Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline In Vivo

Michael Sachs,Courtney Onodera,Kathryn Blaschke,Kevin T Ebata,Jun S Song,Miguel Ramalho-Santos

doi:10.1016/j.celrep.2013.04.032

Michael Sachs, Courtney Onodera + Show 4 more

Open Access

https://doi.org/10.1016/j.celrep.2013.04.032

Copy DOI

Journal: Cell Reports	Publication Date: May 30, 2013
Citations: 155	License type: cc-by

Affiliation: University of California, San Francisco

Abstract

Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 "orphan" bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance.

Highlights

Pluripotency is dependent on the maintenance of a proper epigenetic landscape (GasparMaia et al, 2011; Orkin and Hochedlinger, 2011)
In this work we describe an optimized Chromatin Immunoprecipitation (ChIP) protocol suitable for low numbers of cells and its use to examine the genome-wide and gene-specific distribution of H3K4me3 and H3K27me3 in mouse primordial germ cells (PGCs)
We report that developmental regulatory genes remain bivalent and transcriptionally silent in vivo in PGCs, but not in adjacent somatic cells, throughout E11.5-E13.5, in a manner highly similar to cultured embryonic stem cells (ESCs)

Summary

Introduction

Pluripotency is dependent on the maintenance of a proper epigenetic landscape (GasparMaia et al, 2011; Orkin and Hochedlinger, 2011). Bivalent domains, which are defined by the paradoxical coexistence of a permissive histone mark (H3K4me3) and a repressive mark (H3K27me3), are thought to play an important role in pluripotency by keeping developmental genes in a silenced state poised for activation upon differentiation (Azuara et al, 2006; Bernstein et al, 2006). To characterize the chromatin state of PGCs, we developed a low cell number Chromatin Immunoprecipitation (ChIP) for the analysis of histone marks using less than 10,000 cells per IP without the need for carrier chromatin or pre-amplification Using this technique, we performed ChIP-Seq and ChIP-qPCR to show that bivalent domains are present at developmental regulatory genes at multiple stages of PGC development

Results

Discussion

Experimental Procedures