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:BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen genes, including angiopoietin-1 (ANG1), a secreted angiogenic factor, are corepressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3D culture. BRCA1, CtIP, and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in the ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells that form a capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization, and overexpressed ANG1. These results suggest that, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment.
Project description:BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen of genes including angiopoietin-1 (ANG1), a secreted angiogenic factor, are co-repressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3-D culture. BRCA1, CtIP and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells forming capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization and overexpressed ANG1. These results suggest, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment. Keywords: MCF10A, human mammary epithelial cells, 3-D Matrigel, 15 h, BRCA1-RNAi
Project description:BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen of genes including angiopoietin-1 (ANG1), a secreted angiogenic factor, are co-repressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3-D culture. BRCA1, CtIP and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells forming capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization and overexpressed ANG1. These results suggest, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment. Keywords: MCF10A, human mammary epithelial cells, 3-D Matrigel, 15 h, CtIP-RNAi
Project description:The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions.
Project description:The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions.
Project description:The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions.
Project description:The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions.
Project description:The pathways used by cells to transition from undifferentiated, pluripotent gene expression programs into cell type-specific gene expression programs are incompletely understood. Here we show that the transcription factor Oct1/Pou2f1 recruits histone lysine demethylase complexes to allow for correct induction of silent, developmental lineage-specific genes and “canalize” developmental progression. Using mesodermal differentiation of inducible-conditional Oct1 knockout embryonic stem cells and single-cell gene expression profiling, we show that the potential to progress efficiently through mesodermal development is impaired in the Oct1 deficient condition. Oct1 deficient cells fail to form late presomitic mesoderm and early somite stage populations, and show “leaky” developmental trajectories with inappropriate lineage branching and accumulation of poorly differentiated cells that retain gene expression and metabolic hallmarks of pluripotency. Oct1 directly binds and regulates genes critical for developmental regulation, including genes encoding mesoderm-specific master regulators and components of chromatin regulatory complexes. Cells lacking Oct1 fail to positively resolve gene bivalency and activate gene expression by removing inhibitory H3K27me3 chromatin marks at mesoderm-specific genes. The Oct1 protein interacts with and recruits UTX to lineage-specific bivalent/poised targets, explaining the failure of Oct1 deficient cells to remove H3K27me3. Ectopic Oct1 expression improves the ability of cells to differentiate accurately under mesoderm lineage-inducing conditions.
Project description:BRCA1 exerts transcriptional repression through interaction with CtIP in the C-terminal BRCT domain and ZBRK1 in the central domain. A dozen of genes including angiopoietin-1 (ANG1), a secreted angiogenic factor, are co-repressed by BRCA1 and CtIP based on microarray analysis of mammary epithelial cells in 3-D culture. BRCA1, CtIP and ZBRK1 form a complex that coordinately represses ANG1 expression via a ZBRK1 recognition site in ANG1 promoter. Impairment of this complex upregulates ANG1, which stabilizes endothelial cells forming capillary-like network structure. Consistently, Brca1-deficient mouse mammary tumors exhibit accelerated growth, pronounced vascularization and overexpressed ANG1. These results suggest, besides its role in maintaining genomic stability, BRCA1 directly regulates the expression of angiogenic factors to modulate the tumor microenvironment. Experiment Overall Design: MCF10A cells seeded at 5x105 cells/60mm plate were infected with adenoviral luciferase- or BRCA1-RNAi in duplicate at 20 MOI for 24h. Infected cells were re-seeded at 5x105 cells in a 60mm plate pre-coated with Growth Factor Reduced Matrigel and covered with the growth medium containing 2% Matrigel at 37°C for 15h. RNA was extracted and quality assessed. cDNA synthesized from the harvested RNA was biotin-labeled, hybridized onto Affymetrix HG U133 PLUS 2.0 array (54,676 genes) and stained with streptavidin-phycoerythrin. The hybridized array was analyzed using GeneChip® Scanner 3000 and GCOS 1.2 software (Affymetrix) at the UCI Microarray Core service.