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DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia.

Chen CW, Koche RP, Sinha AU, Deshpande AJ, Zhu N, Eng R, Doench JG, Xu H, Chu SH, Qi J, Wang X, Delaney C, Bernt KM, Root DE, Hahn WC, Bradner JE, Armstrong SA - Nat. Med. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

ABSTRACT
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.

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Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. (a–c) Heatmaps showing ChIP-seq signal of (a) H3K79me2 and Sirt1, and (b,c) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with (a,b) sh-LUC or (c) sh-Sirt1. Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). (d,e) Boxplots showing changes in ChIP-seq signal of (d) Sirt1 and (e) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
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Figure 3: Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. (a–c) Heatmaps showing ChIP-seq signal of (a) H3K79me2 and Sirt1, and (b,c) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with (a,b) sh-LUC or (c) sh-Sirt1. Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). (d,e) Boxplots showing changes in ChIP-seq signal of (d) Sirt1 and (e) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; *P < 0.001 to MLL-AF9 targets using Welch’s t-test.

Mentions: To investigate the mechanisms by which Sirt1 participates in silencing gene expression after Dot1L inhibition, we performed chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) for Sirt1 and H3K79me2 in mouse MLL-AF9 leukemia cells. We observed that EPZ4777 treatment increased the Sirt1 protein occupancy around the transcriptional start site, or TSS, of about one third of annotated genes in the mouse genome (Fig 3a and Supplementary Figs. 4–6). Interestingly, genes that were occupied by the Sirt1 protein after Dot1L inhibition highly correlated with the initial levels of H3K79me2 (positively correlated with actively expressed genes) around their promoter proximal regions. Because Sirt1 is able to deacetylate multiple lysine positions in histones, we further performed ChIP-seq for H3K9ac, a chromatin substrate of Sirt1 that is highly associated with the TSS of actively transcribed genes, in mouse MLL-AF9 leukemia cells. We found that the distribution of H3K9ac at the active TSS overlapped with the loci where the recruitment of Sirt1 was observed after suppression of Dot1L (Fig. 3b). Concomitant with the increased Sirt1 occupancy on the active TSS, we observed reduced H3K9ac on these loci after DOT1L inhibitor treatment (Fig. 3b). Importantly, while depletion of Sirt1 minimally affected the level and distribution of H3K9ac under normal conditions, it completely blocked the loss of H3K9ac from the active TSS induced by DOT1L inhibitor treatment (Fig. 3c). These results suggest that a function of Dot1L activity is to antagonize Sirt1-dependent deacetylation of H3K9 on the actively expressed genes.


DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia.

Chen CW, Koche RP, Sinha AU, Deshpande AJ, Zhu N, Eng R, Doench JG, Xu H, Chu SH, Qi J, Wang X, Delaney C, Bernt KM, Root DE, Hahn WC, Bradner JE, Armstrong SA - Nat. Med. (2015)

Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. (a–c) Heatmaps showing ChIP-seq signal of (a) H3K79me2 and Sirt1, and (b,c) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with (a,b) sh-LUC or (c) sh-Sirt1. Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). (d,e) Boxplots showing changes in ChIP-seq signal of (d) Sirt1 and (e) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
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Related In: Results  -  Collection

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Figure 3: Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. (a–c) Heatmaps showing ChIP-seq signal of (a) H3K79me2 and Sirt1, and (b,c) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with (a,b) sh-LUC or (c) sh-Sirt1. Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). (d,e) Boxplots showing changes in ChIP-seq signal of (d) Sirt1 and (e) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; *P < 0.001 to MLL-AF9 targets using Welch’s t-test.
Mentions: To investigate the mechanisms by which Sirt1 participates in silencing gene expression after Dot1L inhibition, we performed chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) for Sirt1 and H3K79me2 in mouse MLL-AF9 leukemia cells. We observed that EPZ4777 treatment increased the Sirt1 protein occupancy around the transcriptional start site, or TSS, of about one third of annotated genes in the mouse genome (Fig 3a and Supplementary Figs. 4–6). Interestingly, genes that were occupied by the Sirt1 protein after Dot1L inhibition highly correlated with the initial levels of H3K79me2 (positively correlated with actively expressed genes) around their promoter proximal regions. Because Sirt1 is able to deacetylate multiple lysine positions in histones, we further performed ChIP-seq for H3K9ac, a chromatin substrate of Sirt1 that is highly associated with the TSS of actively transcribed genes, in mouse MLL-AF9 leukemia cells. We found that the distribution of H3K9ac at the active TSS overlapped with the loci where the recruitment of Sirt1 was observed after suppression of Dot1L (Fig. 3b). Concomitant with the increased Sirt1 occupancy on the active TSS, we observed reduced H3K9ac on these loci after DOT1L inhibitor treatment (Fig. 3b). Importantly, while depletion of Sirt1 minimally affected the level and distribution of H3K9ac under normal conditions, it completely blocked the loss of H3K9ac from the active TSS induced by DOT1L inhibitor treatment (Fig. 3c). These results suggest that a function of Dot1L activity is to antagonize Sirt1-dependent deacetylation of H3K9 on the actively expressed genes.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus