ABSTRACT: Site-specific reactivity of PCK1 to increasing concentrations of acetyl-CoA. Acetylation stoichiometry is calculated and second-order rate constants are derived.
Project description:Quantitative MS analysis of acetylation in yeast using SILAC labeling and MaxQuant. Download Index of Raw files first. We used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. We used stable isotope labeling with amino acids in cell culture to quantify differences in protein, acetylation, and phosphorylation abundance by MS. Proteins from whole cell lysates were digested to peptides and acetylated peptides enriched using a polyclonal anti-acetyllysine antibody. Peptide fractions were analyzed by reversed-phase liquid chromatography coupled to high resolution liquid chromatography‐tandem mass spectrometry (LC-MS/MS) and raw MS data were computationally processed using MaxQuant.
Project description:Acetyl-Coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on ATP-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants, demonstrate the dynamic changes of H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.
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.
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 direct 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. Global survey of gene expression in CAD cells and differentiated CAD neurons following lentiviral knockdown of ACSS2 or ATP citrate lyase (ACL) (and control = scramble hairpin); survey of hippocampal gene expression changes associated with retrieval of fear memory, after ACSS2-AAV knockdown or in EGFP-AAV control (comparison of 0h vs. 1h post-memory retrieval).
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 direct 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.
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.
Project description:We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild type and sirtuin deacetylase CobB-deficient E. coli. We further assayed stoichiometry in phophotransacetylase (pta) and acetate kinase (ackA) mutant strains in the presence and absence of the sirtuin inhibitor nicotinamide. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurs at very low stoichiometry (median 0.04%). Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Similar to sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry at hundreds of sites in WT, pta, and ackA cells. These results suggest an evolutionarily conserved function of sirtuin deacetylases in suppressing low stoichiometry acetylation.
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 direct regulator of histone acetylation in neurons and of mammalian spatial object 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, and these hippocampal genes are pre-bound by ACSS2. These results reveal a unique connection between cellular metabolism, gene regulation, and neural plasticity, establishing a link between generation of acetyl-CoA “on-site” at chromatin of critical neuronal genes.
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 direct regulator of histone acetylation in neurons and of mammalian spatial object 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, and these hippocampal genes are pre-bound by ACSS2. These results reveal a unique connection between cellular metabolism, gene regulation, and neural plasticity, establishing a link between generation of acetyl-CoA “on-site” at chromatin of critical neuronal genes.
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 direct regulator of histone acetylation in neurons and of mammalian spatial object 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, and these hippocampal genes are pre-bound by ACSS2. These results reveal a unique connection between cellular metabolism, gene regulation, and neural plasticity, establishing a link between generation of acetyl-CoA “on-site” at chromatin of critical neuronal genes.