Project description:Kaiso (ZBTB33) is a transcription factor involved in mitotic clonal expansion and tumorigenesis in association with APC loss of heterozygosity. ENCODE data show strong overlap of the Kaiso promoter binding cite (eKBS) and many other transcription factors, including BRCA1. Here we sought to determine whether BRCA1 is a component of the Kaiso enhanceosome that regulates gene transcription. Using proximal ligation assays (PLA), immunoprecipitation followed by mass spectrometry, luciferase assays, and ChIP-seq experiments we evaluated the association between BRCA1 and Kaiso. Kaiso nuclear extract immunoprecipitation experiments revealed that Kaiso associates strongly with genes involved in RNA splicing and processing. When Kaiso was not cross linked to DNA, BRCA1 was not detected among Kaiso binding proteins. However, overexpression of BRCA1 increased Kaiso-mediated gene transcription in luciferase assays in a Kaiso-dependent manner. Comparison of BRCA1 ChIP-seq and Kaiso ChIP-seq data from HCT116 cells revealed both BRCA1 and Kaiso commonly bind to the promoters of 379 genes. The most enriched term associated with these genes where BRCA1 and Kaiso bind their promoters is metabolism of RNA. Disease processes associated with these BRCA1/Kaiso gene promoters indicate that BRCA1 is functionally linked to a Kaiso-directed program of RNAP2-mediated gene transcription and likely associated with colorectal cancer development and maintenance.
Project description:Cortical interneurons display a remarkable diversity in their morphology, physiological properties and connectivity. Elucidating the molecular determinants underlying this heterogeneity is essential for understanding interneuron development and function. We discovered that alternative splicing differentially regulates the integration of somatostatin- and parvalbumin-expressing interneurons into nascent cortical circuits through the cell-type specific tailoring of mRNAs. Specifically, we identified a role for the activity-dependent splicing regulator Rbfox1 in the development of cortical interneuron subtype specific efferent connectivity. Our work demonstrates that Rbfox1 mediates largely non-overlapping alternative splicing programs within two distinct but related classes of interneurons.
Project description:BCLAF1 is a serine-arginine (SR) protein implicated in transcriptional regulation and mRNA splicing. We have recently identified BCLAF1 as part of a novel mRNA splicing complex that is recruited to different genetic promoters by the breast cancer susceptiblity protein, BRCA1 in response to DNA damage. This ChIP-chip study was designed to identify genes/promoters regulated by the BRCA1/BCLAG1 mRNA splicing complex by identifying promoters bound by BCLAF1 in the absense and presense of BRCA1 in control cells and cells treated with etoposide to induce DNA damage. This study includes tripicate BCLAF1 ChIP-chip experiments in untreated and etoposide treated (1uM 16 hours) control cells (siGFP) and cells depleted of BRCA1 (siBRCA1). Chromatin Immunoprecipitaitons were performed in triplicate with BCLAF1 antibodies in control 293T cells transfected with siGFP siRNAs and BRCA1 siRNAs (siBRCA1 to deplete BRCA1). Immunoprecipitated genomic DNA was labelled with Cy3 and Input genomic DNA was labelled with Cy5 and hybridized to NimbleGen human 3x720k RefSeq promoter arrays to identify BCLAF1 boundgenomic DNA regions.
Project description:BCLAF1 is a serine-arginine (SR) protein implicated in transcriptional regulation and mRNA splicing. We have recently identified BCLAF1 as part of a novel mRNA splicing complex that is recruited to different genetic promoters by the breast cancer susceptiblity protein, BRCA1 in response to DNA damage. This ChIP-chip study was designed to identify genes/promoters regulated by the BRCA1/BCLAG1 mRNA splicing complex by identifying promoters bound by BCLAF1 in the absense and presense of BRCA1 in control cells and cells treated with etoposide to induce DNA damage. This study includes tripicate BCLAF1 ChIP-chip experiments in untreated and etoposide treated (1uM 16 hours) control cells (siGFP) and cells depleted of BRCA1 (siBRCA1).
Project description:The tumor suppressor BRCA1 regulates DNA damage responses and multiple other processes. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal RING domain. Here we show that BRCA1 promotes oxidative metabolism via degradation of Oct1, a transcription factor with pro-glycolytic/tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells towards a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNAseq confirms the deregulation of metabolic genes. BRCA1 mediates direct Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenografts. Oct1 protein levels correlate positively with tumor aggressiveness, and inversely with BRCA1, in primary breast cancer samples. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism.
Project description:We describe the use of saturation genome editing to make and measure the effect of BRCA1 variants on protein function and splicing. We find the results accurately predict the clinical effects of variants.
Project description:Multiple RNA processing events including transcription, mRNA splicing and export are delicately coordinated by the TREX complex. As one of the essential subunits, DDX39B couples the splicing and export machineries by recruiting ALYREF onto mRNA. In this study, we further explore the functions of DDX39B in handling damaged DNA, and unexpectedly find that DDX39B facilitates DNA repair by homologous recombination through upregulating BRCA1. Specifically, DDX39B binds to and stabilizes BRCA1 mRNA. DDX39B ensures ssDNA formation and RAD51 accumulation at DSB sites by maintaining BRCA1 levels. Without DDX39B being present, ovarian cancer cells exhibit hypersensitivity to DNA-damaging chemotherapeutic agents like platinum or PARPi. Moreover, DDX39B-deficient mice show embryonic lethality or developmental retardation, highly reminiscent of those lacking BRCA1. High DDX39B expression is correlated with worse survival in ovarian cancer patients. Thus, DDX39B suppression represents a rational approach for enhancing the efficacy of chemotherapy in BRCA1-proficient ovarian cancers.