EZH2 inhibitor efficacy in non-Hodgkin lymphoma does not require suppression of H3K27 mono-methylation [ChIP-Seq]
ABSTRACT: Here we report the discovery of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, their application across a large lymphoma cell panel and their efficacy in GCBDLBCL xenograft models. Baseline ChIP-seq measurement of KARPAS-422 cell line H3K27me3 levels, without treatment. Two samples -- H3K27me3 and Input included as control.
The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of hi ...[more]
Project description:Examine the distribution of KDM1A and the histone H3K4me2 mark on chromatin following treatment with two distinct classes of KDM1A inhibitors - an irreversible inhibitor (RN-1) and a reversible inhibitor (GSK690) 15 samples total from two treated cell lines - 9 from Kasumi-1 and 6 from SKNO-1. Controls included were input DNA isolated from the treated cells.
Project description:Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Phenotypic effects are preceded by the direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4 and the subsequent down-regulation of the IRF4 transcriptional program. Ectopic expression of IRF4 antagonizes the phenotypic effects of CBP/EP300 bromodomain inhibition and prevents the suppression of the IRF4 target c-MYC. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network. A total of 13 ChIP-seq samples were sequenced. Samples were treated with control (DMSO) or test compound (2.5 uM SGC-CBP30 or 0.25uM CPI267203) for 6 hours. Signal from input samples was included to subtract background signal from each ChIP-seq sample. Antibodies used were against p300, H3K18ac, H3K27ac, or BRD4.
Project description:Native-ChIP was performed to determine whether cross-linked ChIP-Seq enrichments observed at DHS exons result from co-precipitation with proximal promoter. Native ChIP-Seq was performed on H3K4me3 and H3K27me3 histone modifications in human cells
Project description:By using ChIP-seq, we found that loss of BRM activity in developing seedlings leads to ectopic and increased H3K27me3 deposition at several hundred genes, indicating the critical role of BRM in preventing the inappropriate deposition of this histone mark. Removal of CLF in brm mutant could partcially suppress the increased H3K27me3. Examination of H3K27me3 in 14-day-old wt, brm, clf, and brm clf seedlings. Two biological replicates for each one.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. RNA-seq was performed in wild type and Suz12(Bgal/Bgal) ESCs, here used to demonstrate the coverage of the Suz12 gene in mRNA reads.
Project description:Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here we find that the non-canonical PRC1.1 complex, as identified by mass spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34+ cells. Besides a set of genes that is co-targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3 suggesting a gene regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls distinct gene clusters involved in unique cell biological processes required for leukemic cell viability. K562 cells were transduced with lentiviral GFP-fusion vectors encoding GFP-CBX2, PCGF1-GPF, PCGF2-GFP, PCGF4-GFP, GFP-RING1A or GFP-RING1B. K562 cells expressing GFP-fusions at relatively low levels were sorted and expanded and subsequently crosslinked. ChIP reactions were performed using the following antibodies: anti-GFP (ab290, Abcam), anti-H3K27me3 (07-449, Millipore), anti-H3K4me3 (ab8580, Abcam), anti-H2AK119ub (D27C4, Cell Signaling Technology), and anti-KDM2B (ab137547, Abcam). Furthermore, ChIP reactions were performed using primary AML CD34+ and Peripheral Blood (PB) CD34+ cells using anti-H3K27me3 (07-449, Millipore), anti-H3K4me3 (ab8580, Abcam), anti-H2AK119ub (D27C4, Cell Signaling Technology), anti-KDM2B (ab137547, Abcam).
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.
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.
Project description:We report a computational approach for investigation of chromatin state plasticity. We applied this approach to investigate an ENCODE ChIP-seq dataset profiling the genome-wide distribution of H3K27me3 in 19 human cell lines. We found that high plasticity regions (HPRs) can be divided into two functionally and mechanistically distinct groups, consisting of CpG island proximal and distal regions. We identified cell-type specific regulators correlating with H3K27me3 patterns at distal HPRs in ENCODE cell lines. Furthermore, we applied this approach to investigate mechanisms for poised enhancer establishment in primary human erythroid precursors. We predicted and validated a previously unrecognized role of TAL1 in modulating H3K27me3 patterns through interaction with additional cofactors, such as GFI1B. Our integrative approach provides mechanistic insights into chromatin state plasticity and is broadly applicable to other epigenetic marks. Analysis of genomic occupancy of H3K27me3, H3K27ac, GATA1, TAL1/SCL and GFI1B in primary adult human proerythroblasts by ChIP-seq.
Project description:Suz12(Bgal/Bgal) ESCs express a truncated form of Suz12 fused to Beta-galactosidase. These cells maintain a reduced level of H3K27me3 despite this mutation to a core component of PRC2, unlike Eed-/- ESCs whose H3K27me3 is ablated. Two ESC lines mutant in genes of core components of Polycomb Repressive Complex 2 were assessed for H3K27me3 by ChIP-seq, as compared to a wild type ESC line.