ANP32e Binds Histone H2A.Z in a Cell Cycle-Dependent Manner and Regulates Its Protein Stability in the Cytoplasm.

ANP32e, a chaperone of H2A.Z, is receiving increasing attention because of its association with cancer growth and progression. An unanswered question is whether ANP32e regulates H2A.Z dynamics during the cell cycle; this could have clear implications for the proliferation of cancer cells. We confirmed that ANP32e regulates the growth of human U2OS cancer cells and preferentially interacts with H2A.Z during the G1 phase of the cell cycle. Unexpectedly, ANP32e does not mediate the removal of H2A.Z from chromatin, is not a stable component of the p400 remodeling complex and is not strongly associated with chromatin. Instead, most ANP32e is in the cytoplasm. Here, ANP32e preferentially interacts with H2A.Z in the G1 phase in response to an increase in H2A.Z protein abundance and regulates its protein stability. This G1-specific interaction was also observed in the nucleoplasm but was unrelated to any change in H2A.Z abundance. These results challenge the idea that ANP32e regulates the abundance of H2A.Z in chromatin as part of a chromatin remodeling complex. We propose that ANP32e is a molecular chaperone that maintains the soluble pool of H2A.Z by regulating its protein stability and acting as a buffer in response to cell cycle-dependent changes in H2A.Z abundance.

The Role of the Histone Variant H2A.Z in Metazoan Development.

During the emergence and radiation of complex multicellular eukaryotes from unicellular ancestors, transcriptional systems evolved by becoming more complex to provide the basis for this morphological diversity. The way eukaryotic genomes are packaged into a highly complex structure, known as chromatin, underpins this evolution of transcriptional regulation. Chromatin structure is controlled by a variety of different epigenetic mechanisms, including the major mechanism for altering the biochemical makeup of the nucleosome by replacing core histones with their variant forms. The histone H2A variant H2A.Z is particularly important in early metazoan development because, without it, embryos cease to develop and die. However, H2A.Z is also required for many differentiation steps beyond the stage that H2A.Z-knockout embryos die. H2A.Z can facilitate the activation and repression of genes that are important for pluripotency and differentiation, and acts through a variety of different molecular mechanisms that depend upon its modification status, its interaction with histone and nonhistone partners, and where it is deposited within the genome. In this review, we discuss the current knowledge about the different mechanisms by which H2A.Z regulates chromatin function at various developmental stages and the chromatin remodeling complexes that determine when and where H2A.Z is deposited.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- or C-termini with segmental isotope labelling for NMR spectroscopy. With access to these precisely-defined variants, we show that PTMs in both the N- and C-termini cause changes in the chemical shifts and conformational populations in regions distant from the PTM sites; up to 50-60 residues upstream of the PTM site. The PTMs investigated had only minor effects on binding of HMGN1 to nucleosome core particles, suggesting that they have other regulatory roles. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the role of PTMs with atomic precision, from both structural and functional perspectives.

Sequential Chromatin Immunoprecipitation to Identify Heterotypic Nucleosomes.

Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.

Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells.

Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.

Short Histone H2A Variants: Small in Stature but not in Function.

The dynamic packaging of DNA into chromatin regulates all aspects of genome function by altering the accessibility of DNA and by providing docking pads to proteins that copy, repair and express the genome. Different epigenetic-based mechanisms have been described that alter the way DNA is organised into chromatin, but one fundamental mechanism alters the biochemical composition of a nucleosome by substituting one or more of the core histones with their variant forms. Of the core histones, the largest number of histone variants belong to the H2A class. The most divergent class is the designated "short H2A variants" (H2A.B, H2A.L, H2A.P and H2A.Q), so termed because they lack a H2A C-terminal tail. These histone variants appeared late in evolution in eutherian mammals and are lineage-specific, being expressed in the testis (and, in the case of H2A.B, also in the brain). To date, most information about the function of these peculiar histone variants has come from studies on the H2A.B and H2A.L family in mice. In this review, we describe their unique protein characteristics, their impact on chromatin structure, and their known functions plus other possible, even non-chromatin, roles in an attempt to understand why these peculiar histone variants evolved in the first place.

Gene editing of the multi-copy H2A.B gene and its importance for fertility.

BACKGROUND: Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. RESULTS: Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription-splicing territories and changes to pre-mRNA splicing are observed. CONCLUSION: We have produced the first H2A.B knockout mouse using the TALEN approach.

RChIP-Seq: Chromatin-Associated RNA Sequencing in Developmentally Staged Mouse Testes.

Chromatin is a dynamic macromolecular structure comprised of histones and a wealth of non-histone proteins. Recently, it has become clear that RNA is also an integral component of chromatin playing an important role in regulating its structure and function. Central to the understanding of RNA function is the ability to identify and genomically map interactions between chromatin components and RNA.Here, we describe a new method, RChIP-seq (RNA-associated-Chromatin-Immuno Precipitation followed by next-generation sequencing) that allows the identification of RNA species that are directly bound to specific components of chromatin in the mouse testis.

The interplay between H2A.Z and H3K9 methylation in regulating HP1α binding to linker histone-containing chromatin.

One of the most intensively studied chromatin binding factors is HP1α. HP1α is associated with silenced, heterochromatic regions of the genome and binds to H3K9me3. While H3K9me3 is necessary for HP1α recruitment to heterochromatin, it is becoming apparent that it is not sufficient suggesting that additional factors are involved. One candidate proposed as a potential regulator of HP1α recruitment is the linker histone H1.4. Changes to the underlying make-up of chromatin, such as the incorporation of the histone variant H2A.Z, has also been linked with regulating HP1 binding to chromatin. Here, we rigorously dissected the effects of H1.4, H2A.Z and H3K9me3 on the nucleosome binding activity of HP1α in vitro employing arrays, mononucleosomes and nucleosome core particles. Unexpectedly, histone H1.4 impedes the binding of HP1α but strikingly, this inhibition is partially relieved by the incorporation of both H2A.Z and H3K9me3 but only in the context of arrays or nucleosome core particles. Our data suggests that there are two modes of interaction of HP1α with nucleosomes. The first primary mode is through interactions with linker DNA. However, when linker DNA is missing or occluded by linker histones, HP1α directly interacts with the nucleosome core and this interaction is enhanced by H2A.Z with H3K9me3.

The Histone Variant H2A.Z Is a Master Regulator of the Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a profound example of cell plasticity that is crucial for embryonic development and cancer. Although it has long been suspected that chromatin-based mechanisms play a role in this process, no master regulator that can specifically regulate EMT has been identified to date. Here, we show that H2A.Z can coordinate EMT by serving as either an activator or repressor of epithelial or mesenchymal gene expression, respectively. Following induction of EMT by TGF-ß, we observed an unexpected loss of H2A.Z across both downregulated epithelial and upregulated mesenchymal promoters. Strikingly, the repression of epithelial gene expression was associated with reduction of H2A.Z upstream of the transcription start site (TSS), while the activation of mesenchymal gene expression was dependent on removal of H2A.Z downstream of the TSS. Therefore, the ability of H2A.Z to regulate EMT is dependent on its position, either upstream or downstream of the TSS.

SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation.

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.

A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B.

The replacement of histone H2A with its variant forms is critical for regulating all aspects of genome organisation and function. The histone variant H2A.B appeared late in evolution and is most highly expressed in the testis followed by the brain in mammals. This raises the question of what new function(s) H2A.B might impart to chromatin in these important tissues. We have immunoprecipitated the mouse orthologue of H2A.B, H2A.B.3 (H2A.Lap1), from testis chromatin and found this variant to be associated with RNA processing factors and RNA Polymerase (Pol) II. Most interestingly, many of these interactions with H2A.B.3 (Sf3b155, Spt6, DDX39A and RNA Pol II) were inhibited by the presence of endogenous RNA. This histone variant can bind to RNA directly in vitro and in vivo, and associates with mRNA at intron-exon boundaries. This suggests that the ability of H2A.B to bind to RNA negatively regulates its capacity to bind to these factors (Sf3b155, Spt6, DDX39A and RNA Pol II). Unexpectedly, H2A.B.3 forms highly decompacted nuclear subdomains of active chromatin that co-localizes with splicing speckles in male germ cells. H2A.B.3 ChIP-Seq experiments revealed a unique chromatin organization at active genes being not only enriched at the transcription start site (TSS), but also at the beginning of the gene body (but being excluded from the +1 nucleosome) compared to the end of the gene. We also uncover a general histone variant replacement process whereby H2A.B.3 replaces H2A.Z at intron-exon boundaries in the testis and the brain, which positively correlates with expression and exon inclusion. Taken together, we propose that a special mechanism of splicing may occur in the testis and brain whereby H2A.B.3 recruits RNA processing factors from splicing speckles to active genes following its replacement of H2A.Z.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C.

Histone variants at the transcription start-site.

The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief.

Histone variant selectivity at the transcription start site: H2A.Z or H2A.Lap1.

Considerable attention has been given to the understanding of how nucleosomes are altered or removed from the transcription start site of RNA polymerase II genes to enable transcription to proceed. This has led to the view that for transcriptional activation to occur, the transcription start site (TSS) must become depleted of nucleosomes. However, we have shown that this is not the case with different unstable histone H2A variant-containing nucleosomes occupying the TSS under different physiological settings. For example, during mouse spermatogenesis we found that the mouse homolog of human H2A.Bbd, H2A.Lap1, is targeted to the TSS of active genes expressed during specific stages of spermatogenesis. On the other hand, we observed in trophoblast stem cells, a H2A.Z-containing nucleosome occupying the TSS of genes active in the G 1 phase of the cell cycle. Notably, this H2A.Z-containing nucleosome was different compared with other promoter specific H2A.Z nucleosomes by being heterotypic rather than being homotypic. In other words, it did not contain the expected two copies of H2A.Z per nucleosome but only one (i.e., H2A.Z/H2A rather than H2A.Z/H2A.Z). Given these observations, we wondered whether the histone variant composition of a nucleosome at an active TSS could in fact vary in the same cell type. To investigate this possibility, we performed H2A.Z ChIP-H2A reChIP assays in the mouse testis and compared this data with our testis H2A.Lap1 ChIP-seq data. Indeed, we find that different promoters involved in the expression of genes involved in distinct biological processes can contain either H2A.Z/H2A or H2A.Lap1. This argues that specific mechanisms exist, which can determine whether H2A.Z or H2A.Lap1 is targeted to the TSS of an active gene.

Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics.

Although it has been clearly established that well-positioned histone H2A.Z-containing nucleosomes flank the nucleosome-depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in mouse trophoblast stem cells, the amount of histone H2A.Z at promoters decreased during S phase, coinciding with homotypic (H2A.Z-H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z-H2A). To our surprise these nucleosomes remained heterotypic at M phase. At the TSS, we identified an unstable heterotypic histone H2A.Z-containing nucleosome in G1 phase that was lost after DNA replication. These dynamic changes at the TSS mirror a global expansion of the NDR at S and M phases, which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of histone H2A.Z at promoters, histone H2A.Z is targeted to the centromere when mitosis begins.

New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?

The compaction of genomic DNA into chromatin has profound implications for the regulation of key processes such as transcription, replication and DNA repair. Nucleosomes, the repeating building blocks of chromatin, vary in the composition of their histone protein components. This is the result of the incorporation of variant histones and post-translational modifications of histone amino acid side chains. The resulting changes in nucleosome structure, stability and dynamics affect the compaction of nucleosomal arrays into higher-order structures. It is becoming clear that chromatin structures are not nearly as uniform and regular as previously assumed. This implies that chromatin structure must also be viewed in the context of specific biological functions.

Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer.

Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.

A unique H2A histone variant occupies the transcriptional start site of active genes.

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.