The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 59 donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy.

The asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) acts as a repressive mark that antagonizes trimethylation of H3 lysine 4. Here we report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7 and present in euchromatic regions. Profiling of H3-tail interactors by SILAC MS revealed that H3R2me2s excludes binding of RBBP7, a central component of co-repressor complexes Sin3a, NURD and PRC2. Conversely H3R2me2s enhances binding of WDR5, a common component of the coactivator complexes MLL, SET1A, SET1B, NLS1 and ATAC. The interaction of histone H3 with WDR5 distinguishes H3R2me2s from H3R2me2a, which impedes the recruitment of WDR5 to chromatin. The crystallographic structure of WDR5 and the H3R2me2s peptide elucidates the molecular determinants of this high affinity interaction. Our findings identify H3R2me2s as a previously unknown mark that keeps genes poised in euchromatin for transcriptional activation upon cell-cycle withdrawal and differentiation in human cells.

The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter. MatP displaces wild-type MukBEF complexes from ter, thereby facilitating their association with ori, and limiting the availability of topoisomerase IV (TopoIV) at ter. Displacement of MukBEF is impaired when MukB ATP hydrolysis is compromised and when MatP is absent, leading to a stable association of ter and MukBEF. Impairing the TopoIV-MukBEF interaction delays sister ter segregation in cells lacking MatP. We propose that the interplay between MukBEF and MatP directs chromosome organization in relation to MukBEF clusters and associated topisomerase IV, thereby ensuring timely chromosome unlinking and segregation.

Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor—Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.

Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM) and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors. E cotropic viral integration site 1 (EVI1) is a zinc finger (ZNF) transcription factor (TF), and its overexpression in myeloid leukemia (1-3) and epithelial cancers (1, 2, 4-9) has been extensively studied and correlated with adverse patient outcome (7, 10, 11). EVI1 also controls several aspects of embryonic development, including hematopoiesis, angiogenesis, and heart and neural development (12). Three major alternative splice forms of the MDS1 and EVI1 complex locus (MECOM) locus have been identified, including EVI1, EVI1Δ324, and MDS1-EVI1. The PRDI-BF1 and RIZ homology domain containing Myelodysplastic syndrome (MDS)1-EVI1 isoform acts as a tumor suppressor gene, whereas the shorter isoforms, EVI1 and EVI1Δ324, that lack this domain display oncogenic functions (13). The most oncogenic isoform, EVI1, encodes a 1,051-aa protein containing two DNA binding ZNF domains of seven and three motifs. The N-terminal ZNF domain binds to a GATA-like consensus motif (14), whereas the distal ZNF domain binds to an v-ets erythroblastosis virus E26 oncogene homolog (ETS)-like motif (15). EVI1Δ324 lacks ZNFs motifs 6 and 7, which prevents its binding to GATA-like sites.Despite its discovery in 1988 (16, 17), very few EVI1 target genes have been identified, and most of these loci are known to be bound by EVI1 . This lack of knowledge regarding the genes transcriptionally regulated by EVI1 has precluded a complete understanding of EVI1's role in development and cancer. In general, these biological processes are triggered by gene expression changes coordinate...

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Neuronal excitotoxicity induced by aberrant excitation of glutamatergic receptors contributes to brain damage in stroke. Here we show that tau-deficient (tau−/−) mice are profoundly protected from excitotoxic brain damage and neurological deficits following experimental stroke, using a middle cerebral artery occlusion with reperfusion model. Mechanistically, we show that this protection is due to site-specific inhibition of glutamate-induced and Ras/ERK-mediated toxicity by accumulation of Ras-inhibiting SynGAP1, which resides in a post-synaptic complex with tau. Accordingly, reducing SynGAP1 levels in tau−/− mice abolished the protection from pharmacologically induced excitotoxicity and middle cerebral artery occlusion-induced brain damage. Conversely, over-expression of SynGAP1 prevented excitotoxic ERK activation in wild-type neurons. Our findings suggest that tau mediates excitotoxic Ras/ERK signaling by controlling post-synaptic compartmentalization of SynGAP1.