Project description:Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.
Project description:The recognition of modified histones by "reader" proteins constitutes a key mechanism regulating gene expression in the chromatin context. Compared with the great variety of readers for histone methylation, few protein modules that recognize histone acetylation are known. Here, we show that the AF9 YEATS domain binds strongly to histone H3K9 acetylation and, to a lesser extent, H3K27 and H3K18 acetylation. Crystal structural studies revealed that AF9 YEATS adopts an eight-stranded immunoglobin fold and utilizes a serine-lined aromatic "sandwiching" cage for acetyllysine readout, representing a novel recognition mechanism that is distinct from that of known acetyllysine readers. ChIP-seq experiments revealed a strong colocalization of AF9 and H3K9 acetylation genome-wide, which is important for the chromatin recruitment of the H3K79 methyltransferase DOT1L. Together, our studies identified the evolutionarily conserved YEATS domain as a novel acetyllysine-binding module and established a direct link between histone acetylation and DOT1L-mediated H3K79 methylation in transcription control.
Project description:Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.
Project description:Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.
Project description:We discovered a unique expression pattern of two histone methyltransferases Suv39h1 and Suv39h2 during 3T3-L1 adipogenesis, both of which preferentially catalyse the formation of H3K9 dimethylation (H3K9me2) and further H3K9 trimethylation (H3K9me3), a transcriptional repressive mark. The expression of Suv39h1 and Suv39h2 displayed a sharp increase at the early stage of 3T3-L1 differentiation, which peaked after differentiation induction, and then declined towards later stage of differentiation, suggesting a key role for these two histone methyltransferases in adipogenesis. Indeed, inactivating Suv39h1 or Suv39h2 via lentiviral shRNA knockdown inhibited adipogenesis, while overexpressing Suv39h1 promoted adipogenesis. Notably, overexpressing or knocking down Suv39h1 in 3T3-L1 cells was associated with reciprocal changes in the expression of Wnt10a, an anti-adipogenic regulator. Further, Wnt10a knockdown largely prevented the inhibitory effect of Suv39h1 on adipogenesis, indicating Wnt10a as a downstream target mediating Suv39h1's action in adipogenesis. Mechanistically, our comprehensive approaches involving ChIP, co-immunoprecipitation and pyrosequencing analysis demonstrated that Suv39h1 may regulate Wnt10a expression via H3K9 methylation and interaction with DNA methyltransferase 1 (DNMT1) at the Wnt10a promoter, resulting in altered DNA methylation at the promoter. We conclude that Suv39h promotes adipogenesis by epigenetically down-regulating Wnt10a expression via H3K9me3 and DNA methylation at the Wnt10a promoter.Abbreviated title: Suv39h and 3T3-L1 Adipogenesis.
Project description:Previous studies have shown that CCAAT/enhancer-binding protein ? (C/EBP?) plays a very important role during adipocyte terminal differentiation and that AP-2? (activator protein 2?) acts as a repressor to delay the expression of C/EBP?. However, the mechanisms by which AP-2? prevents the expression of C/EBP? are not fully understood. Here, we present evidence that Suv39h1, a histone H3 lysine 9 (H3K9)-specific trimethyltransferase, and G9a, a euchromatic methyltransferase, both interact with AP-2? and enhance AP-2?-mediated transcriptional repression of C/EBP?. Interestingly, we discovered that G9a mediates dimethylation of H3K9, thus providing the substrate, which is methylated by Suv39h1, to H3K9me3 on the C/EBP? promoter. The expression level of AP-2? was consistent with enrichment of H3K9me2 and H3K9me3 on the C/EBP? promoter in 3T3-L1 preadipocytes. Knockdown of Suv39h markedly increased C/EBP? expression and promoted adipogenesis. Conversely, ectopic expression of Suv39h1 delayed C/EBP? expression and impaired the accumulation of triglyceride, while simultaneous knockdown of AP-2? or G9a partially rescued this process. These findings indicate that Suv39h1 enhances AP-2?-mediated transcriptional repression of C/EBP? in an epigenetic manner and further inhibits adipocyte differentiation.