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PDHE1α, PARylation, Acetyl-CoA, Acetylation, Histones, DNA damage repair

ABSTRACT: Chromatin relaxation is a prerequisite step that allows the DNA repair machinery to access double-strand breaks (DSBs), and local histones around the DNA breaks suffer from prompt acetylation changes. However, an intriguing question remains as to where the large demands of acetyl-CoA is precisely produced promptly. Here, we report that pyruvate dehydrogenase 1α (PDHE1α) catalyzes pyruvate metabolism to provide acetyl-CoA promptly in response to DNA damage. PDHE1α was quickly recruited to chromatin in a PARylation-dependent manner, which further drove acetyl-CoA generation to support local chromatin acetylation around the DSB regions. In turn, this process increased the formation of relaxed chromatin to benefit repair factor loading, thus promoting DSB repair to ultimately maintain genome stability and contribute to the resistance of cancer cells to DNA-damaging treatments in vitro and in vivo. In accordance with this, blocking PARylation-based PDHE1α chromatin recruitment markedly attenuated chromatin relaxation and the DSB repair efficiency, resulting in genome instability and restoration of radiosensitivity. Collectively, these findings reveal an undescribed mechanism that underlies site-directed acetyl-CoA generation involving chromatin-associated PDHE1α and its instrumental function in DNA repair and regulating local chromatin acetylation around DSB sites.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jun Zhang

LAB HEAD: Jun Zhang

PROVIDER: PXD040676 | Pride | 2023-07-27

REPOSITORIES: Pride

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PARylated PDHE1α generates acetyl-CoA for local chromatin acetylation and DNA damage repair

Project description:Chromatin relaxation is a prerequisite step for allowing DNA repair machinery to access double-strand breaks (DSBs), and local histones around the DNA breaks suffer from prompt acetylation changes. However, an intriguing question remains as to where the quick and robust acetyl-CoA is produced. Here, we report that pyruvate dehydrogenase 1α (PDHE1α) catalyzes pyruvate metabolic processes and provides acetyl-CoA promptly in response to DNA damage. PDHE1α was quickly recruited to chromatin in a PARylation-dependent manner, which further drove the local acetyl-CoA generation to support local chromatin acetylation around the DSB regions. In turn, this process increased the formation of relaxed chromatin to benefit repair factor loading, thus promote DSB repair to ultimately maintain genome stability and contribute to the resistance of cancer cells to DNA-damaging treatments in vitro and in vivo. Consistent with this, blocking PARylation-based PDHE1α chromatin recruitment markedly attenuated chromatin relaxation and the DSB repair efficiency, and resulted in genome instability and radio re-sensitivity. Collectively, these findings identified an undescribed mechanism that underlies site-directed acetyl-CoA generation involving chromatin-associated PDHE1α and its instrumental function in DNA repair and regulating local chromatin acetylation around DSB sites. The findings provide potential routes to disrupt a key mechanism of cancer cell resistance to genotoxic damage.

2023-07-26 | GSE237929 | GEO

Harnessing Ionic Selectivity In Acetyltransferase Chemoproteomic Probes

Project description:Chemical proteomics (chemoproteomics) has emerged as a powerful strategy for the high-throughput annotation of protein function. This approach involves using an active site probe to capture an enzyme class of interest from cells, allowing the biological activity of multiple enzymes within that family to be assessed in parallel. Coenzyme A based chemoproteomic probes may have utility in studying the pharmacological interactions of additional enzyme classes, beyond histone acetyltransferases. However, one challenge in applying this approach is how to differentiate a ‘true hit’ from background. Many histone acetyltransferases utilize an ordered binding mechanism, with acetyl-CoA binding first, which allows their chemoproteomic capture to be competed by pre-incubating lysates with acetyl-CoA. However, for acetyltransferases that don’t exhibit this binding mode, or which bind to a different ligand, acetyl-CoA competition may not be evident, limiting our ability to discern selective chemoproteomic enrichment from noise. In this study, two physiochemically distinct capture probes were used to enrich proteins to distinguish true hits from background and identify new targets selectively enriched by CoA-based probes, thus expanding the scope of protein-ligand interactions open to chemoproteomic interrogation.

2021-08-19 | PXD020856 | Pride

Gene expression in experimental aortic coarctation and repair: candidate genes for therapeutic intervention?

Project description:The objective of the current investigation was to use microarray techniques to quantify differentially expressed genes (DEGs) in the upstream aorta subjected to high arterial BP after surgical induction of CoA, and restoration of normal arterial BP after its correction. DEGs may offer additional insight into potential mechanisms of persistent CV morbidity despite successful surgical repair. Male New Zealand white rabbits ~10 weeks old and weighing ~1.0 kg randomly underwent proximal dAo CoA. A 20 mmHg BP gradient was imposed using silk (permanent) or Vicryl (degradable) suture to mimic untreated CoA and surgically corrected CoA, respectively. Rabbits develop a pronounced stenosis and accompanying elevated BP as a stimulus for arterial remodeling within one week. Degradation of Vicryl suture in the corrected group restores aortic diameter close to normal, but with modest residual narrowing. Non-experimental rabbits were designated as a control group. This results in a statistically significant increase in mean, systolic and pulse BP proximal to the coarctation for CoA as compared to both control and corrected rabbits. A ~4 mm circumferential region from the proximal aorta between the coarctation site and left subclavian artery was excised at harvest (32 weeks of age) and frozen. Frozen samples (n=4/group) were shipped overnight to Arraystar, Inc (Rockville, MD) for microarray analysis.

2015-07-10 | E-GEOD-70687 | biostudies-arrayexpress

Nucleoside-Diphosphate Kinase 1 and 2 (Nme1/2) are major cellular co-enzyme A/acetyl-CoA binding factors

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2023-08-01 | PXD029159 | Pride

Acetyl-CoA metabolism by ACSS2 regulates neuronal histone acetylation and long-term memory [ChIP-seq]

Project description:Metabolic production of acetyl-CoA has been linked to histone acetylation and gene regulation, however the mechanisms are largely unknown. We show that the metabolic enzyme acetyl-CoA synthetase 2 (ACSS2) is a critical and directchum regulator of histone acetylation in neurons and of long-term mammalian memory. We observe increased nuclear ACSS2 in differentiating neurons in vitro. Genome-wide, ACSS2 binding corresponds with increased histone acetylation and gene expression of key neuronal genes. These data indicate that ACSS2 functions as a chromatin-bound co-activator to increase local concentrations of acetyl-CoA and to locally promote histone acetylation for transcription of neuron-specific genes. Remarkably, in vivo attenuation of hippocampal ACSS2 expression in adult mice impairs long-term spatial memory, a cognitive process reliant on histone acetylation. ACSS2 reduction in hippocampus also leads to a defect in upregulation of key neuronal genes involved in memory. These results reveal a unique connection between cellular metabolism and neural plasticity, and establish a link between generation of acetyl-CoA and neuronal chromatin regulation. Genome-wide examination of histone H3 and H4 acetylation, as well as ACSS2 binding, in undifferentiated CAD cells and differentiated CAD neurons; background adjusted by H3 ChIP or Input.

2017-05-01 | E-GEOD-76850 | biostudies-arrayexpress

Cohesin profiling at DSBs

Project description:Chromatin undergoes major remodeling around DNA double strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit from the cohesin complex, previously characterized as required for repair by homologous recombination. We found that the recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs. In addition, we show that the cohesin complex, which was also recently proposed to be a key player in chromosome organisation and chromatin looping, controls gammaH2AX distribution within domains. Indeed, as we reported for transcription, cohesin binding antagonizes gammaH2AX spreading. Remarkably, depletion of cohesin leads to an increase of gammaH2AX at cohesin-bound genes (revelead by gammaH2AX mapping in upon SCC1 siRNA), associated with a decrease in their expression level after DSB induction. Thus our study identifies a novel role for the cohesin complex in protecting the genes located in gammaH2AX domains from both gammaH2AX spreading and transcriptional shut-down after DSB induction.

2012-01-01 | E-TABM-1164 | biostudies-arrayexpress

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Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection, and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.

2014-03-23 | E-MTAB-1241 | biostudies-arrayexpress

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Project description:Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here we show that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3) /MLL4 complex display impaired histone methylation (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus leading to defective immunoglobulin class-switching. We also show that PTIP accumulation at DSBs contributes to class-switch recombination (CSR) and genome stability independently from Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR, and suggest a non-redundant role for the MLL3/MLL4 complex in altering antibody effector function. Genome-wide analysis of histone modifications, PTIP, and Pol II in PTIP-WT and PTIP-KO mouse activated B cells.

2010-08-25 | E-GEOD-20852 | biostudies-arrayexpress

Engineering acetyl-CoA supply: Functional expression of a bacterial pyruvate-dehydrogenase complex in the cytosol of Saccharomyces cerevisiae

Project description:The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl-CoA is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of a pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required the simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2 and E3 subunits, as well as genes involved in lipoylation of E2 and addition of lipoate to growth media. A strain lacking ACS, that expressed these E. faecalis genes, grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial micro-organisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. For both strains - mutant strain IMY104 and reference strain CEN.PK113-7D' three independent chemostat cultures were performed. Each of the chemosta was sampled for transcriptome analysis. Samples were processed as described below.

2014-11-04 | E-GEOD-59814 | biostudies-arrayexpress

Global Genome-Nucleotide Excision Repair is Organised into Domains Promoting Efficient DNA Repair in Chromatin

Project description:The rates at which lesions are removed by DNA repair can vary widely throughout the genome with important implications for genomic stability. We measured the distribution of nucleotide excision repair (NER) rates for UV induced lesions throughout the yeast genome. By plotting these repair rates in relation to all ORFs and their associated flanking sequences, we reveal that in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organised within the genome. We compared genome-wide DNA repair rates in wild type and in RAD16 deleted cells, which are defective in the global genome-NER (GG-NER) sub-pathway, demonstrating how this alters the normalâ¨distribution of NER rates throughout the genome. We examine the genomic locations of global genome NER factor binding in chromatin before and after UV irradiation, and reveal that GG-NER is organized and initiated from specific locations. By controlling the chromatin occupancy of the histone acetyl transferase Gcn5, the GG-NER complex regulates the histone H3 acetylation status and chromatin structure in the vicinity of these genomic sites to promote the efficient DNA repair of UV induced lesions. This demonstrates that chromatin remodeling during the GG-NER process is organized into domains in the genome. Importantly, we demonstrate that deleting the histone modifier GCN5, an accessory factor required for chromatin remodeling during GG-NER, significantly alters the genomic distribution of NER rates. These observations could have important implications for the effect of histone and chromatin modifiers on the distribution of genomic mutations acquired throughout the genome.

2016-07-21 | E-MTAB-4641 | biostudies-arrayexpress

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