Project description:The E1A binding protein P300 (EP300, also known as p300, lysine acetyltransferase 3B or KAT3B) and its close paralogue CREB-binding protein (CREBBP, aka CBP or KAT3A) possess intrinsic histone acetyltransferase (HAT) activity that can act on both histone and non-histone proteins. P300 and CBP are composed of multiple conserved protein domains, many of which are in proximity to the HAT domain modulating its catalytic activity. Here we show that the TAZ2 domain regulates the intrinsic catalytic activity of p300 in vitro and histone acetylation and chromatin accessibility in cells. Our study extends the knowledge of p300 self-regulation and provides new therapeutic stratagies for human cancers with corresponding p300/CBP mutations.
Project description:The E1A binding protein P300 (EP300, also known as p300, lysine acetyltransferase 3B or KAT3B) and its close paralogue CREB-binding protein (CREBBP, aka CBP or KAT3A) possess intrinsic histone acetyltransferase (HAT) activity that can act on both histone and non-histone proteins. P300 and CBP are composed of multiple conserved protein domains, many of which are in proximity to the HAT domain modulating its catalytic activity. Here we show that the TAZ2 domain regulates the intrinsic catalytic activity of p300 in vitro and histone acetylation and chromatin accessibility in cells. Our study extends the knowledge of p300 self-regulation and provides new therapeutic stratagies for human cancers with corresponding p300/CBP mutations.
Project description:The E1A binding protein P300 (EP300, also known as p300, lysine acetyltransferase 3B or KAT3B) and its close paralogue CREB-binding protein (CREBBP, aka CBP or KAT3A) possess intrinsic histone acetyltransferase (HAT) activity that can act on both histone and non-histone proteins. P300 and CBP are composed of multiple conserved protein domains, many of which are in proximity to the HAT domain modulating its catalytic activity. Here we show that the TAZ2 domain regulates the intrinsic catalytic activity of p300 in vitro and histone acetylation and chromatin accessibility in cells. Our study extends the knowledge of p300 self-regulation and provides new therapeutic stratagies for human cancers with corresponding p300/CBP mutations.
Project description:p300 is a histone acetyltransferase that associates with crucial biological processes. p300 acetylates all four histones in the nucleosome, a basic unit of chromatin, and alters chromatin structure and dynamics. In this study, we performed structural and biochemical analysis to understand the nucleosome binding by p300. Crosslinking mass spectrometry suggests that the p300 catalytic core binds to nucleosomes in multiple binding forms to acetylate different histone tails.
Project description:Murine small intestinal organoids were cultured in the presence or absence of 3µM inhibitor I-CBP112 (PubChem CID 90488984), which targets E1A Binding Protein P300 (Ep300) and Creb-binding protein (Crebbp, Cbp). Organoids were grown in culture media containing EGF, Noggin and R-spondin (ENR), media was changed after 48h, and organoids were harvested after 96h.
Project description:The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1? and HIF-2? binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (?CH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of ~35-50% of global HIF-1?-responsive gene expression, whereas another HIF-transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSAS) accounts for ~70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription. Experiment Overall Design: Three separate affymetrix experiments using mouse embryonic fibroblasts derived from embryos bearing the ?CH1 mutation in p300 and/or CBP and treated with hypoxia or combinations of dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) are described (GSE3195, GSE3196 and GSE3296). Samples are not directly comparable between experiments because of differences in experiment design and Affymetrix chips used.
Project description:To determine whether CBP/P300 HAT activity is essential for leukemia cell maintenance, we utilized the selective acetyltransferase inhibitor A485. Mass spectrometry analysis of post-translational modifications showed that A485 treatment of K/C-III-expressing HEK293T cells for 6 and 12 hours significantly reduced multiple histone acetylation marks.
Project description:Prolactinomas are the most common functional pituitary adenomas, with dopamine receptor agonists (DAs) as the first-line therapy. However, 10–30% of patients develop DA resistance, and the underlying mechanisms remain incompletely elucidated.Clinical samples were collected to analyze p300 expression. Experiments including immunofluorescence, immunohistochemistry, gene editing, Western blot, co-immunoprecipitation, ChIP-qPCR, RNA/CUT&Tag sequencing, flow cytometry, Seahorse assay, and mass spectrometry were performed to explore the synergistic anti-tumor mechanism of upregulating/activating p300 combined with DAs. DAs downregulated p300 by inhibiting the cAMP-PKA-CREB pathway. Upregulating/activating p300 synergized with DAs to promote mitochondrial ROS elevation and cell apoptosis, which depends on p300’s histone acetyltransferase (HAT) domain mediating histone H3 lysine 18 lactylation (H3K18la). p300-mediated H3K18la significantly promoted intracellular ROS accumulation by upregulating Ndufs7 (promoting mitochondrial ROS production) and Washc1 (inhibiting mitophagy). YF-2, a p300 HAT domain activator, exhibited synergistic anti-tumor activity with DAs.This study reveals the p300-H3K18la- mitophagy-ROS regulatory axis, providing a new target and strategy for treating DA-resistant prolactinomas.
Project description:Prolactinomas are the most common functional pituitary adenomas, with dopamine receptor agonists (DAs) as the first-line therapy. However, 10–30% of patients develop DA resistance, and the underlying mechanisms remain incompletely elucidated.Clinical samples were collected to analyze p300 expression. Experiments including immunofluorescence, immunohistochemistry, gene editing, Western blot, co-immunoprecipitation, ChIP-qPCR, RNA/CUT&Tag sequencing, flow cytometry, Seahorse assay, and mass spectrometry were performed to explore the synergistic anti-tumor mechanism of upregulating/activating p300 combined with DAs. DAs downregulated p300 by inhibiting the cAMP-PKA-CREB pathway. Upregulating/activating p300 synergized with DAs to promote mitochondrial ROS elevation and cell apoptosis, which depends on p300’s histone acetyltransferase (HAT) domain mediating histone H3 lysine 18 lactylation (H3K18la). p300-mediated H3K18la significantly promoted intracellular ROS accumulation by upregulating Ndufs7 (promoting mitochondrial ROS production) and Washc1 (inhibiting mitophagy). YF-2, a p300 HAT domain activator, exhibited synergistic anti-tumor activity with DAs.This study reveals the p300-H3K18la- mitophagy-ROS regulatory axis, providing a new target and strategy for treating DA-resistant prolactinomas.
Project description:The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the HAT domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.
