Enzymes catalyzing CpG methylation in DNA, including DNMT1 and DNMT3A/B, are indispensable for mammalian tissue development and homeostasis 1-4. They are also implicated in human developmental disorders and cancers 5-8 , supporting a critical role of DNA methylation during cell fate specification and maintenance. Recent studies suggest that histone posttranslational modifications (PTMs) are involved in specifying patterns of DNMT localization and DNA methylation at promoters and actively transcribed gene bodies 9-11. However, mechanisms governing the establishment and maintenance of intergenic DNA methylation remain poorly understood. Germline mutations in DNMT3A define Tatton-Brown-Rahman syndrome (TBRS), a

Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, S464N). Mammalian Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as establishment of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis and cardiogenesis. Vangl proteins are thought to assemble with other PCP proteins (Dvl, Pk) to form membrane-bound PCP signaling complexes that provide polarity information to the cell. In the present study, we show that Vangl1 is expressed exclusively at the plasma membrane of transfected MDCK cells, where it is targeted to the basolateral membrane. Experiments with an inserted exofacial HA epitope indicate that the segment delimited by the predicted transmembrane domains 1 and 2 is exposed to the extracellular milieu. Comparative studies of the Lp-associated pathogenic mutation D255E indicate that the targeting of the mutant variant at the plasma membrane is greatly reduced; the mutant variant is predominantly retained intracellularly in endoplasmic reticulum (ER) vesicles colocalizing with the ER marker calreticulin. In addition, the D255E variant shows drastically reduced stability with a half-life of approximately 2 h, compared to >9 h for its wild type counterpart and is rapidly degraded in a proteasome-dependent and MG132 sensitive pathway. These findings highlight a critical role for D255 for normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at that site. Our study provide an experimental framework for the analysis of human VANGL mutations recently identified in familial and sporadic cases of spina bifida.

SUMMARY Chromatin dysregulation has emerged as an important mechanism of oncogenesis. To develop targeted treatments, it is important to understand the transcriptomic consequences of mutations in chromatin modifier genes. Recently, mutations in the histone methyltransferase gene nuclear receptor binding SET domain protein 1 (NSD1) have been identified in a subset of common and deadly head and neck squamous cell carcinomas (HNSCCs). Here, we use genome-wide approaches and genome editing to dissect the downstream effects of loss of NSD1 in HNSCC. We demonstrate that NSD1 mutations are responsible for loss of intergenic H3K36me2 domains, followed by loss of DNA methylation and gain of H3K27me3 in the affected genomic regions. In addition, those regions are enriched in cis -regulatory elements, and subsequent loss of H3K27ac correlates with reduced expression of their target genes. Our analysis identifies genes and pathways affected by the loss of NSD1 and paves the way to further understanding the interplay among chromatin modifications in cancer.

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While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChIP-seq of H3K27ac, H3K4me1, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.

In the mouse, the loop-tail mutation (Lp) causes a very severe neural tube defect, which is caused by mutations in the Vangl2 gene. In mammals, Vangl1 and Vangl2 code for integral membrane proteins that assemble into asymmetrically distributed membrane complexes that establish planar cell polarity in epithelial cells and that regulate convergent extension movements during embryogenesis. To date, VANGL are the only genes in which mutations cause neural tube defects in humans. Three independently arising Lp alleles have been described for Vangl2: D255E, S464N, and R259L. Here we report a common mechanism for both the naturally occurring Lp (S464N) and a novel ENU-induced mutation Lp(m2Jus)(R259L). We show that the S464N and R259L variants stably expressed in polarized MDCK kidney cells fail to reach the plasma membrane, their site for biological function. The mutant variants are retained intracellularly in the endoplasmic reticulum, colocalizing with ER chaperone calreticulin. Furthermore, the mutants also show a dramatically reduced half-life of ∼3 h, compared to ∼22 h for the wild-type protein, and are rapidly degraded in a proteasome-dependent and MG132-sensitive fashion. Coexpressing individually the three known allelic Lp variants with the wild-type protein does not influence the localization of the WT at the plasma membrane, suggesting that the codominant nature of the Lp trait in vivo is due to haploid insufficiency caused by a partial loss of function in a gene dosage-dependent pathway, as opposed to a dominant negative phenotype. Our study provides a biochemical framework for the study of recently identified mutations in hVANGL1 and hVANGL2 in sporadic or familial cases of neural tube defects.

The discovery of H3K27M mutations in pediatric gliomas marked a new chapter in cancer epigenomics. Numerous studies have investigated the effect of this mutation on H3K27 trimethylation, but only recently have we started to realize its additional effects on the epigenome. Here, we use isogenic glioma H3K27M +/À cell lines to investigate H3K27 methylation and its interaction with H3K36 and H3K9 modifications. We describe a ''step down'' effect of H3K27M on the distribution of H3K27 methylation: me3 is reduced to me2, me2 is reduced to me1, whereas H3K36me2/3 delineates the boundaries for the spread of H3K27me marks. We also observe a replacement of H3K27me2/3 silencing by H3K9me3. Using a computational simulation, we explain our observations by reduced effectiveness of PRC2 and constraints imposed on the deposition of H3K27me by antagonistic H3K36 modifications. Our work further elucidates the effects of H3K27M in gliomas as well as the general principles of deposition in H3K27 methylation.

Epigenetic modifications on the chromatin do not occur in isolation. Chromatin associated proteins and their modification products form a highly interconnected network, and disturbing one component may rearrange the entire system. We see this increasingly clearly in epigenetically dysregulated cancers. It is important to understand the rules governing epigenetic interactions. Here, we use the mouse embryonic stem cell (mESC) model to describe in detail the relationships within the H3K27-H3K36-DNA methylation subnetwork. In particular, we focus on the major epigenetic reorganization caused by deletion of the histone 3 lysine 36 methyltransferase NSD1, which in mESCs deposits nearly all of the intergenic H3K36me2. Although disturbing the H3K27 and DNA methylation (DNAme) components also affects this network to a certain extent, the removal of H3K36me2 has the most drastic effect on the epigenetic landscape, resulting in full intergenic spread of H3K27me3 and a substantial decrease in DNAme. By profiling DNMT3A and CHH methylation (mCHH), we show that H3K36me2 loss upon Nsd1-KO leads to a massive redistribution of DNMT3A and mCHH away from intergenic regions and towards active gene bodies, suggesting that DNAme reduction is at least in part caused by redistribution of de novo methylation. Additionally, we show that pervasive acetylation of H3K27 is regulated by the interplay of H3K36 and H3K27 methylation. Our analysis highlights the importance of H3K36me2 as a major determinant of the developmental epigenome and provides a framework for further consolidating our knowledge of epigenetic networks.