Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells

In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers

<div><p>YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or <i>YAP1</i> amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.</p></div

YAP1 binding sites largely overlap in cancer cell lines from distinct lineages.

<p>(A) Correlation between SF268 and NCI-H2052 YAP1 ChIP-seq samples. (B) Genomic views of YAP1 shared, SF268-, NCI-H2052 and IMR90-specific regions. (C) Correlation between SF268 and IMR90 YAP1 ChIP-seq samples. (D) H3K27ac ChIP enrichment at YAP1 peak regions (centered on peak summit) that are shared, SF268-specific or IMR90-specific.</p

siRNAs.

<p>siRNAs.</p

Primers used for RT-qPCR.

<p>Primers used for RT-qPCR.</p

Luciferase reporters (Fig 2I).

<p>Luciferase reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g002" target="_blank">Fig 2I</a>).</p

siRNAs (Fig 3A).

<p>siRNAs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g003" target="_blank">Fig 3A</a>).</p

Primers used for ChIP-qPCR.

<p>Primers used for ChIP-qPCR.</p

TEAD single and double motifs occur within most YAP1 binding sites.

<p>(A and B) Enrichment of (A) TEAD and (B) AP-1 motifs in YAP1 peaks. Full list provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.s017" target="_blank">S4 Table</a>. (C) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without TEAD and AP-1 motifs. (D) Number of TEAD motifs in YAP1 peaks. (E) Enrichment of TEAD double motif with several spacer lengths in YAP1 peaks. (F) Sequence conservation of YAP1 peak regions. (G) Sequence conservation of TEAD single and double motifs in YAP1 peak regions. (H) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without single/double TEAD motifs. (I) Luciferase reporter assay for two YAP1 binding regions with either intact double motif or with single or double mutations. Relative luciferase activity represents the ratio of Firefly and Renilla luciferase activity for each sample. The red line indicates the highest mean activity of the two negative control regions. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

Luciferase reporters (Fig 4E).

<p>Luciferase reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g004" target="_blank">Fig 4E</a>).</p