ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomeredysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.

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 ...

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