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

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive sarcoma with recurrent loss-of-function alterations in polycomb-repressive complex 2 (PRC2), a histone-modifying complex involved in transcriptional silencing. To understand the role of PRC2 loss in pathogenesis and identify therapeutic targets, we conducted parallel global epigenomic and proteomic analysis of archival formalin-fixed, paraffinembedded (FFPE) human MPNST with and without PRC2 loss (MPNST LOSS vs. MPNST RET). Loss of PRC2 resulted in increased histone posttranslational modifications (PTM) associated with active transcription, most notably H3K27Ac and H3K36me2, whereas repressive H3K27 di-and trimethylation (H3K27me2/3) marks were globally lost without a compensatory gain in other repressive PTMs. Instead, DNA methylation globally increased in MPNST LOSS. Epigenomic changes were associated with upregulation of proteins in growth pathways and reduction in IFN signaling and antigen presentation, suggesting a role for epigenomic changes in tumor progression and immune evasion, respectively. These changes also resulted in therapeutic vulnerabilities. Knockdown of NSD2, the methyltransferase responsible for H3K36me2, restored MHC expression and induced interferon pathway expression in a manner similar to PRC2 restoration. MPNST LOSS were also highly sensitive to DNA methyltransferase and histone deacetylase (HDAC) inhibitors. Overall, these data suggest that global loss of PRC2mediated repression renders MPNST differentially dependent on DNA methylation to maintain transcriptional integrity and makes them susceptible to therapeutics that promote aberrant transcription initiation. Significance: Global profiling of histone PTMs and protein expression in archival human MPNST illustrates how PRC2 loss promotes oncogenesis but renders tumors vulnerable to pharmacologic modulation of transcription. See related commentary by Natarajan and Venneti, p. 3172

EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and increased levels of an alternatively spliced Mdm2 transcript lacking exon 3. Exon skipping in Mdm2 was also increased in O9–1 mouse neural crest cells after siRNA knock-down of Eftud2 and in MFDM patient cells. Moreover, we found increased nuclear P53, higher expression of P53-target genes, and increased cell death. Finally, overactivation of the P53 pathway in Eftud2 knockdown cells was attenuated by overexpression of non-spliced Mdm2, and craniofacial development was improved when Eftud2-mutant embryos were treated with Pifithrin-α, an inhibitor of P53. Thus, our work indicates that the P53-pathway can be targeted to prevent craniofacial abnormalities and shows a previously unknown role for alternative splicing of Mdm2 in the etiology of MFDM.

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal specific expression and requirement for Eftud2 during craniofacial development. We used RT‐PCR and in situ hybridization to examine expression of Eftud2 during embryogenesis. Using CRISPR/Cas9 we designed guide RNAs to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic day (E) 7.5 and 8.5 of mouse development, Eftud2 was highly expressed in both ectodermal and mesodermal components of the developing head and craniofacial region, by E9.5 Eftud2 was more broadly expressed, in the body wall and developing heart. Eftud2 del heterozygous mice and embryos were viable and fertile and showed a 38% and a 30% reduction of Eftud2 mRNA and protein expression, respectively. Before the onset of organogenesis, Eftud2 del heterozygous embryos had reduced number of somites when compared to their wild type litter mates, indicating a delay in development. Noticeably, RNA sequencing revealed that Eftud2 was the only transcript significantly affected in these heterozygous mice. Eftud2 del homozygous mutant embryos did not implant and failed to grow and hatch ex vivo. Next, we used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Eftud2 flox heterozygous embryos carrying the Wnt‐Cre2 transgene were normal. However, Eftud2 flox homozygous mutant embryos that also carry the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting as early as E9.5. By E11.5, most of these embryos showed an open neural tube, all embryos showed exencephaly at E14.5. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel's cartilage, and abnormal inner ear development. Crosses with the Rosa26R mice reporter line, revealed reduced neural crest cell migration into the pharyngeal region at E10.5. Since the mutation designed is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial during embryogenesis. Future studies are focused on determining the molecular and transcriptional basis of MFDM using this mouse model. Support or Funding Information Canadian Institutes of Health Research This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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