Project description:We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma. 10 DLBCL cell lines (7 mutant and 3 wild type EZH2), that were differentially sensitive to GSK126 in proliferation assays, were treated for 72 hours, in duplicate (n=2), with either DMSO (vehicle) or 500nM of GSK126, a potent selective EZH2 inhibitor. EZH2 mutant cell lines are Pfeiffer, KARPAS-422, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB and SU-DHL-4. EZH2 wildtype cell lines are HT, OCI-LY-19 and Toledo.
Project description:Analysis of the RNA-seq data performed in IR vs NIR hematopoietic stem cells show the loss of the TNF_via_NFKB signature. We showed that the loss of this signature could be associated with H3K9me3 loss at specific retrotransposable elements . To validate this association, we tested if TNFa treatment before irradiation was able to prevent IR-effect on H3K9me3 loss at retrotransposable elements. For this purpose, we treated mice with TNFa 1h before irradiation (IR_TNF) and performed H3K9me3 cut&tag experiments on hematopoietic stem cells 1 month after irradiation and compared them to hematopoietic stem cells sorted from non irradiated mice (NIR) and from non-treated irradiated mice (IR).
Project description:Using UNC0638 and genetic assays to inhibit EHMT1/2 and derepress fetal hemoglobin in adult hematopoietic cells. ChIP-seq for 2 histone modifications and total H3 (for quantitative normalization) in biological triplicates of cord blood, adult bone marrow, and UNC0638-treated adult bone marrow.
Project description:Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modelled this leukemic evolution through stepwise gene editing of GATA1s, SMC3+/- and MPLW515K providing 20 different trisomy or disomy 21 iPSC clones. Cut&Tag against GATA1 was performed on CD41+CD42+ megakaryocytes obtained after 18 days of differentiation of the IPSC clones. 500,000 CD41+CD42+ MK sorted cells were used to analyze GATA1 and GATA1s chromatin occupancy using the CUT&Tag-IT Assay Kit (Active Motif) according to the manufacturer recommendations. Briefly, cells were bound to Concanavalin A-Coated Beads and incubated with primary anti-GATA1 antibody in buffer with Protease Inhibitor Cocktail and 5% digitonine overnight at 4°C under rotation. The Guinea Pig anti-rabbit secondary antibody was incubated in Dig-Wash buffer for 1 hour at RT under rotation. After 3 washes, the CUT&Tag-IT™ Assembled pA-Tn5 Transposomes (1:100) were added for 1 hour at RT under rotation and tagmentation was performed during 1 hour at 37°C. DNA was purified and libraries were generated by PCR. The final libraries were purified, pooled together in equal concentrations and subjected to paired-end sequencing (100 cycles: 2x50) in Novaseq-6000 sequencer (Illumina) at Gustave Roussy.
Project description:To define the gene profile altered by EZH2 and H3K27me3 in response to IFNg, we performed several microarrays in primary ovarian cancer cells transfected with shEZH2 or treated with GSK126. We found that 155 and 124 genes were altered by shEZH2 and GSK126 treatment, respectively, and 20 genes were increased or decreased by both shEZH2 and GSK126 treatment. Primary ovarian cancer cells were pretreated with GSK126, or transduced with a shEZH2 lentivirus, and stimulated with IFNg for 24 hours. RNA were isolated by Rneasy kit according to the manufacturer's protocol, and applied for affymetrix microarray at University of Michigan DNA Sequencing Core
Project description:Contrasting H3K9Me3 binding site number and localisation between activated and quiescent hepatic stellate cells to identify regulation of gene repression.
Project description:Sox2 is a master transcriptional regulator of embryonic development. Having found that Sox2 interacts with RNA-binding proteins, we designed an experiment to discover RNAs associated with Sox2 and other pluripotency factors. Briefly, we used RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to sequence transcriptomes enriched for RNAs associated with Klf4, Nanog, Oct4, Sox2 and Suz12. For completeness, this submission includes all RIP-seq data from this study, although some of the data was only used for exploratory analyses. Such analysis indicated that it was important to include input samples for each of the the cell lines that were to be compared, to account for differences in gene expression (e.g. between J1-birA with and without bioSox2), and that overnight IP was more informative than 3 h IP. Therefore, the analysis described in the associated manuscript used samples from experiment batch 3 and 4 only (samples 11-18).
Project description:Cervical cancer (CC) remains a major cause of cancer-related mortality, particularly in regions with limited screening access, despite being highly preventable and treatable when detected early. MSX1, a homeobox transcription factor with dual roles as a tumor suppressor and oncogene, has an unclear role in CC pathogenesis. This study reveals that MSX1 acts as a tumor-promoting factor in CC, with de novo expression observed in precancerous lesions but absent in normal cervical epithelium. MSX1 enhances clonogenicity and migrationin cervical cancer cells, driven by epithelial-to-mesenchymal transition (EMT) induction. Mechanistically, MSX1 activates RHO/RAC/CDC42 cytoskeletal signaling pathways, with FOS—a downstream RHO effector—identified as a key mediator of CC aggressiveness. Targeting RHO signaling or FOS reverses MSX1-driven aggressive phenotypes, while proteasomal degradation of MSX1 reduces chemoresistance. These findings highlight MSX1’s critical role in CC progression and suggest its potential as a therapeutic target. The study underscores MSX1’s involvement in key oncogenic pathways, offering new insights for developing targeted therapies in cervical cancer.
Project description:Macrophages play a key role in both innate and adaptive immunity, but our knowledge on the changes in transcription regulation that occurs during their differentiation from monocytes is still limited. In this study, we used a meta-analysis followed by a systems biology approach for the identification of differentially expressed genes between monocytes and macrophages and possible regulators of these changes in transcription. Based on the pattern of gene expression change, transcription regulator analysis predicted a decrease in Enhancer of Zeste homolog 2 (EZH2), a histone 3 lysine 27 methyl transferase, activity after differentiation of monocytes into macrophages. This inhibition was validated by a significant decrease in trimethylated H3K27 during differentiation of both human primary monocytes into macrophages and the THP-1 cell line into macrophage-like cells. Overexpressing EZH2 during differentiation of monocytes and THP-1 cells obstructs cellular adhesion, thus preventing the first step in differentiation. Another facet of macrophage differentiation is the cessation of proliferation, and inhibition of EZH2 by the small molecule inhibitor GSK126 in THP-1 cells indeed impedes proliferation. This study shows an important part for epigenetic changes during monocyte differentiation. It highlights the role of EZH2 activity behind the changes needed in adhesion and proliferation mechanisms for macrophage formation. THP-1s were treated with the EZH2 inhibitor GSK126 for phenotypic and genotypic analysis.