The Histone Methyltransferase KMT2B Is Required for RNA Polymerase II Association and Protection from DNA Methylation at the MagohB CpG Island Promoter

KMT2B (MLL2/WBP7) is a member of the MLL subfamily of H3K4-specific histone lysine methyltransferases (KMT2) and is vital for normal embryonic development in the mouse. To gain insight into the molecular mechanism underlying KMT2B function, we focused on MagohB, which is controlled by a CpG island promoter. We show that in cells lacking Mll2—the gene encoding KMT2B—the MagohB promoter resides in inaccessible chromatin and is methylated. To dissect the molecular events leading to the establishment of silencing, we performed kinetic studies in Mll2-conditional-knockout embryonic stem cells. KMT2B depletion was followed by the loss of the active chromatin marks and progressive loss of RNA polymerase II binding with a concomitant downregulation of MagohB expression. Once the active chromatin marks were lost, the MagohB promoter was rapidly methylated. We demonstrate that in the presence of KMT2B, neither transcription elongation nor RNA polymerase II binding is required to maintain H3K4 trimethylation at the MagohB promoter and protect it from DNA methylation. Reexpression of KMT2B was sufficient to reinstate an active MagohB promoter. Our study provides a paradigm for the idea that KMT2 proteins are crucial components for establishing and maintaining the transcriptionally active and unmethylated state of CpG island promoters

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development

Regulation of CpG island promoters by the histone methyltransferase MLL2

MLL2 is a H3K4 specific HMT which vital for normal embryonic development in the mouse. Little is known on how MLL2 is recruited to its target genes and activates transcription. To gain insight into the molecular mechanism underlying MLL2 function, we focused on a known MLL2 target gene: Magoh2. This gene is controlled by a CpG island promoter and is ubiquitously expressed. Our results demonstrate that in the absence of MLL2, the Magoh2 promoter is methylated and Magoh2 is transcriptionally silenced. The Magoh2 promoter adopts the active conformation only in the presence of MLL2. Pol II is lost from the Magoh2 promoter in the absence of MLL2, resulting in Magoh2 down-regulation. We observed loss of H3K4me$_3$and H3K9ac and relocation of a nucleosome over the promoter, coinciding with the onset of DNA methylation. Use of ORB and a-amanitin demonstrated that neither transcription nor the presence of Pol II are required for the maintenance of H3K4me$_3$. Magoh2 silencing can be overcome by re-introducing full-length MLL2. We investigated the role of MLL2 in haemopoiesis and demonstrated that MLL2 is vital for macrophage differentiation from embryoid bodies. MLL2 may be required for correct upregulation of Flk1 and generation of haem angioblast cells. When M//2 was deleted in haemangioblasts, the haemopoietic transcriptional program was perturbed suggesting that MLL2 may also play a role at this later developmental stage

TET2 regulates immune tolerance in chronically activated mast cells

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Platelet function is modified by common sequence variation in megakaryocyte super enhancers

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.status: publishe

Platelet function is modified by common sequence variation in megakaryocyte super enhancers

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.</p