Gene expression changes upon Dot1L knockdown and Dot1L inhibitor treatment during reprogramming
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29253 | GEO | 2012/03/01
SECONDARY ACCESSION(S): PRJNA139971
REPOSITORIES: GEO
Similar Datasets
Project description:Through a loss-of-function approach, we identified that inhibition of the histone methyltransferase, Dot1L, accelerated somatic cell reprogramming, significantly increased the yield of induced pluripotent stem (iPS) cell colonies, and substituted for Klf4 and c-Myc in the reprogramming cocktail. To understand the mechanism by which Dot1L inhibition results in these phenotypes, we carried out gene expression profiling using Affymetrix microarrays. GSM723207-GSM723224: Embryonic stem cell-derived fibroblasts (dH1fs) were retrovirally transduced in culture with vectors expressing either a control shRNA or an shRNA targeting Dot1L. These cells were then superinfected with either Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses or Oct4, Sox2 and c-Myc (OSM) retroviruses. Total RNA was harvested 6 days later. There were three biologic replicates for each condition. GSM880675-GSM880682: dH1fs were treated with 10uM Dot1L inhibitor (EPZ004777) and then superinfected with Oct4, Sox2, Klf4 and c-Myc (OSKM) retroviruses. Total RNA was harvested 6 days later. There were two biologic replicates for each condition.
2012-03-01 | E-GEOD-29253 | biostudies-arrayexpress
Project description:Epigenetic regulators have important roles during embryonic development as well as somatic cell reprogramming. We previously showed that inhibition of DOT1L, the histone H3 lysine 79 methyltransferase, increases the efficiency of reprogramming via regulation of lineage specific genes. However, the role of DOT1L-interacting proteins in reprogramming remains unknown. In this study, DOT1L interactors were identified using the BioID method in which a promiscuous BirA ligase (BirA*) was employed to biotinylate DOT1L-proximal proteins. The resulting interaction candidates were investigated for their effects on reprogramming. Candidate genes were knocked-down in human fibroblasts via shRNAs followed by reprogramming. Our results indicated that knock-down of AF10 (MLLT10), significantly increased the iPSC generation efficiency, suggesting that it acts as a barrier to reprogramming similar to DOT1L. This finding was verified by CRISPR/Cas9 mediated knockout of AF10. Overexpression of AF10 reversed the effect of AF10 knockout and decreased reprogramming efficiency. To determine how AF10 silencing changes the gene expression, RNA-sequencing was performed on human fibroblasts undergoing reprogramming. AF10 suppression resulted in downregulation of fibroblast-specific genes and accelerated the activation of pluripotency-related genes. Our analysis also demonstrated that silencing of AF10 results in gene expression changes similar to DOT1L inhibition during reprogramming. Taken together, this study uncovered AF10 as a novel barrier to reprogramming and contributed to our understanding of epigenetic mechanisms that maintain cell identity.
2020-11-09 | GSE161043 | GEO
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
Project description:During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as Nanog occurs after the mesenchymal to epithelial transition (MET). Here we report that both adult stem cells (neural stem cells- NSCs) and differentiated cells (astrocytes) of the neural lineage can activate Nanog in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the Nanog+Ecadherin+ populations expressed stabilization markers and had upregulated several cell cycle genes; and were transgene independent. Inhibition of Dot1L activity, which has previously been shown to increase MET, enhanced both the numbers of Nanog+ and Nanog+E-cadherin+ colonies, suggesting a MET independent pathway for activation of Nanog in NSCs. Expressing Sox2 in MEFs prior to reprogramming does not alter the ratio of Nanog colonies that express E-cadherin obtained. Taken together these results provide a novel pathway for reprogramming taken by cells of the neural lineage. Neural Stem Cells (NSCs) were induced to reprogram by the induction of Oct4, Sox2, c-Myc and Klf4. Reprogramming colonies that expressed either Nanog alone (N+E-) or Nanog and E-cadherin (N+E+) were sorted by flow cytometry and used as input for RNA-sequencing. There are 3 replicates each for the N+E- and N+E+ samples.
2016-02-18 | E-GEOD-76130 | biostudies-arrayexpress
Project description:We present a robust serum-free system for the rapid and efficient reprogramming of mouse somatic cells by Oct4, Sox2 and Klf4. The elimination of fetal bovine serum and oncogene c-Myc allowed reprogramming cells to be detected as early as Day 2 and reached greater than 10% of the population at Day 7 post retroviral transduction. The resulting iPS colonies were isolated with high efficiency to establish pluripotent cell lines. Based on this method, we further developed iPS-SF1 as a dedicated reprogramming medium for chemical screening and mechanistic investigations. Comparasion of iPS cell lines derived from serum-free culture condition, MEF cells cultured in serum and serum-free condition and ES cell lines was shown.
2009-03-18 | GSE15267 | GEO
Project description: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.
2021-04-30 | GSE160580 | GEO
Project description:E-cadherin upregulation is an early event of reprogramming of fibroblasts to induce pluripotent stem cells (iPS). Knocking down of E-cadherin by shRNA impairs iPS generation, though some colonies with great morphorlogical difference to shRNA control colonies remain. To illustrate the molecular and functional difference between shECAD iPS clones and shRNA control iPS clones, three respective iPS clones (shECAD 4,8,9 and Ctrl 2,3,4) were derived and DNA microarrays were run to analyze the transcriptional profile of these clones. OG2 MEFs were infected with Sox2, Klf4, Oct4 and c-Myc (SKOM) plus either Luciferase shRNA (shLUC) or E-cadherin shRNA (shECAD) retrovirus. At Day 6 post infection cells were split onto feeder cells. Several colonies from SKOM+shLuc and SKOM+shECAD were picked out at Day 14 post infection respectively and three cell lines were established, namely Ctrl 2,3,4 for SKOM+shLuc iPS and shECAD 4,8,9 for SKOM+shECAD iPS. All clones were maintained on feeder cells in mESC medium. RNA were extracted from these six cell lines and DNA microarrays were run to analyze the transcriptional profile.
2010-06-08 | E-GEOD-21062 | biostudies-arrayexpress
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
Project description:Since the initial discovery that OCT4, SOX2, KLF4 and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to do so. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlie the induction of pluripotency, We analyzed 3 arrays from human fibroblats; 1 array from 4F iPSC; 1 array from 4F iPSC; 1 array for GATA3SOX2, KLF4 and cMYC iPS; 1 array for GATA3vP16,SOX2VP16, KLF4 and cMYC iPS;1 array for GATA3vP16SOX2VP16, KLF4 and cMYC iPS; 1 array for GATA3vP16SOX2VP16, KLF4 and cMYC iPS; 1 array for hES4;1 array for hES10; 1 arrays for iPS generated with OCT4, ZIC2, ZNF521, ASCL1, HESX1,FOXD5,KLF4 and cMYC; 1 arrays for iPS generated with OCT4, ZIC2, ZNF521, ASCL1, HESX1,FOXD5,KLF4 and cMYC; 1 array for iPS generated with OCT4, SOX1, KLF4 and cMYC;1 array for iPS generated with OCT4, SOX1, KLF4 and cMYC;1 array for iPS generated with OCT4, ZNF521, KLF4 and cMYC: 1 array for iPS generated with OCT4, SOX3, KLF4 and cMYC
2013-12-31 | E-GEOD-48275 | biostudies-arrayexpress
Project description:Many repetitive DNA elements are packaged in heterochromatin, but depend on occasional transcription to maintain long-term silencing. The factors that promote transcription of repeat elements in heterochromatin are largely unknown. Here, we show that DOT1L, a histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), is required for transcription of major satellite repeats to maintain pericentromeric heterochromatin (PCH), and that this function is essential for preimplantation development. DOT1L is a transcriptional activator at single-copy genes but does not have a known role in repeat element transcription. We show that H3K79me3 is specifically enriched at repetitive elements, that loss of DOT1L compromises pericentromeric major satellite transcription, and that this function depends on interaction between DOT1L and the chromatin remodeler SMARCA5. DOT1L inhibition causes chromosome breaks and cell cycle defects, and leads to embryonic lethality. Together, our findings uncover a vital new role for DOT1L in transcriptional activation of heterochromatic repeats.
2023-05-26 | PXD028824 | Pride