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Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability., (© 2021. The Author(s).)

Linker histones play essential roles in the regulation and maintenance of the dynamic chromatin structure of higher eukaryotes. The influence of human histone H1.0 on the nucleosome structure and biophysical properties of the resulting chromatosome were investigated and compared with the 177-bp nucleosome using Cryo-EM and SAXS. The 4.5 Å Cryo-EM chromatosome structure showed that the linker histone binds at the nucleosome dyad interacting with both linker DNA arms but in a tilted manner leaning towards one of the linker sides. The chromatosome is laterally compacted and rigid in the dyad and linker DNA area, in comparison with the nucleosome where linker DNA region is more flexible and displays structural variability. In solution, the chromatosomes appear slightly larger than the nucleosomes, with the volume increase compared to the bound linker histone, according to solution SAXS measurements. SAXS X-ray diffraction characterisation of Mg-precipitated samples showed that the different shapes of the 177 chromatosome enabled the formation of a highly ordered lamello-columnar phase when precipitated by Mg 2+ , indicating the influence of linker histone on the nucleosome stacking. The biological significance of linker histone, therefore, may be affected by the change in the polyelectrolyte and DNA conformation properties of the chromatosomes, in comparison to nucleosomes.

Emerging evidence suggests that histone variants are novel epigenetic regulators of memory, whereby histone H2A.Z suppresses fear memory. However, it is not clear if altered fear memory can also modify risk for PTSD, and whether these effects differ in males and females. Using conditional-inducible H2A.Z knockout (cKO) mice, we showed that H2A.Z binding is higher in females and that H2A.Z cKO enhanced fear memory only in males. However, H2A.Z cKO improved memory on the non-aversive object-in-place task in both sexes, suggesting that H2A.Z suppresses non-stressful memory irrespective of sex. Given that risk for fear-related disorders, such as PTSD, is biased toward females, we examined whether H2A.Z cKO also has sex-specific effects on fear sensitization in the stress-enhanced fear learning (SEFL) model of PTSD, as well as associated changes in pain sensitivity. We found that H2A.Z cKO reduced stress-induced sensitization of fear learning and pain responses preferentially in female mice, indicating that the effects of H2A.Z depend on sex and the type of task, and are influenced by history of stress. These data suggest that H2A.Z may be a sex-specific epigenetic risk factor for PTSD susceptibility, with implications for developing sex-specific therapeutic interventions.

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.

The mRNA processing and export factor, Iws1, interacts with the histone H3/H4 chaperone, Spt6 (Supt6 in mouse gene ontology) and recruits the lysine methyltransferase, Setd2, to chromatin to regulate H3K36me3. This recruitment is known to be crucial for pre-mRNA splicing and Iws1 has been shown to interact with REF1/Aly to mediate mRNA export. However, the role of this complex has not yet been examined in embryonic development. Here, we show that knockdown of either Iws1 or Supt6 blocked embryo development, primarily at the 8/16-cell stage, indicating that Iws1 and Supt6 are crucial for mouse preimplantation development. In the knockdown embryos, we observed decreases in pre-mRNA splicing, mRNA export and the expression of the lineage-specific transcription factor, Nanog. We found that either Iws1 or Supt6 are required for H3K36 trimethylation and that concurrent knockdown of both Iws1 and Supt6 blocks embryonic development at the 2-cell stage. We show that H3K36me3 is modulated by the Pi3k/Akt pathway, as inhibition of this pathway reduced the global level of H3K36me3 while activation of the pathway increased the level of this modification in 2-cell embryos. We observed that Iws1 interacts with nuclear Akt in early embryos, and herein propose that Akt modulates H3K36me3 through interaction with Iws1. Together, our results indicate that the Iws1 and Supt6 play crucial roles in embryonic genome activation, lineage specification, and histone modification during mouse early development.

Linker histone H1 is a master regulator of higher order chromatin structure, but its involvement in the DNA damage response and repair is unclear. Here, we report that linker histone H1.2 is an essential regulator of ataxia telangiectasia mutated (ATM) activation. We show that H1.2 protects chromatin from aberrant ATM activation through direct interaction with the ATM HEAT repeat domain and inhibition of MRE11-RAD50-NBS1 (MRN) complex-dependent ATM recruitment. Upon DNA damage, H1.2 undergoes rapid PARP1-dependent chromatin dissociation through poly-ADP-ribosylation (PARylation) of its C terminus and further proteasomal degradation. Inhibition of H1.2 displacement by PARP1 depletion or an H1.2 PARylation-dead mutation compromises ATM activation and DNA damage repair, thus leading to impaired cell survival. Taken together, our findings suggest that linker histone H1.2 functions as a physiological barrier for ATM to target the chromatin, and PARylation-mediated active H1.2 turnover is required for robust ATM activation and DNA damage repair.

Broad domain promoters and super enhancers are regulatory elements that govern cell-specific functions and harbor disease-associated sequence variants. These elements are characterized by distinct epigenomic profiles, such as expanded deposition of histone marks H3K27ac for super enhancers and H3K4me3 for broad domains, however little is known about how they interact with each other and the rest of the genome in three-dimensional chromatin space. Using network theory methods, we studied chromatin interactions between broad domains and super enhancers in three ENCODE cell lines (K562, MCF7, GM12878) obtained via ChIA-PET, Hi-C, and Hi-CHIP assays. In these networks, broad domains and super enhancers interact more frequently with each other compared to their typical counterparts. Network measures and graphlets revealed distinct connectivity patterns associated with these regulatory elements that are robust across cell types and alternative assays. Machine learning models showed that these connectivity patterns could effectively discriminate broad domains from typical promoters and super enhancers from typical enhancers. Finally, targets of broad domains in these networks were enriched in disease-causing SNPs of cognate cell types. Taken together these results suggest a robust and unique organization of the chromatin around broad domains and super enhancers: loci critical for pathologies and cell-specific functions.

Epigenetic mechanisms regulate the formation, consolidation and reconsolidation of memories. However, the signaling path from neuronal activation to epigenetic modifications within the memory-related brain circuit remains unknown. We report that learning induces long-lasting histone modifications in hippocampal memory-activated neurons to regulate memory stability. Neuronal activity triggers a late-onset shift in Nrxn1 splice isoform choice at splicing site 4 by accumulating a repressive histone marker, H3K9me3, to modulate the splicing process. Activity-dependent phosphorylation of p66α via AMP-activated protein kinase recruits HDAC2 and Suv39h1 to establish repressive histone markers and changes the connectivity of the activated neurons. Removal of Suv39h1 abolished the activity-dependent shift in Nrxn1 splice isoform choice and reduced the stability of established memories. We uncover a cell-autonomous process for memory preservation in which memory-related neurons initiate a late-onset reduction of their rewiring capacities through activity-induced histone modifications.

Regions of hypoxia occur in most solid tumors, and they are associated with a poor prognostic outcome. Despite the absence of detectable DNA damage, severe hypoxia (<0.1% O2) induces a DNA damage response, including the activation of p53 and subsequent induction of p53-dependent apoptosis. Factors affecting hypoxia-induced p53-dependent apoptosis are unclear. Here we asked whether H3K9me3, through mediating gene repression, could regulate hypoxia-induced p53-dependent apoptosis. Under hypoxic conditions, increases in H3K9me3 occur in an oxygen-dependent but HIF-1-independent manner. We demonstrate that under hypoxic conditions, which induce p53 activity, the negative regulator of p53, APAK, is repressed by increases in H3K9me3 along the APAK loci. APAK repression in hypoxia is mediated by the methyltransferase SETDB1 but not Suv39h1 or G9a. Interestingly, increasing hypoxia-induced H3K9me3 through pharmacological inhibition of JMJD2 family members leads to an increase in apoptosis and decreased clonogenic survival and again correlates with APAK expression. The relevance of understanding the mechanisms of APAK expression regulation to human disease was suggested by analysis of patients with colorectal cancer, which demonstrates that high APAK expression correlates with poor prognosis. Together, these data demonstrate the functional importance of H3K9me3 in hypoxia, and they provide a novel mechanistic link between H3K9me3, p53 and apoptosis in physiologically relevant conditions of hypoxia.

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Postmitotic neurons are subject to a vast array of environmental influences that require the nuclear integration of intracellular signaling events to promote a wide variety of neuroplastic states associated with synaptic function, circuit formation, and behavioral memory. Over the last decade, much attention has been paid to the roles of transcription and chromatin regulation in guiding fundamental aspects of neuronal function. A great deal of this work has centered on neurodevelopmental and adulthood plasticity, with increased focus in the areas of neuropharmacology and molecular psychiatry. Here, we attempt to provide a broad overview of chromatin regulation, as it relates to central nervous system (CNS) function, with specific emphasis on the modes of histone posttranslational modifications, chromatin remodeling, and histone variant exchange. Understanding the functions of chromatin in the context of the CNS will aid in the future development of pharmacological therapeutics aimed at alleviating devastating neurological disorders.

How do drugs of abuse, such as cocaine, cause stable changes in neural plasticity that in turn drive long-term changes in behavior? What kind of mechanism can underlie such stable changes in neural plasticity? One prime candidate mechanism is epigenetic mechanisms of chromatin regulation. Chromatin regulation has been shown to generate short-term and long-term molecular memory within an individual cell. They have also been shown to underlie cell fate decisions (or cellular memory). Now, there is accumulating evidence that in the CNS, these same mechanisms may be pivotal for drug-induced changes in gene expression and ultimately long-term behavioral changes. As these mechanisms are also being found to be fundamental for learning and memory, an exciting new possibility is the extinction of drug-seeking behavior by manipulation of epigenetic mechanisms. In this review, we critically discuss the evidence demonstrating a key role for chromatin regulation via histone acetylation in cocaine action.

While chromatin modifications can offer a useful readout for enhancer activities, it is less clear whether these modification marks are a cause or consequence of transcription factor occupancy and enhancer activation. We have examined in details the temporal events of acetyltransferase associations and histone acetylations at different regulatory regions of the Myod1 locus. Our studies demonstrate that the histone acetyltransferase (HAT) p300 is stepwise enriched at distinct Myod1 regulatory regions during myogenic differentiation. This enrichment of p300 is associated with increased histone acetylation in a discrete pattern. Inhibition of p300 HAT activity impedes myogenic differentiation, which is coupled with decreased histone acetylation at specific Myod1 regulatory regions. We show for the first time that p300 is directly involved in the early regulation of Myod1 enhancer, and provide molecular insights into how p300 HAT activity and histone acetylation are related to enhancer activation and, consequently, gene transcription.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT(2A) receptor-dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

Once thought to be transcriptional noise, large non-coding RNAs (lncRNAs) have recently been demonstrated to be functional molecules. The cell-type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of lncRNAs in embryonic stem (ES) cells, lineage-restricted neuronal progenitor cells, and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methylation patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these lncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of the protein-coding genes.

Lung cancer is the leading cause of cancer deaths. Despite optimal diagnosis and early treatment, many patients die of recurrent disease. There are no sufficiently useful biomarkers to predict the risk of tumor recurrence. Here, we show that expression of histone macroH2A1.1 and macroH2A2 predicts lung cancer recurrence, identifying these histone variants as a novel tool for an improved risk stratification of cancer patients. Moreover, macroH2A isoforms are highly expressed in cells undergoing senescence, a known antitumor mechanism, suggesting macroH2A1.1 may be a useful biomarker for senescent cells in tumors.

Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G(2)/M phase. Lack of G(2)/M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.

That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.

The eukaryotic genome is organized into functionally and structurally distinct domains, representing regulatory units for gene expression and chromosome behavior. DNA sequences that mark the border between adjacent domains are the insulators or boundary elements, which are required in maintenance of the function of different domains. Some insulators need others enable to play insulation activity. Chromatin domains are defined by distinct sets of post-translationally modified histones. Recent studies show that these histone modifications are also involved in establishment of sharp chromatin boundaries in order to prevent the spreading of distinct domains. Additionally, in some loci, the high-order chromatin structures for long-range looping interactions also have boundary activities, suggesting a correlation between insulators and chromatin loop domains. In this review, we will discuss recent progress in the field of chromatin domain boundaries.

Cellular senescence is a signal transduction program leading to irreversible cell cycle arrest. This growth arrest can be triggered by many different mechanisms including recognition by cellular sensors of DNA double-strand breaks leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. Senescence is initiated by the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals (STASIS: stress or aberrant signaling-induced senescence). The process of carcinogenesis involves a series of changes that allow tumor cells to bypass the senescence program. Nevertheless, tumor cells retain the capacity to undergo senescence. Treatment of tumor cells with many conventional anticancer therapies activates DNA damage signaling pathways, which induce apoptosis in some cells and senescence in others. Overexpression of tumor suppressors or inhibition of oncogenes can also induce rapid senescence in tumor cells. Senescent cells, while not dividing, remain metabolically active and produce many secreted factors, some of which stimulate and others inhibit the growth of tumors. The emerging knowledge about the pathways that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

Histone proteins associate with and compact eukaryotic nuclear DNA to form chromatin. The basic unit of chromatin is the nucleosome, which is made up of 146 base pairs of DNA wrapped around two of each of four core histones, H2A, H2B, H3 and H4. Chromatin structure and its regulation are important in transcription and DNA replication. We therefore thought that DNA-damage signalling and repair components might also modulate chromatin structure. Here we have characterized a conserved motif in the carboxy terminus of the core histone H2A from Saccharomyces cerevisiae that contains a consensus phosphorylation site for phosphatidylinositol-3-OH kinase related kinases (PIKKs). This motif is important for survival in the presence of agents that generate DNA double-strand breaks, and the phosphorylation of this motif in response to DNA damage is dependent on the PIKK family member Mec1. The motif is not necessary for Mec1-dependent cell-cycle or transcriptional responses to DNA damage, but is required for efficient DNA double-strand break repair by non-homologous end joining. In addition, the motif has a role in determining higher order chromatin structure. Thus, phosphorylation of a core histone in response to DNA damage may cause an alteration of chromatin structure that facilitates DNA repair.

Histone proteins and the nucleosomes they form with DNA are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications that often occur on tail domains of these proteins has been well documented. Although the function of these highly conserved modifications has remained elusive, converging biochemical and genetic evidence suggests functions in several chromatin-based processes. We propose that distinct histone modifications, on one or more tails, act sequentially or in combination to form a 'histone code' that is, read by other proteins to bring about distinct downstream events.

Eukaryotic genomes are packaged into nucleosomes, which are thought to repress gene expression generally. Repression is particularly evident at yeast telomeres, where genes within the telomeric heterochromatin appear to be silenced by the histone-binding silent information regulator (SIR) complex (Sir2, Sir3, Sir4) and Rap1 (refs 4-10). Here, to investigate how nucleosomes and silencing factors influence global gene expression, we use high-density arrays to study the effects of depleting nucleosomal histones and silencing factors in yeast. Reducing nucleosome content by depleting histone H4 caused increased expression of 15% of genes and reduced expression of 10% of genes, but it had little effect on expression of the majority (75%) of yeast genes. Telomere-proximal genes were found to be de-repressed over regions extending 20 kilobases from the telomeres, well beyond the extent of Sir protein binding and the effects of loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific rather than generally repressive.

The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R. Faast et al., in preparation). To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core.

'The amino termini of histones extend from the nucleosomal core and are modified by acetyltransferases and deacetylases during the cell cycle. These acetylation patterns may direct histone assembly and help regulate the unfolding and activity of genes.

In Xenopus, cells from the animal hemisphere are competent to form mesodermal tissues from the morula through to the blastula stage. Loss of mesodermal competence at early gastrula is programmed cell-autonomously, and occurs even in single cells at the appropriate stage. To determine the mechanism by which this occurs, we have been investigating a concomitant, global change in expression of H1 linker histone subtypes. H1 histones are usually considered to be general repressors of transcription, but in Xenopus they are increasingly thought to have selective functions in transcriptional regulation. Xenopus eggs and embryos at stages before the midblastula transition are deficient in histone H1 protein, but contain an oocyte-specific variant called histone B4 or H1M. After the midblastula transition, histone B4 is progressively substituted by three somatic histone H1 variants, and replacement is complete by early neurula. Here we report that accumulation of somatic H1 protein is rate limiting for the loss of mesodermal competence. This involves selective transcriptional silencing of regulatory genes required for mesodermal differentiation pathways, like muscle, by somatic, but not maternal, H1 protein.

Heterochromatin is a cytologically visible form of condensed chromatin capable of repressing genes in eukaryotic cells. For the yeast Saccharomyces cerevisiae, despite the absence of observable heterochromatin, there is genetic and chromatin structure data which indicate that there are heterochromatin-like repressive structures. Genes experience position effects at the silent mating loci and the telomeres, resulting in a repressed state that is inherited in an epigenetic manner. The histone H4 amino terminus is required for repression at these loci. Additional studies have indicated that the histone H3 N terminus is not important for silent mating locus repression, but redundancy of repressive elements at the silent mating loci may be responsible for masking its role. Here we report that histone H3 is required for full repression at yeast telomeres and at partially disabled silent mating loci, and that the acetylatable lysine residues of H3 play an important role in silencing.

The increasingly detailed biochemical definition of the protein complexes that regulate gene transcription has led to the re-emergence of questions about the role of histones. Much recent evidence suggests that transcriptional activation requires that transcription factors successfully compete with histones for binding to promoters, and that there may be more than one mechanism by which this is achieved.

The approximate shape of the chromatin subunit called the nucleosome is now known, but its internal architecture is not well understood. Recent studies reveal details of the organisation of DNA within the nucleosome, and show that the arginine-rich histones are essential to DNA folding. Nucleosomes or structures related to them seem to be present at points of DNA replication and transcription; interactions within and between nucleosomes are likely to play a critical part in these processes.

Density labelling studies have shown that nascent histones are not mixed with parental histones during the assembly of nucleosome cores. However, experiments in other laboratories, examining histone deposition with respect to newly synthesized DNA, have been interpreted as suggesting that a substantial proportion of core histones (greater than 15%) are randomized at each chromatin replication. The data presented here support our previous results in showing that conservatively assembled nucleosome core histone octamers are conservatively segregated over successive cell generations. It is also shown that the nucleosome cores assembled during 1-beta-D-arabinofuranosylcytosine inhibition of DNA synthesis are conservatively segregated for a minimum of five or six cell generations. These results suggest that the nonrandom assembly of nucleosome cores is not merely a coincidence of the mechanism of histone transport into the nucleus and that the conservative mode of nucleosome segregation is a fundamental feature of chromatin replication, one which is stable to modulations in chromatin packaging.

Active genes are packaged into an altered nucleosome structure forming a chromosomal domain defined by increased sensitivity to nucleases. This structure, reflecting a potential for transcription, contains sites hypersensitive to nuclease digestion adjacent to the coding regions and may also be distinguished by specific non-histone proteins, variant or modified histones or modified DNA. Its formation, by unfolding of a tightly packed chromatin fibre by factors which might affect DNA supercoiling, may be the first step in gene activation.

In eukaryotic cells, DNA is packed into regularly spaced chromatin subunits called nucleosomes. The average distance between nucleosomes (the repeat length) varies in a tissue- and species-specific manner, with values ranging from about 160 to 240 DNA base pairs (bp). Thus, it has been recognized that the repeat length could be one of the factors underlying selective gene expression. In cells growing in culture, the characteristic repeat length for that type of cell seems to arise from an immature chromatin structure in which nucleosomes are initially irregularly spaced or are arranged in small closely packed clusters. At present no in vitro system has been described which is capable of reconstituting the mature physiological nucleosome spacing from purified chromatin components. Moreover, neither the factors necessary for spacing nor the reaction mechanism are known. We describe here an in vitro system that can restore the native subunit spacing in rearranged chromatin samples which have irregularly spaced nucleosomes similar to the situation apparent in newly replicated chromatin.