Sustained activation of AMPK enhances differentiation of human iPSC-derived cardiomyocytes via sirtuin activation
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ABSTRACT: Lee and colleagues demonstrate that sustained activation of AMPK enhances differentiation of iPSC-derived cardiomyocytes. Sustained AMPK activation decreased histone acetylation at known target sites for nuclear-localized sirtuins, suggesting that AMPK activation enhances sirtuin activity. AMPK-induced sirtuin-mediated deacetylation of histone proteins may regulate chromatin accessibility and enhance cardiomyocyte differentiation.
Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity. Six factors and Input ChIP-seq samples were analyzed in Saccharomyces eerevisiae.
Project description:Sirtuins are NAD+-dependent deacylases with diverse enzymatic activities and substrates. They were implicated in regulation of cancer progression, neurodegeneration, aging, and viral infection. Sirtuin-2 localizes to the cytoplasm and is known to deacetylate alpha-tubulin. Current knowledge on sirtuin-2 interactions and its effect on gene expression in normal cells is very limited. We used proteomics approach to define its interactions and its effect on global protein levels in human fibroblasts.
Project description:Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD+-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 co-localises with the other Sirtuins at silent regions (cen, mat, tel, rDNA) and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function whereas Hst4 represses amino acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 5’ mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation. Keywords: chromatin; fission yeast; gene silencing; retrotransposon; Sirtuin; HDAC
Project description:Protein acetylation is a posttranslational modification that orchestrates gene regulation and various biological processes, such as fungal pathogenesis. One promising strategy for treating fungal infections is targeting the enzymes that regulate protein acetylation. Sirtuins, an NAD+-dependent lysine deacetylase, have been described as histone deacetylase and as regulators of secondary metabolism in various Aspergillus spp. However, the roles of sirtuin remain unclear. We employed a comprehensive set of experimental techniques, including gene deletion, phenotyping, in vivo virulence assays, metabolome analysis, transcriptome analysis, and acetylome analysis. Our findings reveal that sirtuins in A. fumigatus are intricately involved in crucial cellular processes such as cell wall integrity, secondary metabolite (SM) production, protease secretion, thermotolerance, and virulence. Notably, among the six sirtuins studied, AFSirE exerts a major influence on the phenotype of A. fumigatus. Furthermore, through acetylome analysis, we identified 42 and 260 proteins exhibiting differential acetylation in the AfSirE and SIRTko strains, respectively. Transcriptome data obtained from RNA sequencing (RNA-seq) demonstrated that sirtuins play a regulatory role in the expression of genes associated with SM production, cell wall component biosynthesis, and a variety of virulence factors.
Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity.
Project description:A longevity gene, sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress and exercise.The objective is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults.
Project description:Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.
Project description:Lithocholic acid (LCA), accumulated in mammals during calorie restriction (CR), can activate AMP-activated protein kinase (AMPK) and slow down ageing1. However, how LCA is signalled to activate AMPK and elicit the biological effects is unclear. Here, we show that LCA can enhance the activity of sirtuins (SIRTs) to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), thereby triggering AMPK activation via the lysosomal glucose-sensing pathway. Through proteomic analysis of SIRT1-coimmunoprecipitated proteins, we identify that TUB-like protein 3 (TULP3), a sirtuin-interacting protein2, is an LCA receptor, and demonstrate that the LCA-bound TULP3 allosterically activates SIRTs. Activated SIRTs then deacetylate the V1E1 subunit of v-ATPase on K52, K99 and K191 residues. Muscle-specific expression of the 3KR mutant of V1E1, mimicking the deacetylated state, dominantly activates AMPK and rejuvenates muscles in aged mice. Moreover, LCA depends on the TULP3 homologues tub-1 and ktub to activate AMPK and extend lifespan and healthspan in nematodes and flies, respectively. Our study thus elucidates that LCA triggers the TULP3-sirtuin-v-ATPase-AMPK route to manifest benefits of calorie restriction. This project contains the MS data for the modification of each of 21 v-ATPase subunits and the interacting protein of SIRT1 we identified. It also contains the validation data for MEF cell lines with bile acid receptor knockout.