Similar Datasets
Project description:Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage
| PRJNA549946 | ENA
Project description:Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.
| S-EPMC7737631 | biostudies-literature
Project description:CREB Coactivators CRTC2 and CRTC3 Modulate Bone Marrow Hematopoiesis
| PRJNA407926 | ENA
Project description:Intramyocardial Sprouting Tip Cells Specify Coronary Arterialization
| PRJNA925482 | ENA
Project description:The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8 that interacts with the TATA-binding protein. SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast. In addition, both SAGA and SLIK perform chromatin modifications and the two complexes have been speculated to uniquely contribute to transcription regulation. To test the respective contribution of SAGA vs. SLIK in transcription regulation, we assayed the chromatin modifying functions of SAGA vs. SLIK, revealing identical kinetics on minimal substrates in vitro. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (KD = 10-17 nM) and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of Spt8 subunit in SLIK neither drive any major conformational differences in its structure compared to SAGA, nor significantly affect HAT, DUB or DNA binding activities in vitro.
2021-04-19 | PXD024640 | Pride
Project description:Arsenic exposure is a global health problem. Millions of people encounter arsenic through contaminated drinking water, consumption, and inhalation. The arsenic response locus in budding yeast is responsible for the detoxification of arsenic and its removal from the cell. This locus constitutes a conserved pathway ranging from prokaryotes to higher eukaryotes. The goal of this study was to identify how the arsenic response locus is regulated in an arsenic dependent manner. An affinity enrichment strategy called CRISPR-Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) was used that provides for the proteomic characterization of a given locus. CRISPR-ChAP-MS was applied to the arsenic response locus and uncovered 40 nuclear-annotated proteins showing enrichment. Functional assays, identified the histone acetyltransferase activity of SAGA and the ATPase chromatin remodeling activity of SWI/SNF to be required for activation of the locus. Furthermore, SAGA and SWI/SNF were both found to specifically organize the chromatin structure at the arsenic response locus for activation of gene transcription. This study provides the first proteomic characterization of an arsenic response locus and key insight into the mechanism of transcriptional activation that is necessary for detoxification of arsenic from the cell.
2020-05-07 | PXD009902 | Pride
Project description:We set out to develop an unbiased, genome-wide method to screen for proteins that locate to the OCT4 proximal enhancer (PE) in hESCs; for this purpose, we developed an optimized locus-specific proteomics approach in hESCs. First, we designed TALEN (Transcription Activator-Like Effector Nuclease) constructs to target the sequences that are near the OCT4 PE, located in a region of DNaseI hypersensitivity. TALEN constructs with the highest cutting efficiency were chosen for locus-specific proteomics. We then made modifications to the original TALEN protein to transform it into a catalytically-dead TALE (dTALE) protein that is optimized for locus-specific proteomics in hESCs via three steps: 1) The nuclease-domain FokI at the C-terminus was replaced by a GFP (green fluorescence protein); 2) a 3X FLAG tag at the N-terminus was included for following pull-down analysis; 3) the existing CMV promoter was replaced with an EF1alpha promoter that has robust expression in hESCs. This dTALE protein could then be chemically crosslinked to the OCT4 locus together with all the other proteins that bind to the locus. We verified that dTALE protein binds to the targeted locus by chromatin immunoprecipitation (ChIP)-qPCR. Following crosslinking, chromatin was sheared, and all the associated proteins were immunoprecipitated using an anti-FLAG antibody. Immunoprecipitation pulled down the dTALE protein as well as other proteins and complexes that are also attached to that region. Crosslinking was then reversed, and the samples were subjected to mass spectrometry to enable generation of a list of proteins that potentially bind to OCT4 PE locus.
2019-01-07 | PXD010963 | Pride
Project description:OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage
| PRJNA774795 | ENA
Project description:Transcription factors require coactivators and corepressors to modulate transcription in mammalian cells. The vitamin D receptor (VDR) utilizes coactivators and corepressors to gain tight control over the activity of a diverse set of genes that can regulate calcium transport, slow proliferation and promote immune responses. We have recently established the VDR/RXR cistrome in human colon cancer cells and have linked these binding sites to the genes that are regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In additional studies described herein, we demonstrate that the coactivators SRC1, CBP and MED1 are recruited to upregulated genes to facilitate transcription as expected. SRC1 was the most highly correlated to VDR/RXR binding (50%). However, we also found that corepressor molecules such as NCoR and SMRT were present along with SRC1, CBP or MED1 at these 1,25(OH)2D3 activated gene enhancers. Interestingly, genome-wide NCoR binding mimicked VDR binding by increasing its association with VDR binding in response to 1,25(OH)2D3 treatment. Overall, these data indicate a complex role for corepressor and coactivator complexes in the activation or active repression of 1,25(OH)2D3 responsive genes. 5 coregulators are analyzed by ChIP-seq. The Input from GSE31939 was used as the normalizing factor for all transcription factor IPs. If lanes had more than 1 replicate, these lanes were combined for greater read depth.
2012-09-05 | E-GEOD-39277 | biostudies-arrayexpress
Project description:Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1α. We could then efficiently knockdown PGC-1β expression by shRNA expression. Loss of PGC-1α did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1α or PGC-1β coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1α and PGC-1β were deficient. These data show that PGC-1α is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis. Affymetrix microarray analysis of total RNA from wt, PGC-1α KO and PGC-1α KO cells expressing an RNAi specific for PGC-1β knockdown was performed. Of the 461 mitochondrial genes analyzed, 181 were found to be at least 20% different between wt and defective PGC-1α and β adipocytes (p < 0.05). More than 85% of these genes were downregulated in cells deficient for PGC-1alpha and PGC-1beta. Keywords: Analysis of mitochondrial gene expression
2006-07-06 | GSE5042 | GEO