Project description:To dissect Cohesin-independent mechanisms that compartmentalize the mammalian genome, we combined super-resolution imaging and high-resolution chromatin interactome, chromatin binding and accessibility and RNA sequencing analysis. We found that the bromodomain and extra-terminal domain (BET) family protein BRD2 is one key regulator to promote compartmental interactions in the absence of Cohesin. We also identified competitive, rather than cooperative relationship between BET family proteins in shaping the 3D genome organization. This study uncovers new mechanisitc insights on the 3D organization of the mammalian genome.
Project description:we combined Assay for Transposase-Accessible Chromatin and lattice light-sheet PALM microscopy (3D ATAC-PALM) to selectively image key features of the 3D accessible genome in single cells. We found that accessible chromatin domains (ACDs) form spatially segregated clusters in the nucleus. Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced extensive 3D spatial mixing of ACD clusters and reduced physical separation between ACDs within chromosomes. Experimental perturbations and modeling suggest that both weak, multivalent, dynamic protein-protein interactions together with loop extrusion influence ACD organization. Live-cell studies suggest that ACD clustering regulates transcription factor binding site distribution, target search kinetics and binding dynamics. Here we report the ATAC-seq results from Tn5 PA549 and nextera Tn5 upon various chemical and genetic perturbations.
Project description:We analyzed a role of Brd2 protein in transcription and alternative splicing. 289 genes change alternative splicing after Brd2 knockdown and 1459 genes alter gene expression compared to cells treated with negative control siRNA. 6 samples (3 independent samples of cells treated with Brd2 siRNA, 3 samples of cells treated with negative control siRNA)
Project description:We analyzed a role of Brd2 protein in transcription and alternative splicing. 289 genes change alternative splicing after Brd2 knockdown and 1459 genes alter gene expression compared to cells treated with negative control siRNA.
Project description:The dual bromodomain protein Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. In transgenic mice, constitutive lymphoid expression of Brd2 causes a malignancy most similar to human diffuse large B cell lymphoma. We compare the genome-wide transcriptional expression profiles of these lymphomas with those of proliferating and resting normal B cells. Transgenic tumors reproducibly show differential expression of a large number of genes important for cell cycle control and lymphocyte biology; expression patterns are either tumor-specific or proliferation-specific. Several of their human orthologs have been implicated in human lymphomagenesis. Others correlate with human disease survival time. BRD2 is underexpressed in some subtypes of human lymphoma and these subtypes display a number of similarities to the BRD2-mediated murine tumors. We illustrate with a high degree of detail that cancer is more than rampant cellular proliferation, but involves the additional transcriptional mobilization of many genes, some of them poorly characterized, which show a tumor-specific pattern of gene expression. Experiment Overall Design: 26 samples are included in this series. Sporadic murine E-mu-BRD2 mediated lymphomas are divided into three classes by disease severity and compared to either resting or mitogen-activated B Cells. The resting and activated B Cells are either wildtype or E-mu-BRD2 transgenic. All samples are on the FVB wildtype background.
Project description:Spatial genome organization is essential to direct fundamental DNA-templated biological processes (e.g. transcription, replication, and repair), but the 3D in situ nanometer-scale structure of accessible cis-regulatory DNA elements within the crowded nuclear environment remains elusive. Here, we combined the recently developed Assay for Transposase-Accessible Chromatin with visualization (ATAC-see), PALM super-resolution imaging and lattice light-sheet microscope (a method termed 3D ATAC-PALM) to selectively image and quantitatively analyze key features of the 3D accessible genome in single cells. 3D ATAC-PALM reveals that accessible chromatin are non-homogeneously organized into spatially segregated clusters or accessible chromatin domains (ACDs). To directly link imaging and genomic data, we optimized multiplexed imaging of 3D ATAC-PALM with Oligopaint DNA-FISH, RNA-FISH and protein fluorescence. We found that ACDs colocalize with active chromatin and enclose transcribed genes. By applying these methods to analyze genetically purterbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight the 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.
Project description:Role of the bromodomain and extraterminal motif (BET) protein BRD2 in CTCF chromatin occupancy, tested by CRISPR/Cas9-mediated depletion of BRD2 in GATA1-null erythroblasts expressing an inducible GATA1-ER fusion (G1E-ER4). Pharmacologic inhibitors of the BET (bromodomain and extraterminal motif) family of proteins are being explored for the treatment of various diseases, including cancer, yet the individual functions of BET proteins remain unclear. Here we find that BRD2 co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites, whereas BRD2 is dispensable for CTCF occupancy. Genome editing at a CTCF/BRD2 co-occupied site reveals a functional boundary element that upon perturbation results in transcriptional misregulation. Single-molecule RNA FISH reveals that either site-specific CTCF loss or BRD2 depletion increases the correlation in expression of two genes flanking the boundary. Together these findings indicate that BRD2 supports chromatin boundary activity in a CTCF-dependent manner and suggest that pharmacologic BET inhibitors influence gene expression in part by perturbing chromatin domain boundary function.