Abstract
Embryonic stem (ES) cells exhibit general characteristics of open chromatin, a state that may be necessary for ES cells to efficiently self-renew while remaining poised for differentiation. Histone H3K4 and H3K9 trimethylation associate as a general rule, with open and silenced chromatin, respectively, for ES cell pluripotency maintenance. However, how histone modifications are regulated to maintain open chromatin in ES cells remains largely unknown. Here, we demonstrate that trithorax protein Ash2l, homologue of the Drosophila Ash2 (absent, small, homeotic-2) protein, is a key regulator of open chromatin in ES cells. Consistent with Ash2l being a core subunit of mixed lineage leukemia methyltransferase complex, RNAi knockdown of Ash2l was sufficient to reduce H3K4 methylation levels and drive ES cells to a silenced chromatin state with high H3K9 trimethylation. Genome-wide ChIP-seq analysis indicated that Ash2l is recruited to target loci through two distinct modes and enriched at a family of genes implicated in open chromatin regulation, including chromatin remodeler Cdh7, transcription factor c-Myc, and H3K9 demethylase Kdm4c. Our results underscore the importance of Ash2l in open chromatin regulation and provide insight into how the open chromatin landscape is maintained in ES cells.
Highlights
The trithorax protein Ash2l is a core component of the mixed lineage leukemia (MLL) complex essential for H3K4 methylation and mouse embryonic development
Ash2l Is Important for Embryonic stem (ES) Cell Pluripotency—The global chromatin structure of ES cells is characterized as relatively open and devoid of heterochromatin [8, 11, 48], with discrete regions of trimethylated H3K27 and H3K4 bivalency associated with essential developmental regulatory genes (21, 23–25, 49 –51)
Given the importance of Ash2l in catalytic methylation of H3K4 by the MLL histone methyltransferase complex and in embryonic development [34, 38], we hypothesized that Ash2l may play an important role in determining the chromatin status of ES cells and in turn maintain ES cell self-renewal
Summary
The trithorax protein Ash2l is a core component of the MLL complex essential for H3K4 methylation and mouse embryonic development. Ash2l Essential for ES Cell Pluripotency and Open Chromatin been proposed as a poised state for developmentally important genes [23,24,25], one that allows rapid activation of such genes during embryonic differentiation. Kdm4c, a histone demethylase that catalyzes the removal of the H3K9me mark, regulates global H3K9me levels and is required for maintained pluripotency in mouse ES cells [27]. It is clear that a multitude of chromatin remodeling proteins participate in the dynamic process of histone mark establishment and regulation, the mechanisms that maintain high H3K4me and low H3K9me in ES cells remain unclear. Nome ChIP-seq analysis revealed multiple potential targets of Ash2l that may participate in open chromatin regulation These findings indicate that Ash2l is required for ES cell pluripotency and the global, active chromatin modifications associated with ES cells. Our work suggests that a regulatory network of specific epigenetic factors interact and sustain the open chromatin state of ES cells
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