Highlights d A modified CUT&RUN method allows chromatin profiling from small numbers of cells d Transcription factors are profiled from single cells and blastocyst-stage embryos

SUMMARY During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI is dependent on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and -independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.DOI: http://dx.doi.org/10.7554/eLife.21964.001

SummaryThe Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin regulatory complex that functions as a transcriptional co-repressor in metazoans. The NuRD subunit MBD3 is essential for targeting and assembly of a functional NuRD complex as well as embryonic stem cell (ESC) pluripotency. Three MBD3 isoforms (MBD3A, MBD3B, and MBD3C) are expressed in mouse. Here, we find that the MBD3C isoform contains a unique 50-amino-acid N-terminal region that is necessary for MBD3C to specifically interact with the histone H3 binding protein WDR5. Domain analyses of WDR5 reveal that the H3 binding pocket is required for interaction with MBD3C. We find that while Mbd3c knockout ESCs differentiate normally, MBD3C is redundant with the MBD3A and MBD3B isoforms in regulation of gene expression, with the unique MBD3C N terminus required for this redundancy. Together, our data characterize a unique NuRD complex variant that functions specifically in ESCs.

BackgroundDifferential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.ResultsHere we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.ConclusionsUsing an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1104) contains supplementary material, which is available to authorized users.