Histone H3.3 incorporation provides a unique and functionally essential telomeric chromatin in embryonic stem cells

Abstract

Little is known about the telomere chromatin dynamics of embryonic stem (ES) cell. Here, we demonstrate localization of histone H3.3 at interphase telomeres and enrichment of Ser31-phosphorylated H3.3 at metaphase telomeres in pluripotent mouse ES cells. Upon differentiation, telomeric H3.3S31P signal decreases, accompanied by increased association of heterochromatin repressive marks and decreased micrococcal nuclease sensitivity at the telomeres. H3.3 is recruited to the telomeres at late S/G2 phase, coinciding with telomere replication and processing. RNAi-depletion of H3.3 induces telomere-dysfunction phenotype, providing evidence for a role of H3.3 in the regulation of telomere chromatin integrity in ES cells. The distinctive changes in H3.3 distribution suggests the existence of a unique and functionally essential telomere chromatin in ES cells that undergoes dynamic differentiation-dependent remodeling during the process of differentiation.[Supplemental material is available online at www.genome.org.]Pluripotent embryonic stem (ES) cells possess an unlimited capacity for self-renewal and the potential to differentiate into multiple lineages. Upon cellular differentiation, they undergo dramatic morphological and molecular changes through selective silencing and activation of specific genes. Recent studies have pointed to epigenetic phenomena as having fundamental roles in the differentiation process (Martens et al. 2005;Meshorer et al. 2006). Examples are the differentiation-dependent increase in the silenced chromatin marks, including H3K9me3, H3K20me2, H4K20me3, and H3K27me3, and a decrease in the level of acetylated H3 and H4. The hyperdynamic binding of structural chromatin proteins is also a hallmark of ES cells, but not of cells that are already lineage committed.While epigenetic factors are essential for maintaining pluripotency, the capacity for continual telomere renewal is also critical. In vertebrates, telomeres consist of tandem arrays of TTAGGG repeats that are bound by a specialized multiprotein complex ''shelterin.'' Telomere length is maintained by telomerase, a reverse transcriptase that adds telomere repeats de novo after each cell division. High telomerase activity is a key factor that maintains telomere self-renewal and proliferative capacity in ES cells. Mammalian telomeres contain epigenetic markers such as H3K9me3 and H4K20me3 that are characteristic of silenced chromatin, and DNA hypermethylation at subtelomeric regions (Garcia-Cao et al. 2004;Gonzalo et al. 2006). Loss of these modifications results in defective telomere functions as shown by aberrantly increased telomere length and chromosomal instability, indicating that these repressive markers are essential for the formation of a compacted telomere chromatin and the regulation of telomere length.The nucleosome consists of an octamer of four core histones, H2A, H2B, H3, and H4, wrapped inside 146 bp of dsDNA. Adjacent nucleosomes are connected via linker DNA bound by the linker histone H1. Higher-order organization of this basic ...

Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres

“…In differentiated cells, H3.3 has been found to localize to pericentric chromatin (10,27). Recent work performed in murine embryonic fibroblasts (MEFs) found that H3.3 is deposited at pericentric DNA repeats in an ATRX-Daxx-dependent manner (24) ATRX-Daxx also localizes to PML bodies, and ATRX has been shown to colocalize with heterochromatin on both the inactive-X chromosome and Y-chromosome in mice (28,29).…”

“…The three amino acid variations in the histone fold have been shown to be necessary for H3.3 replication-independent incorporation into chromatin (5,6). Higher eukaryotes utilize separate chaperones and deposition pathways for the different histone H3 variants, and previous work identified two major pathways: replication-coupled deposition of H3.1/H3.2 by the CAF1 complex, and replication-independent deposition of H3.3 by the HIRA complex (6)(7)(8) While originally associated with euchromatic sites of active transcription, H3.3 has recently been found associated with regulatory elements and constitutive heterochromatin at telomeres (9)(10)(11)(12). We previously found that HIRA is required for localization of H3.3 to actively transcribed regions, while ATRX is essential for H3.3 incorporation at telomeres.…”

The double face of the histone variant H3.3

“…Unexpectedly, enrichment of H3.3 was recently also observed in regions of the genome that should be transcriptionally silent. Indeed, H3.3 accumulation is found at telomeres and pericentric heterochromatin in mouse ES cells and mouse embryonic fibroblasts (MEF), respectively ( Figure 3) [24,[48][49][50]. Of note, these accumulations could either reflect more loading or less removal of H3.3 at these loci as compared with other places in the genome.…”

Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells

“…Interestingly, these results are in line with the "active" pattern of PTMs found on H3.3 (Hake et al 2006;Loyola et al 2006) and with the fact that deposition of H3.3 is typically associated with transcriptional activation (Ahmad and Henikoff 2002;Chow et al 2005;Wirbelauer 2005;Mito et al 2005;Jin et al 2009). However, this does not exclude localization of H3.3 in pericentromeric or telomeric regions, as demonstrated in proliferating cells (Wong et al 2008;Goldberg et al 2010;Drane et al 2010;Corpet et al 2014). These regions localize outside of SAHF in senescent cells.…”