Histone H2A variant

Function of yeast H2A variants

Silencing at mating locus HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. Mutants of Sir1p were generated that are recruited to the silencer but are unable to silence, and these mutants were used to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing (Dhillon, 2000).

Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA. Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA. The variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA. This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization. H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation. These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure (Santisteban, 2000).

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state (Meneghini, 2003).

Incorporation of yeast H2A variants into chromatin

The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. How histone variants such as H2AZ are incorporated into nucleosomes has been obscure. This study has found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays. Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes. These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery (Mizuguchi, 2004).

The condensation of eukaryotic DNA in arrays of nucleosomes has a profound effect on gene function. To counteract constraints imposed by nucleosome structure, cells deploy two major classes of multiprotein enzymes, which covalently modify the nucleosome core histones or catalyze nucleosome mobility in an adenosine triphosphate (ATP)-dependent fashion. Much of the current understanding of these processes is derived from analyses of nucleosomal histones that represent the major histone species within cells. However, the existence of minor histone variants, encoded by distinct, nonallelic genes, has long been recognized. Recent studies have revealed that variants of histone H2A and histone H3 play important roles not only in gene expression but also in the repair of DNA breaks and the assembly of chromosome centromeres. These advances establish a third mechanism of chromatin reconfiguration, raising fundamental questions about the stability of nucleosomes in nonreplicative phases of the cell cycle and the cellular machinery responsible for incorporating histone variants into nucleosomes (Mizuguchi, 2004).

The histone H2A.F/Z (H2AZ) variant is a functionally distinct, highly conserved histone subgroup that likely represents a separate evolutionary lineage of histone H2A proteins. Histone H2AZ replaces the major histone H2A in a fraction of the nucleosomes isolated from chromatin and reconstitutes a similar though nonidentical structure to the canonical nucleosome. There is growing evidence that the incorporation of H2AZ has an important influence on gene expression. Moreover, H2AZ in transcriptionally active domains near yeast telomeres and flanking the HMR mating-type locus functions as a buffer to gene silencing caused by the spread of heterochromatin proteins. This study shows that Saccharomyces cerevisiae Swr1, an uncharacterized member of the Swi2/Snf2 family of chromatin remodeling adenosine triphosphatases (ATPases), is contained in a multicomponent protein complex that catalyzes H2AZ-specific histone exchange (Mizuguchi, 2004).

In budding yeast, the paralogous genes SWR1 and INO80 are distinguished from other members of the SWI2/SNF2 family by an insertion that splits the conserved ATPase domain into two segments. In contrast to Ino80, little is known about Swr1. To enable purification of native Swr1, a triple Flag epitope tag was engineered at its C terminus and the function of Swr1-Flag verified in vivo. Immunopurification from whole-yeast extracts has revealed over 12 proteins associating with Swr1-Flag. This assembly, named the SWR1 complex, sediments as a peak during glycerol gradient centrifugation and exhibits nucleosome-stimulated ATPase activity. Components of the SWR1 complex were identified by peptide microsequencing with the use of mass spectrometry. Several subunits-Act1, Arp4, Rvb1, and Rvb2-are common to the INO80 complex and other chromatin remodeling complexes. Arp6 is an actin-related protein, Yaf 9 is the yeast counterpart of the human leukemogenic protein AF9, and the remaining SWR1 subunits-Vps72, Vps71, Swc1, Aor1, and God1-are uncharacterized database entries (Mizuguchi, 2004).

Several small polypeptides that copurified with the SWR1 and INO80 complexes were identified as histones. Although the association of histones with chromatin remodeling complexes could be due to nonspecific binding of chromatin, the persistence of histone binding upon nuclease digestion suggested otherwise. Among the histones copurifying with the SWR1 complex, a substantial number of tryptic peptides are derived from histone Htz1, the H2AZ variant in yeast (Mizuguchi, 2004).

Purified Htz1 associates with SWR1 complex. To confirm the physical interaction between yeast Htz1 and the SWR1 complex, native Htz1-Flag was immunopurified from whole yeast extracts. It was found that Htz1 copurifies with its partner histone H2B and many other polypeptides, including all components of the SWR1 complex. Glycerol gradient centrifugation separated the free Htz1-H2B dimer (fraction 5) from two additional assemblies: the SWR1 complex (fraction 22) and a complex that was named NAP-Z, containing the nucleosome assembly protein Nap1 and other polypeptides. The bulk of soluble Htz1 is distributed between free Htz1-H2B dimers and Nap1-associated Htz1-H2B dimers. The SWR1 complex contains only a minor portion of the soluble Htz1-H2B dimer population (Mizuguchi, 2004).

Swr1 and Htz1 functions overlap in vivo. To determine the biological relevance of the interaction between Htz1 and the SWR1 complex, the growth phenotypes of mutants was analyzed. The swr1 null or catalytic site mutant is viable and sensitive to caffeine and the alkylating agent methyl methanesulfonate (MMS) and weakly sensitive to ultraviolet (UV) irradiation. The htz1 mutant is also viable, sensitive to caffeine and MMS, and moderately sensitive to UV, implicating a role for both proteins in DNA damage repair and other metabolic activities. However, the htz1 mutant is more sensitive than swr1 to diminished deoxynucleotide triphosphate pools caused by the ribonucleotide reductase inhibitor hydroxyurea (HU). The swr1 phenotypes were rescued by the wild-type gene but not by a mutant carrying a Lys727Gly727 (K727G) substitution in the ATP-binding motif of Swr1, indicating that ATP-use is crucial for its in vivo function (Mizuguchi, 2004).

Genome-wide transcription profiles were examined for swr1 and htz1 mutants. Complementary DNA hybridization to yeast whole-genome microarrays revealed that 71 out of 162 Swr1-activated genes are also activated by Htz1 (44% overlap). Of 77 genes repressed by Swr1, 29 are also repressed by Htz1 (38% overlap). The incompleteness of the overlaps suggests a degree of functional independence for the two genes, or it may reflect an inherent limitation of microarray analyses. By contrast, 48 out of 446 Ino80-activated genes are also activated by Htz1 (11% overlap), and 72 out of 779 Ino80-repressed genes are also repressed by Htz1 (9% overlap). The Swr1-activated, but not Ino80-activated, genes are highly represented near yeast telomeres, consistent with the chromosomal locations of Htz1-activated genes. No preferential location of Htz1- or Swr1-repressed genes at yeast telomeres was found (Mizuguchi, 2004).

Chromatin binding of Htz1 in vivo requires Swr1. The physical and functional connections between Swr1 and Htz1 suggested that the SWR1 complex might facilitate the assembly or remodeling of variant nucleosomes containing Htz1, leading to changes in transcription of targeted genes. Accordingly, the in vivo binding of Htz1 at various chromosomal locations in wild-type and swr1 mutant cells was compared with the use of formaldehyde cross-linking and chromatin immunoprecipitation (ChIP). Htz1 is known to bind preferentially near telomeres and at regions flanking the silent mating type locus, where it antagonizes the effects of gene silencing by heterochromatin. At 3 kb from the telomere, binding of Htz1 at the RDS1 promoter was lost in the swr1 null or catalytic site mutant. This cannot be attributed to a failure to express Htz1 in the swr1 mutant. Loss of Htz1 binding in swr1 also occurs at the ADE17 promoter, 415 kb from the telomere. Likewise, a loss of Htz1 binding is observed at CAR2 and LSM3 coding regions, 65 kb from the telomere (Mizuguchi, 2004).

How might Swr1 be used for the incorporation of Htz1 in chromatin? It is proposed that the ATP-driven disruption of conventional nucleosomes by the SWR1 complex leads to displacement of histone H2A (probably as the H2A-H2B dimer) and replacement with Htz1 (Htz1-H2B) in a histone dimer exchange reaction. Accordingly, immobilized nucleosome arrays were prepared with the use of DNA bound to magnetic beads and a recombinant yeast nucleosome assembly system. Incubation with the SWR1 complex, a source of Htz1-H2B dimers, and ATP revealed a striking transfer of Htz1-Flag to immobilized nucleosomes. Either free Htz1-H2B dimers or dimers associated with the NAP-Z complex could be transferred by the SWR1 enzyme, indicating that Nap1 functions as an escort rather than part of the histone transfer machinery. The reaction proceeds efficiently at room temperature with catalytic levels of the SWR1 complex (one enzyme to 50 nucleosomes) and stoichiometric amounts of Htz1-H2B, resulting in substantial transfer of Htz1-Flag to the immobilized nucleosome array in 60 min (77% transfer of free Htz1-H2B dimers and 57% transfer of Nap1-associated dimers). Transfer of Htz1 is readily observed within 5 min and is specific for nucleosomal templates, because none occurs on immobilized, naked DNA. Moreover, the SWR1 complex transfers Htz1-H2B selectively over H2A-H2B, as shown by weak transfer (11%) when tagged H2A-H2B dimers are substituted for tagged Htz1-H2B in an otherwise identical reaction and by the excess of competing H2A-H2B (ninefold) required to reduce transfer of tagged Htz1-H2B by half (Mizuguchi, 2004).

Although maximal incorporation of Htz1 into immobilized chromatin requires exogenous ATP, transfer could be reproducibly detected in the absence of ATP. This was also observed when ATP was substituted with adenosine diphosphate, guanosine triphosphate, and nonhydrolyzable ATP analogs. Such transfer of Htz1 likely results from endogenous ATP bound to the purified SWR1 complex, because transfer was not detectable when an intact enzyme complex carrying a mutation in the ATP binding site of Swr1 was substituted for the wild-type complex. Other complexes of the SWI/SNF family were also evaluated. At equimolar or greater levels to the SWR1 complex, purified ISW1 (imitation switch), SWI/SNF, RSC (remodel the structure of chromatin), and INO80 complexes transferred Htz1 weakly despite having higher ATPase activities (Mizuguchi, 2004).

To address the fate of histone H2A in the nucleosome upon transfer of Htz1 by SWR1, the products of the transfer reaction were analyzed with the use of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (which allows preferential visualization of yeast histones H2A and H2B). Of the four core histones, only histone H2A was substantially displaced (60%) from immobilized nucleosome arrays upon transfer of Htz1. By contrast, the level of histone H2B (and H3 and H4) remained essentially unchanged. Hence, it is concluded that the SWR1 complex catalyzes displacement of histone H2A and replacement with Htz1 in a bona fide histone exchange reaction. To verify the native conformation of nucleosome core particles produced by the histone replacement reaction, mononucleosomes were reconstituted on a 201-base pair (bp) DNA fragment with the use of bacterially expressed, conventional histone octamers. Mononucleosomes were incubated with SWR1 complex, tagged Htz1-H2B dimers, and ATP, followed by digestion with micrococcal nuclease to release the nucleosome core particle. Native gel electrophoresis and DNA staining showed that the bulk of the core particle population retains native conformation, as judged by electrophoretic mobility. The incorporation of Htz1-Flag in the nucleosome core particle was shown by Western blot analysis of the native gel (Mizuguchi, 2004).

Two chromatin remodeling mechanisms involving alterations in DNA-histone contacts without major perturbation of the histone octamer have been proposed. In the 'sliding' mechanism, the ATP-driven propagation of a local DNA twist or bulge over the histone octamer surface, initiating from the DNA entry or exit positions, causes the octamer to be relocated relative to the DNA sequence. There is general agreement that the comparatively small (0.5 MD) ISWI-containing complexes use a sliding mechanism to shift nucleosome positions within a nucleosome array in vitro. Importantly, remodeling intermediates detected in vivo are consistent with a nucleosome sliding mechanism for yeast ISW2. A more complicated DNA 'looping' mechanism distinguishes the large (1 MD) SWI/SNF and closely related enzymes from ISWI enzymes. The looping mechanism is characterized by peeling of a long DNA segment from one edge of the nucleosome, octamer mobilization, and DNA rewrapping; nucleosomal DNA is globally distorted and becomes more susceptible to restriction nuclease digestion. Both sliding and looping mechanisms apparently involve an ATP-dependent DNA-translocating activity displayed by Swi2/Snf2 family proteins (Mizuguchi, 2004).

The catalytic exchange of nucleosomal H2A for the H2AZ variant by the SWR1 complex suggests a previously unknown mechanism of chromatin remodeling that necessitates the disruption of histone-histone as well as histone-DNA contacts. Assuming the H2A-H2B dimer to be the unit of histone exchange, histone replacement necessitates displacement of two H2A-H2B dimers from the nucleosome core particle. It is proposed that, like SWI/SNF, the SWR1-catalyzed unwrapping of nucleosomal DNA from the entry or exit positions of the nucleosome exposes the DNA-binding surface of an H2A-H2B dimer. This unwrapping should promote the intrinsic tendency of the histone octamer to dissociate into constitutent H2A-H2B dimers and the (H3-H4)2 tetramer. In addition, components of the SWR1 complex could directly provoke dissociation of the first H2A-H2B dimer from the histone octamer. Subsequent release of Htz1-H2B from the SWR1 complex near the vacant site, coupled with relaxation or rewrapping of DNA, would then reassemble a core particle containing one each of H2A-H2B and Htz1-H2B dimers. Structural incompatibility between two heterotypic dimers within the same nucleosome core particle should then facilitate SWR1-catalyzed exchange of the second H2A-H2B dimer in a cooperative fashion (Mizuguchi, 2004).

Other workers have independently found biochemical and genetic interactions between Swr1 and Htz1 or their mammalian orthologs, although the demonstration of SWR1-catalyzed ATP-dependent histone variant exchange in vitro is unprecedented. Furthermore, the transfer of histones per se is not exclusive to ATP-dependent chromatin remodeling enzymes (Mizuguchi, 2004).

The association of histones Htz1-H2B with the purified SWR1 complex suggests that specific SWR1 components are responsible for binding to Htz1. Likely candidates are the actin-related proteins (Arps), some of which have a role in the chromatin remodeling process and exhibit histone-binding activity in vitro. The SWR1 complex contains actin, Arp4, and, uniquely, Arp6, raising the prospect that Arp6 may have a key role in sequestering the Htz1-H2B dimer specifically (Mizuguchi, 2004).

The SWR1-dependent incorporation of Htz1 at either intergenic or transcribed regions suggests that enzyme recruitment to chromatin may involve more than promoter-specific factors and raises the possibility that Htz1 affects both initiation and elongation stages of transcription. Indeed, recent genetic studies underscore a role for Htz1 in transcription elongation. The additional Htz1-interacting proteins identified by mass spectrometry could contribute toward this function. How the presence of one or several Htz1-variant nucleosomes within a conventional nucleosome array affects transcription initiation or elongation is unclear (Mizuguchi, 2004).

The catalysis of H2AZ variant exchange by the SWR1 complex invites speculation on a general mechanism by which histone variants are incorporated into nucleosomes. It is suggested that other ATP-dependent chromatin remodeling complexes might catalyze the incorporation of specific histone variants, although proteins such as chromatin assembly factors may also be important. Moreover, covalent modifications might provide histone signals for the catalytic exchange of histone variants or even of major histones, thereby contributing to mechanisms for histone replacement and nucleosome loss associated with transcription in vivo. The further development of assays to capture the movements of physiologically marked histones should provide insight into the regulation of chromatin dynamics (Mizuguchi, 2004).

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. A complex containing 13 different polypeptides associated with a soluble pool of H2A.Z was identified in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions (Kabor, 2004).

Mechanisms that specify promoter nucleosome location and identity

The chromatin architecture of eukaryotic gene promoters is generally characterized by a nucleosome-free region (NFR) flanked by at least one H2A.Z variant nucleosome. Computational predictions of nucleosome positions based on thermodynamic properties of DNA-histone interactions have met with limited success. This study shows that the action of the essential RSC remodeling complex in S. cerevisiae helps explain the discrepancy between theory and experiment. In RSC-depleted cells, NFRs shrink such that the average positions of flanking nucleosomes move toward predicted sites. Nucleosome positioning at distinct subsets of promoters additionally requires the essential Myb family proteins Abf1 and Reb1, whose binding sites are enriched in NFRs. In contrast, H2A.Z deposition is dispensable for nucleosome positioning. By regulating H2A.Z deposition using a steroid-inducible protein splicing strategy, it was shown that NFR establishment is necessary for H2A.Z deposition. These studies suggest an ordered pathway for the assembly of promoter chromatin architecture (Hartley, 2009).

A striking result presented in this study is that at a majority of promoters, the normal positioning of NFR-flanking nucleosomes requires the essential multisubunit ATP dependent chromatin modeling complex RSC. Such a central role for RSC in generating promoter chromatin architecture is consistent with several of its properties: (1) RSC, unlike most chromatin remodeling enzymes in yeast, is essential for viability; (2) RSC slides nucleosomes in vitro, and (3) RSC is required globally for RNA polymerase II transcription. The current studies are also consistent with a recent lower-resolution study that concluded that RSC affected histone density at a number of promoters. A recent study indicated changes in the positioning nucleosomes at ~12% of promoters in cells lacking the Isw2 chromatin remodeling complex. The primary function of Isw2 appears to be in transcriptional repression and in suppressing antisense transcription. Interestingly, in contrast to RSC, Isw2 appears to move nucleosomes in vivo toward the NFR, raising the possibility that it antagonizes the action of RSC at some promoters. The potential for dynamic involvement of multiple ATPases at promoters further underscores the active nature of mechanisms that position nucleosomes in vivo (Hartley, 2009).

H2A.Z homolog in tetrahymena

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stain at all stages of the life cycle. Micronuclei do not stain during growth or starvation but stain with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappears from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stains micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin (Stargell, 1993).

Structure vertebrate H2A.Z

Activation of transcription within chromatin has been correlated with the incorporation of the essential histone variant H2A.Z into nucleosomes. H2A.Z and other histone variants may establish structurally distinct chromosomal domains; however, the molecular mechanism by which they function is largely unknown. The 2.6 A crystal structure of a nucleosome core particle containing the histone variant H2A.Z is reported in this study. The overall structure is similar to that of the previously reported 2.8 A nucleosome structure containing major histone proteins. However, distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)(2) tetramer. Moreover, H2A.Z nucleosomes have an altered surface that includes a metal ion. This altered surface may lead to changes in higher order structure, and/or could result in the association of specific nuclear proteins with H2A.Z. Finally, incorporation of H2A.Z and H2A within the same nucleosome is unlikely, due to significant changes in the interface between the two H2A.Z-H2B dimers (Suto, 2000).

Functional analysis of vertebrate H2A.Z

H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from their H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle, the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation (Abbott, 2001).

Explaining the determinants involved in regulating the equilibrium between different chromatin structural states is fundamental to understanding differential gene expression. Histone variant H2A.Z is essential to chromatin architecture in higher eukaryotes but its role has not yet been explained. H2A.Z is shown to facilitates the intramolecular folding of nucleosomal arrays while simultaneously inhibiting the formation of highly condensed structures that result from intermolecular association. This makes a case for H2A.Z playing a fundamental role in creating unique chromatin domains poised for transcriptional activation. These results provide new insights into understanding how chromatin fiber dynamics can be altered by core histone variants to potentially regulate genomic function (Fan, 2002).

H2A.Z has been shown to regulate transcription in yeast, and that function resides in its C-terminal region as the reciprocal portion of H2A cannot substitute for the latter. Fusion of a transcriptional activating region to the C-terminal region of H2A, which is substituted for that of H2A.Z, can allow the chimera to fulfil the special role of H2A.Z in positive gene regulation, as well as complement growth deficiencies of htz1delta cells. The 'transcription' function of H2A.Z is linked to its ability to preferentially localize to certain intergenic DNA regions. These results suggest that H2A.Z modulates functional interactions with transcription regulatory components, and thus increases its localization to promoters where it helps poise chromatin for gene activation (Larochelle, 2003).

The histone variant H2A.Z plays an essential role in metazoans but its function remains to be determined. A new inducible RNAi strategy was developed to elucidate the role of H2A.Z in mammalian cell lines. In the absence of H2A.Z, the genome becomes highly unstable and this instability is caused by defects in the chromosome segregation process. Analysis of H2A.Z localization reveals that in these cells it is enriched at heterochromatic foci with HP1alpha on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1alpha-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1alpha. It is concluded that H2A.Z is essential for the accurate transmission of chromosomes (Rangasamy, 2004).

Controlling the degree of higher order chromatin folding is a key element in partitioning the metazoan genome into functionally distinct chromosomal domains. However, the mechanism of this fundamental process is poorly understood. Studies have suggested that the essential histone variant H2A.Z and the silencing protein HP1alpha may function together to establish a specialized conformation at constitutive heterochromatic domains. HP1alpha is a unique chromatin binding protein. It prefers to bind to condensed higher order chromatin structures and alters the chromatin-folding pathway in a novel way to locally compact individual chromatin fibers without crosslinking them. Strikingly, both of these features are enhanced by an altered nucleosomal surface created by H2A.Z (the acidic patch). This shows that the surface of the nucleosome can regulate the formation of distinct higher order chromatin structures mediated by an architectural chromatin binding protein (Fan, 2004).

Critical to vertebrate development is a complex program of events that establishes specialized tissues and organs from a single fertilized cell. Transitions in chromatin architecture, through alterations in its composition and modification markings, characterize early development. A variant of the H2A core histone, H2A.Z, is essential for development of both Drosophila and mice. H2A.Z is required for proper chromosome segregation. Whether H2A.Z has additional specific functions during early development remains unknown. Depletion of H2A.Z by RNA interference perturbs Xenopus laevis development at gastrulation, leading to embryos with malformed, shortened trunks. Consistent with this result, whole embryo in situ hybridization indicates that endogenous expression of H2A.Z is highly enriched in the notochord. H2A.Z modifies the surface of a canonical nucleosome by creating an extended acidic patch and a metal ion-binding site stabilized by two histidine residues. To examine the significance of these specific surface regions in vivo, the consequences were investigated of overexpressing H2A.Z and mutant proteins during X. laevis development. Overexpression of H2A.Z slows development following gastrulation. Altering the extended acidic patch of H2A.Z reverses this effect. Remarkably, modification of a single stabilizing histidine residue located on the exposed surface of an H2A.Z containing nucleosome is sufficient to disrupt normal trunk formation mimicking the effect observed by RNA interference. Taken together, these results argue that key determinants located on the surface of an H2A.Z nucleosome play an important specific role during embryonic patterning and provide a link between a chromatin structural modification and normal vertebrate development (Ridgway, 2004).

H2A.Z and transcriptional regulation

The post-translational modification of histones and the incorporation of core histone variants play key roles in governing gene expression. Many eukaryotic genes regulate their expression by limiting the escape of RNA polymerase from promoter-proximal pause sites. Elongating RNA polymerase II complexes encounter distinct chromatin landscapes that are marked by methylation of lysine residues Lys(4), Lys(79), and Lys(36) of histone H3. However, neither histone methylation nor acetylation directly regulates the release of elongation complexes stalled at promoter-proximal pause sites of the c-myc gene. In contrast, transcriptional activation is associated with local displacement of the histone variant H2A.Z within the transcribed region and incorporation of the major histone variant H2A. This result indicates that transcribing RNA polymerase II remodels chromatin in part through coincident displacement of H2A.Z-H2B dimers and incorporation of H2A-H2B dimers. In combination, these results suggest a new model in which the incorporation of H2A.Z into nucleosomes down-regulates transcription; at the same time it may act as a cellular memory for transcriptionally poised gene domains (Farris, 2005)

Incorporation of H2A.Z into the chromatin of inactive promoters has been shown to poise genes for their expression. This study provides evidence that H2A.Z is incorporated into the promoter regions of estrogen receptor (ERalpha) target genes only upon gene induction, and that, in a cyclic pattern. Moreover, members of the human H2A.Z-depositing complex, p400, also follow the same gene recruitment kinetics as H2A.Z. Importantly, cellular depletion of H2A.Z or p400 leads to a severe defect in estrogen signaling, including loss of estrogen-specific cell proliferation. Incorporation of H2A.Z within the estrogen receptor responsive trefoil factor 1 promoter chromatin allows nucleosomes to adopt preferential positions along the DNA translational axis. Finally, evidence is provided that H2A.Z is essential to allow estrogen-responsive enhancer function. Taken together, these results provide strong mechanistic insight into how H2A.Z regulates ERalpha-mediated gene expression and provide a novel link between H2A.Z-p400 and ERalpha-dependent gene regulation and enhancer function (Gévry, 2009).

Developmental expression and roles of H2A.Z

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1 degrees have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function. A mammalian histone variant, H2A.Z, plays a critical role in early development, and it is concluded that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development (Mattaei, 2001).

Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). Undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric heterochromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, H2A.Z has been shown to directly interact with the pericentric heterochromatin binding protein INCENP. It is proposed that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins (Rangasamy, 2003).

p21 transcription is regulated by differential localization of histone H2A.Z

In yeast cells, H2A.Z regulates transcription and is globally associated within a few nucleosomes of the initiator regions of numerous promoters. H2A.Z is deposited at these loci by an ATP-dependent complex, Swr1.com. H2A.Z suppresses the p53 --> p21 transcription and senescence responses. Upon DNA damage, H2A.Z is first evicted from the p21 promoter, followed by the recruitment of the Tip60 histone acetyltransferase to activate p21 transcription. p400, a human Swr1 homolog, is required for the localization of H2A.Z, and largely colocalizes with H2A.Z at multiple promoters investigated. Notably, the presence of sequence-specific transcription factors, such as p53 and Myc, provides positioning cues that direct the location of H2A.Z-containing nucleosomes within these promoters. Collectively, this study strongly suggests that certain sequence-specific transcription factors regulate transcription, in part, by preferentially positioning histone variant H2A.Z within chromatin. This H2A.Z-centered process is part of an epigenetic process for modulating gene expression (Gévry, 2007).

Eukaryotic DNA is condensed many fold (e.g., 10,000) into chromatin, the basic unit of which contains 146 base pairs (bp) of DNA and an octamer of histone proteins (H2A, H2B, H3, and H4). Due to the high level of compaction, chromatin typically represses certain cellular DNA transactions, including transcription. For successful transcription, it is argued that nucleosomes need to be remodeled or evicted from promoter regions for the transcriptional machinery to be efficiently recruited to a target gene (Gévry, 2007).

The incorporation of histone variants into specific nucleosomes within a promoter region constitutes a mechanism by which promoter region chromatin can become more permissive to transcription initiation and elongation following receipt of a proper physiological cue. One such histone variant is H2A.Z. In Saccharomyces cerevisiae, it can elicit positive effects on gene expression. In addition, H2A.Z regulates genes that are proximal to telomeres and acts as a 'buffer' to antagonize the spread of heterochromatin into euchromatic regions (Meneghini, 2003). Furthermore, recent reports (Guillemette, 2005; Li, 2005; Raisner, 2005; Zhang, 2005) have shown that H2A.Z is preferentially localized within a few nucleosomes of the initiator regions of multiple promoters in the yeast genome. Interestingly, these H2A.Z-rich loci are largely devoid of transcriptional activity, which suggests that the variant histone prepares genes for activation (Guillemette, 2005) and/or operates as a transcriptional repressor. Finally, yeast H2A.Z has been shown to regulate nucleosome positioning, which provides mechanistic insight into how its presence can alter promoter transcriptional state (Gévry, 2007).

An ATP-dependent chromatin remodeling complex that specifically loads H2A.Z onto chromatin and exchanges it with H2A exists in yeast (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). This complex, in which the catalytic subunit is Swr1, also shares essential subunits with the NuA4 histone acetyltransferase complex (Krogan, 2003; Kobor, 2004). In addition to their importance in gene regulation, the Swr1 complex, H2A.Z, and NuA4 are all involved in the regulation of yeast chromosome stability (Krogan, 2004). This is noteworthy because, in mammalian cells, depletion of H2A.Z causes major nuclear and chromosomal abnormalities (Rangasamy, 2004) as witnessed by a high incidence of lagging chromosomes and chromatin bridges (Gevry, 2007).

There are two homologs of Swr1 in human cells: p400/Domino (referred to as p400), and SRCAP. There are also three uncharacterized p400-type SWI2-SNF2 molecules, including hIno80. Members of this family of SWI2/SNF2 chromatin remodeling enzymes each contain a spacer region inserted into the SWI2/SNF2 homology region (Gevry, 2007).

p400 was originally isolated as an E1A-associated protein, and it was also shown to interact with p53, Myc, and SV40 large T antigen. It is also required for E1A to induce p53-mediated apoptosis. SRCAP has been isolated as a CREB-binding protein. While one report shows that both p400 and SRCAP constitute part of the same complex, a recent study shows that SRCAP and p400 exist in distinct complexes with H2A.Z (Jin, 2005; Ruhl, 2006). Recently an SRCAP-containing complex was purified, and it was shown to have the ability to exchange H2A-H2B for H2A.Z-H2B in reconstituted mononucleosomes (Ruhl, 2006). It remains to be determined whether mammalian homolog(s) of Swr1, such as p400 and SRCAP, also catalyze H2A.Z deposition in vivo (Gevry, 2007).

Depletion of p400 elevates p21 synthesis to initiate premature senescence in primary human fibroblasts (Chan, 2005). Senescence has been observed in tissue culture cells as a stable form of cell growth arrest provoked by diverse stresses. Recently, oncogene-induced senescence was shown to occur in various precancerous lesions both in humans and mice, further suggesting that senescence acts as a defense mechanism against malignant cell development. Importantly, the action of p400 at p21 depends on the function of p53, a key regulator of p21 transcription (Gevry, 2007).

Given the possibility of a link between p400 and H2A.Z, it was asked whether H2A.Z is also an important regulator of p21 expression. The results of this effort show that H2A.Z depletion induces p21 expression in a p53-dependent fashion, as well as the premature senescence of primary diploid fibroblasts. Similar to senescence induced by p400 depletion, inactivating p53 or p21 blocked the emergence of certain senescent phenotypes following H2A.Z depletion. In a normal setting, H2A.Z is highly enriched at discrete p53-binding sites that lie within the p21 promoter. This distinctive localization pattern depends on the presence of p53, and was detected at other p53 target gene promoters as well. The presence of p400 is required to localize H2A.Z at those loci, and purified recombinant p400 from insect cells can carry out in vitro exchange of H2A.Z-H2B dimers into chromatin. H2A.Z and p400 localization at the p53-binding sites in p21 is severely diminished following p21 induction, and this process is not dependent on active p21 transcription per se. After H2A.Z and p400 eviction from the p53-binding sites in p21, it was observed that the Tip60 histone acetyltransferase isrecruited to the distal p53-binding site in the promoter to positively regulate p21 expression. Finally, overexpression of Myc, a known suppressor of p21 synthesis, significantly increases H2A.Z localization at the Myc-binding site in the TATA initiator region of the p21 promoter. This observation is consistent with the view that Myc represses p21 expression by preferentially recruiting H2A.Z-containing nucleosome(s) to this element (Gevry, 2007).

Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks

Eukaryotic chromatin is separated into functional domains differentiated by posttranslational histone modifications, histone variants, and DNA methylation. Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are critical for eukaryotic development, and aberrant methylation-induced silencing of tumor suppressor genes is a common feature of human cancer. In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5' ends of genes where it promotes transcriptional competence. How DNA methylation and H2A.Z influence transcription remains largely unknown. This study shows that in the plant Arabidopsis thaliana, regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes in H2A.Z deposition, while mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z17 leads to genome-wide hypermethylation. These findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation (Zioberman, 2008).

H3.3 actively marks enhancers and primes gene transcription via opening higher-ordered chromatin

The histone variants H3.3 and H2A.Z have recently emerged as two of the most important features in transcriptional regulation, the molecular mechanism of which still remains poorly understood. This study investigated the regulation of H3.3 and H2A.Z on chromatin dynamics during transcriptional activation. In vitro biophysical and biochemical investigation showed that H2A.Z promotes chromatin compaction and represses transcriptional activity. Surprisingly, with only four to five amino acid differences from the canonical H3, H3.3 greatly impaires higher-ordered chromatin folding and promotes gene activation, although it has no significant effect on the stability of mononucleosomes. It was further demonstrated that H3.3 actively marks enhancers and determines the transcriptional potential of retinoid acid (RA)-regulated genes via creating an open chromatin signature that enables the binding of RAR/RXR. Additionally, the H3.3-dependent recruitment of H2A.Z on promoter regions results in compaction of chromatin to poise transcription, while RA induction results in the incorporation of H3.3 on promoter regions to activate transcription via counteracting H2A.Z-mediated chromatin compaction. These results provide key insights into the mechanism of how histone variants H3.3 and H2A.Z function together to regulate gene transcription via the modulation of chromatin dynamics over the enhancer and promoter regions (Chen, 2013).

Expression of non-acetylatable H2A.Z in myoblast cells blocks myoblast differentiation through disruption of MyoD expression

H2A.Z is a histone H2A variant that is essential for viability in Tetrahymena, Drosophila and also during embryonic development of mice. Although implicated in diverse cellular processes, including transcriptional regulation, chromosome segregation and heterochromatin formation, its essential function in cells remains unknown. Cellular differentiation is part of the developmental process of multicellular organisms. To elucidate the roles of H2A.Z and H2A.Z acetylation in cellular differentiation, this study examined the effects of expressing wild type (WT) or a non-acetylatable form of H2A.Z in the growth and differentiation of the myoblast C2C12 cell line. Ectopic expression of wild type or mutant H2A.Z resulted in distinct phenotypes in the differentiation of the C2C12 cells and the formation of myotubes. Most strikingly, expression of the H2A.Z non-acetylatable mutant (H2A.Z-Ac-mut) resulted in a complete block of myoblast differentiation. This phenotype was determined to be caused by a loss of MyoD expression in the Ac-mut-expressing cells prior to and post-induction of differentiation. Moreover, chromatin accessibility assays showed that the promoter region of MyoD is less accessible in the differentiation-defective cells. All together, these new findings show that expression of the Ac-mut form of H2A.Z resulted in a dominant phenotype that blocked differentiation due to chromatin changes at the MyoD promoter (Law, 2015).

Histone H2A variant : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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