The histone H3 lysine 36 methyltransferase HypB facilitates embryonic stem cell differentiation

Histone H3 lysine 36 (H3K36) methylation was identified as a conserved modification from yeast to human. In yeast, biochemical characterization of the SET2 protein and genome wide mapping of H3K36me2 and K36me3 indicate that H3K36 methylation functions in transcription elongation through Set2/Rpd3S pathway. \nA number of H3K36 methyltransferases and demethylases have been identified in different species, which underscores the dynamics of H3K36 methylation. As in yeast, H3K36me3 also peaks at the 3’ end of genes in mammals. The genome wide view of H3K36me1 and H3K36me2 is not clear yet. To date, the functional significance of H3K36 methylation remains largely unknown in mammals. \nIn this thesis, homologs of SET2 in mammals, including Nsd1, Nsd2, Nsd3, and HypB, were studied. Nsd proteins displayed weak methyltransferase activity towards histone H3 in vitro. Their target specificities needs to be further analyzed. In vitro, HypB showed strong activity for histone H3 lysine 36. In vivo, H3K36 trimethylation levels were significantly reduced in HypB knock-down cells, indicating that HypB is a major H3K36 trimethyltransferase. Distribution of H3K36 methylation (mono-, di-, and tri-) were analyzed by immunofluorescence both in human and mouse cells. All three methylation states of showed euchromatic distribution, whereas H3K36 mono- and dimethylation also showed heterochromatic enrichment in terminally differentiated NIH3T3 cells as well. In embryonic stem cells, H3K36 methylation showed an inverse correlation with the expression level of Oct4, a stem cell marker, suggesting a potential role of H3K36 methylation in ES cell differentiation. After induction of differentiation by removing LIF or adding retinoic acid to the culture medium, stem cell genes failed to be repressed and lineage specific genes failed to be activated to the same degree in HypB knockdown cell as observed in mock treated ES cells. The presence of H3K36me3 along Oct4 locus was mapped by CHIP. H3K36me3 was highly enriched in the coding region, and was low upstream of the transcription start site in undifferentiated ES cells. During differentiation, however, H3K36me3 decreased on the coding region and increased slightly on enhancer region of Oct4 in the course of Oct4 repression after differentiation. In all, we propose that H3K36me3 is catalyzed by HypB and has an inverse correlation with Oct4 expression. HypB facilitates ES cell differentiation. The molecular mechanism by which HypB facilitates differentiation requires further investigation.