Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant

Abstract

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. Here, we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. We find that the Xnp chromatin remodeler and the Hira histone chaperone independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. We propose that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus.nucleosome assembly | transcription D NA in the eukaryotic nucleus is associated with histone proteins to form nucleosomes, the fundamental units of chromatin. Most nucleosomes are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. These regions are repackaged by replication-independent (RI) nucleosome replacement using the H3.3 histone variant (1, 2). This process results in the enrichment of the H3.3 histone variant at all sites where nucleosomes are unstable or disrupted (3, 4). How H3.3 is delivered to dynamic chromatin sites is unknown. However, biochemical isolation of predeposition complexes has identified shared and distinctive assembly factors that associate with the H3 and H3.3 histones and mediate the replicationcoupled or RI assembly of nucleosomes, respectively (4-6). These factors include histone chaperones and chromatin remodelers that are important for new nucleosome assembly, and might potentially target histones to active chromatin regions. However, mutants in some of these factors have surprisingly limited phenotypes. The Hira chaperone promotes H3.3 deposition at genes (4) but is only essential for H3.3 deposition on sperm chromatin during fertilization (7,8). In Drosophila the ATRX/XNP remodeler homolog Xnp colocalizes with H3.3 in somatic cells, but is not essential (9). In mammals, ATRX/XNP promotes H3.3 deposition only at telomeres and some heterochromatic sequences (4, 6, 10). These results have raised the possibilities that H3.3 assembly factors are redundant or that additional factors involved in the deposition of this histone variant exist. Loss of H3.3 itself can be compensated in somatic cells by the major H3 histone, suggesting that assembly of any nucleosome suffices (11).In this work we use an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to study the mechanism of nuc...

The Drosophila DAXX-Like Protein (DLP) Cooperates with ASF1 for H3.3 Deposition and Heterochromatin Formation

“…Hence, the suppression of H3.3 expression has a profound effect on the DLP distribution pattern, which further confirms the close relationship between these two proteins. The nucleolar staining in H3.3-depleted cells is reminiscent of the one described previously for XNP (23), suggesting that DLP and XNP may have some common target sites in the genome. Indeed, the major site for XNP binding is at the base of the X chromosome, where DLP is found.…”

“…6A). Since the incorporation of H3.3 core is abolished in Hira HR1 ; xnp Ϫ mutant cells (23), this suggests that the contribution of DLP to RI deposition is not equivalent to that of XNP.…”

“…A previously reported model suggested that XNP recruits a predeposition complex containing H3.3/H4 dimers, ASF1 (23), and additional factors. We anticipated that DLP may be such an additional factor, and we investigated the molecular interaction between DLP and ASF1 (41).…”

“…Moreover, DLP is implicated in RI deposition of the histone variant H3.3 and, with ASF1, may constitute the central core of a predeposition complex recruited to chromatin gaps by XNP. The existence of such a complex was recently suggested (23).…”

“…XNP and HIRA bind DNA at genomic gaps when nucleosomes are disassembled, marking the sites for RI assembly. It has been hypothesized that a separate predeposition complex containing anti-silencing factor 1 (ASF1) and H3.3/H4 heterodimers is recruited by XNP and HIRA and that the heterodimers are subsequently incorporated into new nucleosomes (23).…”