Analysis of Human Histone H2AZ Deposition In Vivo Argues against Its Direct Role in Epigenetic Templating Mechanisms

Abstract

Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.Packaging into chromatin is a key distinguishing characteristic of the eukaryotic genome. Chromatin exists in different higher-order structures, which vary in terms of the degree of DNA condensation and methylation, the accessibility of the chromatin for regulatory proteins, and various histone posttranslational modifications (9). Different histone modifications were shown to be associated with different functional states. The extreme density and variety of modifications and their association with various functional states of chromatin have led to the "histone code" hypothesis (14, 38, 41), which postulates that the posttranslational modifications of core histones control association of chromatin with regulatory proteins, which regulate the structure and activity of chromatin.An important aspect of the histone code is its postulated epigenetic role. Currently, the term "epigenetic" is used to describe heritable or stable changes of phenotype that do not depend on changes in primary DNA sequence (34). Chromatin is considered a principal carrier of epigenetic information (9). However, unlike DNA replication, which relies on the complementary base-base recognition (44), the postulated epigenetic templating mechanisms depend on preferential recruitment of the enzymes that deposit particular epigenetic marks (DNA methylation, histone acetylation, or methylation) on chromatin to sites containing the same mark (13,14,28,42).The replacement histone variants add another dimension to the field of histone code studies (16,33,35,43). Unlike their canonical counterparts, the replacement histones are expressed in a replication-independent manner and employ specialized machineries for their deposition into chromatin (21, 24, 40). They can be associated with active or silenced chromatin and are involved in the regulation of gene expression and the organization of chromatin structure (16,17,22). Recent studies also suggest a role for replacement histones in epigenetic templating. One example is provided by neocentromeres in higher eukaryotes. Their formation and propagation do not require a particular DNA sequence (1, 5). CenpA, the specialized version of H3 histone, replaces H3 in the centromeric nucleosomes and likely serves as an epigenetic mark determining the maintenance of the centromere-specific nucleoprotein complex independent of DNA sequence...

The Chromatin Remodeling Protein, SRCAP, Is Critical for Deposition of the Histone Variant H2A.Z at Promoters

“…Several mechanisms might be used to direct SRCAP recruitment to this region, including histone modifications and binding of specific transcription factors. Although triMeH3K4 is found at active promoters and enriched in nucleosomes containing H2A.Z (19), the lack of overlap with the SRCAP binding site within the SP-1 promoter suggests that this modification does not represent a bona fide targeting site for recruitment of SRCAP. Other histone modifications are enriched in nucleosomes containing H2A.Z, including triMeH3K27 and triMeH3K9 (19).…”

“…Although triMeH3K4 is found at active promoters and enriched in nucleosomes containing H2A.Z (19), the lack of overlap with the SRCAP binding site within the SP-1 promoter suggests that this modification does not represent a bona fide targeting site for recruitment of SRCAP. Other histone modifications are enriched in nucleosomes containing H2A.Z, including triMeH3K27 and triMeH3K9 (19). These modifications, however, have been associated with heterochromatin and are unlikely to play a role in targeting SRCAP to active promoters.…”

“…SRCAP Is Recruited Adjacent to the Sites of triMeH3K4 Deposition-Nucleosomes containing H2A.Z have been found to be enriched in the histone modification triMeH3K4 (19). This suggested that the location of triMeH3K4 might provide a signal for recruitment of SRCAP to the SP-1 promoter.…”

“…Histone modifications found in promoters of active genes, e.g. triMeH3K4, acetylated H2B, and acetylated H4 have been reported to be enriched in H2A.Z-containing nucleosomes purified from chicken and HeLa cells (12,13,19). The loss of specific histone acetyltransferases GCN5 and Sas3 has also been reported to reduce H2A.Z deposition (5).…”

Reconciling the positive and negative roles of histone H2A.Z in gene transcription

“…65 This prediction has been invalidated by the fact that hybrid nucleosomes carrying both H2A.Z and H2A were observed both in vivo and in vitro. 66,67 Second, subtle localized changes in the interaction of H2A.Z with H3 would result in a decreased stability of the nucleosome. In vitro studies aimed at investigating the structural impact of the incorporation of H2A.Z on nucleosome dynamics have generated contradictory observations with respect to its stability.…”