed in vivo. Readthrough of this pause is increased by an activator that contains the human heat shock factor 1 (HSF1) transcriptional activation domains. Maximal effect of the activator requires that the system be supplemented with fractions that have hSWI/SNF activity, which has been shown previously to alter nucleosome structure. No significant readthrough is observed in the absence of activator, and neither the activator nor the hSWI/SNF fraction affected elongation on naked DNA; therefore, these results suggest that an activator can cause increased readthrough of promoter-proximal pausing by decreasing the inhibitory effect of nucleosomes on transcriptional elongation.

Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

HSF2, one of the heat shock transcription factors in mammalian cells, is localized to the cytoplasm during normal growth and moves to the nucleus upon activation. Heat shock transcription factors in metazoans contain four hydrophobic heptad repeat sequences, three in the amino terminus and one in the carboxy terminus, which are predicted to form alpha-helical coiled-coil structures analogous to the leucine zipper. Here, we show that point mutations in either of two amino-terminal zippers or in the carboxy-terminal zipper disrupt normal localization of HSF2 and cause it to be constitutively nuclear. We demonstrate further that two sequences immediately surrounding the amino-terminal zipper domain are required for nuclear localization. These sequences fit the consensus for a bipartite nuclear localization signal (NLS). We suggest that interactions between the amino- and carboxy-terminal zippers normally mask the NLS sequences of HSF2 and that these interactions are disrupted upon activation to expose the NLS sequences and allow transport of HSF2 to the nucleus. We conclude that zipper domains can regulate subcellular localization.

The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS. RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes. Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination. Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II. The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled. Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators. These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.

Regulatory factors must contend with chromatin structure to function. Although nucleosome structure and position on promoters can be important in determining factor access, the intrinsic ability of factors to bind to nucleosomal DNA might also play an essential regulatory role. We have used templates where nucleosomes were either randomly positioned or rotationally phased to demonstrate that two transcription factors, heat shock factor (HSF) and GAL4, differ significantly in their ability to bind to nucleosomes. GAL4 was able to bind to nucleosomal templates. Surprisingly, in contrast to its behavior on naked DNA, GAL4 bound better to multiple GAL4 sites than to a single GAL4 site on these templates. HSF alone was not able to bind to nucleosomal templates. HSF was able to bind to nucleosomal templates, however, when the TATA-binding factor TFIID was present. Consequently, binding to nucleosomal templates could be facilitated by adjacent binding of the same protein in the case of GAL4 but required binding of a second protein in the case of HSF. Taken together, these data demonstrate that regulatory factors differ in their inherent ability to bind to nucleosomal templates. These differences are likely to be important to the function of these factors in vivo.

One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the level of appropriately initiated expression from the human heat shock protein 70 (hsp70) promoter. Sequences required for full activation lie upstream of the transcription initiation site and are distinct from sequences necessary for basal expression. These sequences also appear distinct from promoter sequences necessary for heat induction, serum induction, and induction by the papovavirus T antigens. The c-myc protein inhibits appropriately initiated expression from the mouse metallothionein I (MT-I) promoter. A mutation that removes 138 amino acids of exon 2 produces a c-myc gene product that is capable of activating the hsp70 promoter but is no longer capable of inhibiting MT-I expression, suggesting that these two properties reside in different domains of the c-myc protein. Expression from the adenovirus EII promoter is slightly inhibited, while expression from the SV40 early promoter is minimally affected by the c-myc protein. Both the spectrum of promoters regulated by the c-myc protein and the sequence requirements for that regulation differ from those of previously characterized viral trans-activating proteins. The data suggest that the c-myc protein can both stimulate and inhibit transcription from mammalian promoters in a novel manner.