Chem-CRISPR/dCas9 -a chemically induced platform for epigenome editing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE232132 | GEO | 2023/06/09
REPOSITORIES: GEO
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Project description:Epigenetic aberration is one of the major driving factors in the initiation, promotion, and progression of human cancer and it is also often associated with acquired therapeutic resistance. Several chemical epigenetic modulators have been reported. However, results from animal models and clinical trials have indicated that severe on- and off-target toxicity is one of the leading factors behind failures in epidrug development. CRISPR/dCas9 technology provides a powerful tool for precise epigenetic regulation at the single-gene level. This approach requires ectopic expression of exogenous epigenetic modulatory proteins, which cause side effects and technical challenges for preparation and delivery. Recently, we have shown that Cas9 tagging with the peptide motif (FCPF) was recognized by perfluoro biphenyl derivatives. Here, we introduced the FCPF-tag into dCas9 and report a chemically induced platform for epigenome editing (Chem-CRISPR/dCas9FCPF). By conjugating JQ1, a BRD4 inhibitor, to perfluoro biphenyl (JQ1-FCPF), we demonstrate that JQ1-FCPF covalently binds to CRISPR/dCas9 and specifically inhibits BRD4 in proximity to the promoters/enhancers of c-MYC with the guidance of c-MYC sgRNAs. Thus, we achieved epigenetic modulation of c-MYC with high efficiency and specificity.
2023-06-09 | GSE234491 | GEO
Project description:Chem-CRISPR/dCas9 -a chemically induced platform for epigenome editing
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Project description:Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. Here we report the preferential and high sensitivity of TNBCs to BET bromodomain inhibitors such as JQ1 manifested by cell cycle arrest in early G1, apoptosis, and induction of markers of luminal epithelial differentiation in vitro and in vivo. The sensitivity of TNBC and other tumor types to BET inhibition establishes a rationale for clinical investigation, and a motivation to understand mechanisms of resistance. After engendering acquired resistance to BET inhibition in previously sensitive TNBCs, we utilized integrative approaches to identify a unique mechanism of epigenomic resistance to this epigenetic therapy. Resistant cells remain dependent on BRD4, confirmed by RNA interference. However, TNBC cells adapt to BET bromodomain inhibition by re-recruitment of unmutated BRD4 to super-enhancers, now in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify hyper-phosphorylation of BRD4 and strong association with MED1. Together, these studies provide a rationale for BET inhibition in TNBC and argue for combination strategies to anticipate clinical drug resistance. Chem-Seq in parental and JQ1 resistant triple negative breast cancer (TNBC)
2016-01-07 | E-GEOD-63284 | biostudies-arrayexpress
Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition. five samples co-transfected with five uPA sgRNAs and each of the four dCas9 fusions, or control transfection with pUC19 plasmid
2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress
Project description:Epigenetic pathways regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. While chromatin alterations are, in principle, reversible and often amendable to drug intervention, the promise of targeting such pathways therapeutically has been hampered by our limited understanding of cancer-specific epigenetic dependencies. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukemia (AML) – an aggressive hematopoietic malignancy often associated with aberrant chromatin states. By screening a custom shRNA library targeting known chromatin regulators in a genetically defined AML mouse model, we identify the bromodomain-containing protein Brd4 as a critical requirement for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust anti-leukemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation. Extensive evaluation of JQ1-sensitivity in primary human leukemia samples and in established cell lines revealed a broad activity of this compound against diverse AML subtypes. These effects are, at least in part, due to a requirement for Brd4 in maintaining Myc expression and promoting aberrant self-renewal. Together, our results indicate that Brd4 is a promising therapeutic target in AML and identify a small molecule that efficiently targets Myc. These findings also highlight the utility of RNAi screening as a discovery platform for revealing epigenetic vulnerabilities for direct pharmacologic intervention in cancer. In order to understand downstream targets of Brd4, we performed array in murine or human MLL-AF9/NrasG12D cell line under the condition that Brd4 was suppressed by using shRNAs or the small molecule inhibitor JQ1. To test the hypothesis that Myc might be an important target of Brd4, we performed arrary on murine ectopic Myc overexpression MLL-AF9/NrasG12D cell under JQ1 treatment.
2011-08-03 | E-GEOD-29799 | biostudies-arrayexpress
Project description:Proinflammatory stimuli rapidly and globally remodel chromatin landscape, thereby enabling transcriptional responses. Yet, the mechanisms coupling chromatin regulators to the master regulatory inflammatory transcription factor NF-kB remain poorly understood. We report in human endothelial cells (ECs) that activated NF-kB binds to enhancers, provoking a rapid, global redistribution of BRD4 preferentially at super-enhancers, large enhancer domains highly bound by chromatin regulators. Newly established NF-kB super-enhancers drive nearby canonical inflammatory response genes. In both ECs and macrophages BET bromodomain inhibition prevents super-enhancer formation downstream of NF-kB activation, abrogating proinflammatory transcription. In TNFa-activated endothelium this culminates in functional suppression of leukocyte rolling, adhesion and transmigration. Sustained BET bromodomain inhibitor treatment of LDLr -/- animals suppresses atherogenesis, a disease process rooted in pathological vascular inflammation involving endothelium and macrophages. These data establish BET-bromodomains as key effectors of inflammatory response through their role in the dynamic, global reorganization of super-enhancers during NF-kB activation. Chem-Seq for the biotinylated small molecule JQ1 in untreated or TNFalpha treated human endothelial cells
2014-09-29 | E-GEOD-60051 | biostudies-arrayexpress
Project description:Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mTOR inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of BRD4 inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains antiproliferative activity of JQ1 in OS cells. MNNG/HOS cells treated with 7.5mM JQ1, 12.5nM Rapamycin or both were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding control (treated with DMSO).
2015-05-01 | E-GEOD-57203 | biostudies-arrayexpress
Project description:The BET (bromodomain and extra terminal) protein family members including BRD4 bind to acetylated lysines on histones and regulate the expression of important oncogenes, e.g., MYC and BCL2. Here we demonstrate the sensitizing effects of the histone hyperacetylation inducing pan-histone deacetylase inhibitor (HDI) panobinostat (PS) on human AML blast progenitor cells (BPCs) to the BET protein inhibitor JQ1. Treatment with JQ1 but not its inactive enantiomer (R-JQ1) was highly lethal against AML BPCs expressing mutant NPM1c+ with or without co-expression of FLT3-ITD, or AML expressing MLL fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC and BCL2, and reduced their levels in the AML cells. Co-treatment with JQ1 and the HDAC inhibitor panobinostat (PS) synergistically induced apoptosis of the AML BPCs, but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC and BCL2, while increasing p21, BIM and cleaved PARP levels in the AML BPCs. Co-treatment with JQ1 and PS significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (p < 0.01). These findings highlight co-treatment with a BRD4 antagonist and an HDI as a potentially efficacious therapy of AML. Two samples were analyzed (untreated cells, cells treated with JQ1)
2014-01-01 | E-GEOD-51950 | biostudies-arrayexpress
Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
2020-05-01 | GSE129397 | GEO
Project description:Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1 treated parental counterparts. Furthermore, while epithelial-mesenchymal transition (EMT) is observed upon resistance, TGF- induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4 - including AXL and activators of the PI3K pathway - synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC re-activation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.
2021-04-07 | GSE164813 | GEO