Project description:ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4, and Snf2h. The localization patterns of all three proteins significantly overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome-wide, and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Most regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization, and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. We examine the genome-wide location and activity of three remodeler proteins (Brg1, CHD4 and Snf2h)
Project description:ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4, and Snf2h. The localization patterns of all three proteins significantly overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome-wide, and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Most regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization, and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. We examine the genome-wide location and activity of three remodeler proteins (Brg1, CHD4 and Snf2h)
Project description:The ATP dependent chromatin remodeler SMARCAD1 and the co-repressor KAP1 (Trim28/Tif1 beta) interact directly. We wanted to understand the interplay between these two proteins in the context of chromatin; in particular at repetitive sequences. Therefore, we carried out an investigation in E14 ES cells of the genome wide binding behaviour of KAP1 and FLAG-tagged SMARCAD1; both wild-type SMARCAD1 and a catalytically inactive mutant. The E14 cells analysed were either wild-type or depleted for endogenous SMARCAD1 protein. FLAG and KAP1 ChIP was performed on double-cross linked chromatin and sequenced on an Illumina HiSeq1500 with matched input controls. A FLAG antibody precipitation was carried out on cells not expressing FLAG tagged protein as an additional control.
Project description:Expression data from 3134 mouse mammary epithelial cell line -/+ Tet-inducible chromatin remodeler mutant variants ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. We also examined effects of mutant remodeler variants on selective gene expression by global analysis of RNA expression patterns and found more than 800 genes are deregulated by these variants. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes. Flag tagged dominant negative variants of the chromatin remodelers Brg1, Chd4, and Snf2h were each stably integrated into a cell line containg the tetracycline transactivator regulatory system (3134Tet (7110)). The 3134Tet control cells and each of the dominant-negative remodeler cell lines were grown in media with or without tetracycline for 48 hrs to induce expression of the tetracycline transactivator alone (3134Tet cells) or tetracycline transactivator and dominant negative remodeler variant. Following treatment, cells were harvested for RNA extraction and hybridization to Affymetrix Mouse Gene 1.0 ST arrays.
Project description:In order to understand the role of CYP1B1 and of its common polymorphism V432L, we characterized head-and-neck squamous cell carcinoma (HNSCC) cell lines that do not express this cytochrome, and we generated isogenic derivatives of these lines expressing similar levels of the two variant forms of CYP1B1. Our study reveals that the variant (L432) CYP1B1 form is a strong enhancer of cell proliferation, both in vitro and in vivo, and of migration and invasion capacity in vitro. It is also associated to resistance to DNA-damaging agents in vitro and in vivo. Transcriptome analysis by RNA-seq as well as protein blots showed that the cells expressing the variant CYP1B1 genotype presented an accentuated epithelial character as compared to those expressing the wild-type form, in agreement with in silico data extracted from cell line collections. The variant CYP1B1 cell cultures are enriched in ALDH-positive cells as compared to wild-type CYP1B1 cell cultures, which could be related the phenotypic differences detected. Finally, in order to identify associations between germline CYP1B1 polymorphism and patients’ survival), a clinical study was done, including in advanced HNSCC patients treated with chemotherapy and cetuximab. This prospective study revealed that the variant CYP1B1 genotype was associated with poor prognosis and was confirmed in a retrospective validation study.
Project description:High-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy in women.The lack of effective second line therapeutics remains a substantial challenge for BRCA-1/2 wildtype HGSOC patients, and contributes to poor survival rates due to drug resistance. There is a striking need to elucidate and implement new and alternative treatment options for patients with HGSOC. Histone Deacetylases (HDACs) are promising targets in HGSOC treat-ment, however, the mechanism and efficacy of HDAC inhibitors is understudied in HGSOC. In order to consider HDACs as a treatment target, we need to better understand how they are functioning within HGSOC. This includes elucidating HDAC6 protein-protein interactions. In this study, we carried out substrate trapping to elucidate HDAC6-specific interactors in the context of BRCA-1/2 wildtype HGSOC
Project description:Analysis of patient-specific nucleotide variants is a cornerstone of personalised medicine. Although only 2% of the genomic sequence is protein-coding, mutations occurring in these regions have the potential to influence protein structure and may have severe impact on disease aetiology. Of special importance are variants that affect modifiable amino acid residues, as protein modifications involved in signal transduction networks cannot be analysed by genomics. Proteogenomics enables analysis of proteomes in context of patient- or tissue-specific non-synonymous nucleotide variants. Here, we developed a proteogenomics workflow and applied it to study resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in malignant melanoma cell line A375. This approach resulted in high identification and quantification of non-synonymous nucleotide variants and (phospho)proteins. We integrated multi-omic datasets to reconstruct the perturbed signalling networks associated with BRAFi resistance and to predict drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that aurora kinase A (AURKA) inhibition is effective and specific against BRAFi resistant A375 cells. Furthermore, we investigated nucleotide variants that interfere with protein post-translational modification (PTM) status and potentially influence cell signalling. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins; among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.
Project description:The purpose of the study was to identify downstream gene targets regulated by a new alternative splice of BAFF (B-Cell Activating factor belonging to the TNF Family) that we called Delta4-BAFF (because it characterized by an alternative splice of exon 4). To do this we used human burkitt's lymphoma cell line (RAMOS) stably transfected with Delta4-BAFF or stably transfected with Delta4-BAFF mutated on its N-glycosylation site (Delta4-BAFF-N124D). One control was used : RAMOS stably transfected with empty vector (pIRES2-GFP from Clontech)
Project description:A new site of methylation was identified on histone H3 K42 in Saccharomyces cerevisiae. Mutations were engineered at this site to mimic either a constitutively modified state, K42L, or a constitutively unmodified state, K42Q in addition to an alanine substitution. K42A. The effects of these mutations on global transcription was monitored in yeast cells whose sole source of histone H3 was from a plasmid expressing these mutant proteins, and compared to that of an isogenic strain expressing the wild-type histone H3 protein from the same plasmid. Two biological replicates are included for each yeast strains expressing a specific histone H3 mutant. Three reference strains were also analyzed in the same way which expressed the wild-type histone protein. All strains were handled in the same way. A fresh culture of each strain was inoculated from a saturated overnight culture and grown to an OD A600 of approximately 0.7. Cells were harvested and total RNA extracted.
Project description:FUS is one of the pathogenic RNA-binding proteins for amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilized SynGAP mRNA at its 3’UTR and maintained spine maturation and cognitive function in mice. To elucidate whether this mechanism could be pathogenic for ALS, we identified SynGAP 3’UTR variant at the binding site of FUS, different from that in mice, from a multicenter cohort in Japan. Human induced pluripotent stem cells (hiPSC)-derived motor neurons with SynGAP variant showed spine abnormality with aberrant SynGAP splicing. To evaluate how SynGAP variant altered the access of RNA binding proteins to SynGAP 3'UTR, we performed pull down assay by using biotinylated RNA probes with or without the variant.