Probe the function of histone lysine 36 methylation using histone H3 lysine 36 to methionine mutant transgene in mammalian cells

Chondroblastoma is a cartilaginous tumor that typically arises under 25 y of age (80%). Recent studies have identified a somatic and heterozygous mutation at the H3F3B gene in over 90% chondroblastoma cases, leading to a lysine 36 to methionine replacement (H3.3K36M). In human cells, H3F3B gene is one of 2 genes that encode identical H3.3 proteins. It is not known how H3.3K36M mutant proteins promote tumorigenesis. We and others have shown that, the levels of H3K36 di- and tri-methylation (H3K36me2/me3) are reduced dramatically in chondroblastomas and chondrocytes bearing the H3.3K36M mutation. Mechanistically, H3.3K36M mutant proteins inhibit enzymatic activity of some, but not all H3K36 methyltransferases. Chondrocytes harboring the same H3F3B mutation exhibited the cancer cell associated phenotypes. Here, we discuss the potential effects of H3.3K36M mutation on epigenomes including H3K36 and H3K27 methylation and cellular phenotypes. We suggest that H3.3K36M mutant proteins alter epigenomes of specific progenitor cells, which in turn lead to cellular transformation and tumorigenesis.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.     

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons

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Parent-specific complementary patterns of histone H3 lysine 9 and H3 lysine 4 methylation at the Prader-Willi syndrome imprinting center

The Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region, on human chromosome 15q11-q13, exemplifies coordinate control of imprinted gene expression over a large chromosomal domain. Establishment of the paternal state of the region requires the PWS imprinting center (PWS-IC); establishment of the maternal state requires the AS-IC. Cytosine methylation of the PWS-IC, which occurs during oogenesis in mice, occurs only after fertilization in humans, so this modification cannot be the gametic imprint for the PWS/AS region in humans. Here, we demonstrate that the PWS-IC shows parent-specific complementary patterns of H3 lysine 9 (Lys9) and H3 lysine 4 (Lys4) methylation. H3 Lys9 is methylated on the maternal copy of the PWS-IC, and H3 Lys4 is methylated on the paternal copy. We suggest that H3 Lys9 methylation is a candidate maternal gametic imprint for this region, and we show how changes in chromatin packaging during the life cycle of mammals provide a means of erasing such an imprint in the male germline.