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Transcriptional profiling of chemical Dot1L inhibition during reprogramming to induced pluripotent stem cells

ABSTRACT: Three independent transcriptional profiling experiments were performed to ascertain how Dot1L affects cell fate change. 1) In a timecoure ("Tc"), reprogramming mouse embryonic fibroblasts were treated with DMSO control or Dot1L inhibitor (Dot1Li -SGC0946) for 2 or 4 days. Surprisingly, despite the enrichment of H3K79me2 on thousands of actively transcribed genes, Dot1L inhibition (Dot1Li) results in few changes in steady state mRNA levels during reprogramming, the majority of which are spuriously upregulated. 2) To determine if Dot1Li increased reprogramming beyond the mesenchymal to epithelial transition (MET) that occurs early in reprogramming when starting from MEFs, reprogramming cells were sorted ("Sort") for surface CDH1 expression, and then treated with Dot1Li or control. Dot1Li increased reprogramming efficiency of both CDH1- and CDH1+ populations to a similar extent and few genes were DE. 3) To assess if Dot1Li increased pluripotency acquisition beyond CDH1 upregulation, CDH1 or empty vector was expressed ("Exp") in reprogramming cells. CDH1 expression did not initiate MET, or enhance reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE160580 | GEO | 2021/04/30

REPOSITORIES: GEO

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The role of AF10 (Mllt10) in maintaining cell identity (RNA-Seq)

Project description:AF10 is a cofactor of the H3K79 methyltransferase DOT1L. To uncover the role of H3K79me in reprogramming to induced pluripotent stem cells (iPSCs), we bred reprogrammable mice encoding doxycycline inducible Oct4-2A-Klf4-2A-IRES-Sox2-2A-c-Myc (OKSM) transgene with conditional flox (fl)-AF10 (Mllt10). We isolated mouse embryonic fibroblasts (MEFs), deleted AF10 with CRE-recombinase or treated cells with empty vector control, and initiated reprogramming in DOT1L chemical inhibitor SGC0946 or DMSO control. Gene expression was assessed using RNA-Seq and genome wide localization of H3K79me1 and H3K79me2 was determined by ChIP-Seq on day 4 of reprogramming.

2023-04-01 | GSE214139 | GEO

DOT1L protects neural stem cells from ER stress

Project description:In order to understand the role of H3K79me2 and DOT1L during CNS development, we analysed neural stem cells after pharmacological inhibition of DOT1L (5 µM SGC0946). To identify target genes of DOT1L in the cortex, we determined the transcriptome of cortical progenitor cells derived from E14.5 NMRI mice after interference with DOT1L activity for three days in vitro (DIV3). Therefore, three independent experiments were performed (ctrl1 – inh1, ctrl2 – inh2, ctrl3 – inh3).

2015-12-31 | E-MTAB-3521 | biostudies-arrayexpress

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2023-04-01 | GSE214141 | GEO

Genome-wide maps of Dot1l associated epigenetic modifications during pluripotency acquistion

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ChIPSeq of MV4;11 cell treated with DMSO, I-BET, SGC0946 and combination of I-BET and SGC0946

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2016-06-12 | E-GEOD-71776 | biostudies-arrayexpress

RNASeq of MV4;11 cell treated with DMSO, I-BET, SGC0946 and combination of I-BET and SGC0946

Project description:Central to the molecular pathogenesis of MLL leukaemia is the abnormal co-optation of members of transcription complexes including disrupter of telomeric silencing 1-like (DOT1L) and bromodomain containing protein 4 (BRD4). Consequently, targeted therapies against DOT1L and BRD4 are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukaemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation we find that native BRD4 and DOT1L exist in largely separate protein complexes. Genetic disruption or small molecule inhibition of BRD4 and DOT1L shows marked synergistic activity against MLL-FP leukaemia cell lines, primary human leukaemia cells and murine leukaemia models. Mechanistically, we find a previously unrecognised functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in close proximity to superenhancers. DOT1L via H3K79me2 facilitates the deposition of histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide novel insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this poor prognostic disease. RNASeq of MV4;11 cell treated with DMSO, I-BET, SGC0946 and combination of I-BET and SGC0946 in duplicate

2016-06-12 | E-GEOD-71777 | biostudies-arrayexpress

Analysis of H3K79 methylation during reprogramming

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2012-02-22 | E-GEOD-35791 | biostudies-arrayexpress

ChIPSeq of MV4;11 cell treated with SGC0946 for 8 hours and washout

2016-06-12 | E-GEOD-71779 | biostudies-arrayexpress

Inhibition of DOT1L increases osteoclast fusion and resorption and aggravates bone loss

Project description:Osteoclasts are absorptive cells and play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic regulation of osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation during osteoclast formation. DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. DOT1L inhibition also increased osteoclast area and accelerated bone mass reduction in a mouse ovariectomy (OVX) model of osteoporosis. DOT1L inhibitors did not alter osteoblast differentiation in vitro and in vivo. Proteomics data, together with bioinformatics analysis, revealed that DOT1L inhibition altered reactive oxygen species (ROS) generation, autophagy activation, and cell fusion-related protein expression. ROS generation increased, and autophagy activation and cell migration ability enhancement were verified subsequently by flow cytometry and transwell migration assays. DOT1L inhibition increased NFATc1 nuclear translocation and NF-κB activation and strengthend osteoclast fusion and expression of resorption-related protein CD9, and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-mediated H3K79me2 epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.

2018-01-24 | PXD006448 | Pride

Global gene expression analysis of Dot1l-deficient and control intestinal villus cells in mouse

Project description:Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5+ mouse intestinal stem cells and mature intestinal villus epithelium correlated with mRNA expression levels but not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5+ stem cells or in all intestinal epithelial cells eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. Absence of methylated H3K79 did not impair health, intestinal homeostasis or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo. Examination of differential gene expression between Dot1l control (Dot1 f/f) and Dot1l mutant (Villin-Cre, Dot1l f/f) villus cells.

2012-10-19 | E-GEOD-41710 | biostudies-arrayexpress

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