Genome-wide characterizaion of H3K27me3 levels upon inhibition of JMJD3 histone demethylase using a small molecule inhibitor in T cell leukamia
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Panagiotis Ntziachristos
PROVIDER: E-GEOD-56696 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we abrogated the expression of JMJD3 (KDM6B) and UTX (KDM6A) H3K27me3 demethylases in human T-ALL lines and assayed for genome-wide expression changes using RNA sequencing. This piece of data was further integrated to ChIP-Sequencing analysis of H3K27me3 from the same treatment as well as H3K27me3 and JMJD3 genome-wide analysis from treatment of T-ALL lines with the GSKJ4 inhibitor. These results, coupled to genomic analysis of primary samples for the genomic status of the UTX gene in T-ALL, helped us to identify a hitherto unknown role of JMJD3as an oncogenice facilitator in leukemia whereas UTX seems to play a tumor suppressor role. Whole RNA was extracted from 1-5 million T-ALL (lines) cells or primary cells using the RNAeasy kit (Qiagen) according to the manufacturer’s protocol. Poly-A+ (magnetic oligodT-containing beads (Invitrogen)) or Ribominus RNA was used for library preparation. cDNA preparation and strand-specific library construction was performed using the dUTP method. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method. Differential gene expression analysis was performed for each matched knockdown vs control pairs, separately in each biological or technical replicate in each of two cell lines (CUTLL1, CEM). Three types of comparisons were tested: (a) JMJD3 knockdown vs Renilla, (b) JMJD3 knockdown vs UTX knockdown, and (c) UTX knockdown vs Renilla. Analysis was performed using both DEGseq and Cufflinks packages leading to very similar conclusions.
2014-10-01 | E-GEOD-56689 | biostudies-arrayexpress
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we abrogated the expression of JMJD3 (KDM6B) and UTX (KDM6A) H3K27me3 demethylases in human T-ALL lines and assayed for genome-wide changes in H3K27me3 levels. This piece of data was further integrated to expression changes using RNA sequencing in the same cells as well as ChIP-Sequencing analysis of H3K27me3 and JMJD3 genome-wide analysis from treatment of T-ALL lines with the GSKJ4 inhibitor. These results, coupled to genomic analysis of primary samples for the genomic status of the UTX gene in T-ALL, helped us to identify a hitherto unknown role of JMJD3 as an oncogenice facilitator in leukemia whereas UTX seems to play a tumor suppressor role. Half to one million cells were treated with micrococcal nuclease (MNASE) to generate mononucleosomal particles and an adaptation of the Upstate ChIP protocol was used.
2014-10-01 | E-GEOD-56692 | biostudies-arrayexpress
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we assayed for genome-wide localization of JMJD3 enrichment. This piece of data was further integrated to expression changes using RNA sequencing as well as ChIP-Sequencing analysis of H3K27me3 upon genomic/genetic knock-down or chemical inhibition of JMJD3 and UTX. These results, coupled to genomic analysis of primary samples for the genomic status of the UTX gene in T-ALL, helped us to identify a hitherto unknown role of JMJD3 as an oncogenice facilitator in leukemia whereas UTX plays a tumor suppressor role. JMJD3 ChIP: 10 million cells were used for the ChIP and precipitated using 10micrograms of antibody (abgent, AP1022a) against human JMJD3.
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Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we conduct expression analysis in NOTCH1-IC-induced tumors in Utx wild-type (Utx+/+ or Utx+/Y) and knockout (Utx-/Y) background. These results, coupled to genomic analysis of primary samples for the genomic status of the UTX gene in T-ALL, helped us to characterize the hitherto understudied role of Utx as an oncogenic facilitator in leukemia and the contrasting expression signatures between JMJD3 and UTX in this disease. Whole RNA was extracted from 1-5 million primary cells from Notch1-IC-expressing (sorted populations of) mouse T-ALL tumors using the RNAeasy kit (Qiagen) according to the manufacturer’s protocol. Poly-A+ (magnetic oligodT-containing beads (Invitrogen)) or Ribominus RNA was used for library preparation. cDNA preparation and strand-specific library construction was performed using the dUTP method. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method. Differential gene expression analysis was performed between knockout vs wild-type background samples. Analysis was performed using DEGseq package leading to very similar conclusions.
2014-10-01 | E-GEOD-56690 | biostudies-arrayexpress
Project description:The JmjC domain containing protein JMJD3/KDM6B catalyses H3K27me3 and H3K27me2 demethylation. JMJD3 appears to be highly regulated at the transcriptional level and is upregulated in response to diverse stimuli such as differentiation inducers and stress signals. Accordingly, JMJD3 has been linked to the regulation of different biological processes such as differentiation of embryonic stem cells, inflammatory responses in macrophages, and induction of cellular senescence via regulation of the INK4A-ARF locus. Here we show here that JMJD3 interacts with the tumour suppressor protein p53. We find that the interaction is dependent on the p53 tetramerization domain. Following DNA damage, JMJD3 is transcriptionally upregulated and by performing genome-wide mapping of JMJD3, we demonstrate that it binds genes involved in basic cellular processes, as well as genes regulating cell cycle, response to stress and apoptosis. Moreover, we find that JMJD3 binding sites show significant overlap with p53 bound promoters and enhancer elements. The binding of JMJD3 to p53 target sites is increased in response to DNA damage, and we demonstrate that the recruitment of JMJD3 to these sites is dependent on p53 expression. Therefore, we propose a model in which JMJD3 is recruited to p53 responsive elements via its interaction with p53 and speculate that JMJD3 could act as a fail-safe mechanism to remove low levels of H3K27me3 and H3K27me2 to allow for efficient acetylation of H3K27. Examination of JMJD3 and p53 genome-wide binding in untreated BJ cells or cells exposed to DNA damage (IR, 10 Gy)
2014-06-11 | E-GEOD-55912 | biostudies-arrayexpress
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2015-02-16 | E-GEOD-57399 | biostudies-arrayexpress
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we chemically inhibited the H3K27me3 demethylase JMJD3 using the GSKJ4 inhibitor and assayed for genome-wide changes in H3K27me3 and JMJD3 enrichment. This piece of data was further integrated to expression changes using RNA sequencing as well as ChIP-Sequencing analysis of H3K27me3 upon genomic knock-down of JMJD3 and UTX. These results, coupled to genomic analysis of primary samples for the genomic status of the UTX gene in T-ALL, helped us to identify a hitherto unknown role of JMJD3 as an oncogenice facilitator in leukemia whereas UTX seems to play a tumor suppressor role. Histone ChIP: Half to one million cells were treated with micrococcal nuclease (MNASE) to generate mononucleosomal particles and an adaptation of the Upstate ChIP protocol was used.
2014-10-01 | E-GEOD-56693 | biostudies-arrayexpress
Project description:FBXW7 modulates stress response by post-translational modification of HSF1 HSF1 orchestrates the heat-shock response upon exposure to heat stress and activates a transcriptional program vital for cancer cells. In this study we assayed for genome-wide localization of HSF1 enrichment in the HCT116 FBXW7 KO colon cells and their wild type counterpart in untreated cells and upon heat shock. These results revealed that accumulation of nuclear HSF1 in FBXW7 KO cells results in rewiring of the HSF1 transcriptional program. Five million cells were used for the ChIP and precipitated using 5 micrograms of antibody (cell signaling, 4356) against human HSF1
2015-02-20 | E-GEOD-57398 | biostudies-arrayexpress
Project description:HSF1 orchestrates a transcriptional program vital for cancer cells. In this study we assayed for genome-wide localization of HSF1 enrichment in the 451Lu melanoma in untreated cells. These results revealed a transcriptional program enriched for metastasis-related genes. Twenty million cells were used for the ChIP and precipitated using 5 micrograms of antibody (cell signaling, 4356) against human HSF1
2015-02-20 | E-GEOD-63589 | biostudies-arrayexpress
Project description:Inflammatory responses triggered by either microbial or endogenous stimuli rely on a complex transcriptional program that involves the differential expression of hundreds of genes. Jmjd3, a JmjC family histone demethylase (HDM), is quickly induced by the transcription factor NF-kB in response to inflammatory stimuli. Jmjd3 erases a histone mark associated with transcriptional repression and silencing, trimethylated lysine 27 in histone H3 (H3K27me3). Thus, Jmjd3-mediated demethylation of H3K27me3 links inflammation to the control of a histone modification involved in lineage determination, differentiation and tissue homeostasis. However, the specific contribution of Jmjd3 induction to innate immunity and inflammation remains unknown. Here we combined genome-wide mapping and gene knockout studies to investigate this issue. Chromatin immunoprecipitation (ChIP) coupled to ultra high-throughput sequencing (ChIP-Seq) in LPS-stimulated primary mouse macrophages demonstrated that Jmid3 is recruited to a large number of genomic targets with a strong preference for active transcription start sites (TSS). Virtually all Jmjd3-bound TSSs were characterized by high levels of H3K4me3, a marker of gene activity, and high levels of RNA polymerase II (Pol_II). Inducible genes showing a strong increase in H3K4me3 and Pol_II recruitment after endotoxin treatment (including those encoding several cytokines, chemokines and antiviral proteins) were in most cases Jmjd3-associated. In Jmjd3-knockout macrophages, initial RNA_Pol II recruitment and activation of Jmjd3 target genes was unaffected, but RNA_Pol II was prematurely released, thus resulting in non-sustained responses. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analyzed here were uncoupled from measurable effects on this histone mark. Our data indicate that Jmjd3 is the effector of an NF-kB-controlled feed-forward transcriptional loop pervasively sustaining inflammatory transcriptional responses in a manner that is independent of H3K27me3 demethylation, and suggest the possible use of anti-Jmjd3 drugs to dampen pathologic inflammation. Keywords: Epigenetics Genome wide maps of histone demethylase jmjd3, the histone marks H3K4me3 and H3K27me3, and RNA-Polymerase II induction in mouse bone marrow-derived macrophages of two types: (a) untreated and (b) stimulated with lipopolysaccharide and interferon gamma to produce an inflammatory response.
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