Our goal is to understand the mechanisms that govern transcription in the heart during health and disease. Transcription is a highly dynamic process that requires metabolic intermediates for its activation or deactivation, these include: acetyl-Coenzyme A (acetyl-CoA) for histone acetylation, a-ketoglutarate (aKG) as a cofactor for histone and DNA demethylases, and succinyl-CoA (suc-CoA) for histone succinylation, not discounting other acyl-CoAs. Since none of the CoA-linked metabolites could be exported out of the mitochondria, the nucleus, must acquire its acetyl-CoA, for example, mainly via export of citrate from the mitochondria during substrate abundance, which is then converted to acetyl-CoA in the nucleus via ATP citrate lyase. On the other hand, the nucleus?s source of aKG, suc-CoA, or other short-chain acyl-CoAs (e.g. butyryl-CoA, malonyl-CoA, propionyl- CoA?etc.), is not fully accounted for. The other unanswered question, is how are genes selectively activated by specific substrates, and how does this influence an organ?s homeostasis? The dogma has always been that oxidative enzymes and substrate oxidation are specifically confined to the mitochondria. However, in a recent unbiased screen, using chromatin immunoprecipitation and mass spectrometry for discovery of proteins that associate with H2A.Z-bound chromatin in the heart, we uncovered mitochondrial enzymes of the TCA cycle, b-oxidation, and branched-chain amino acid catabolism, in the nucleus, localized to the transcription start sites (TSS) of genes. Recombinant green florescence fusion proteins combined with mutations of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and chromatin binding in both rodent and human cells. More conclusively, chromatin immunoprecipitation-deep sequencing (ChIP-Seq), confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start sites. Finally, knockdown or knockout of H2A.Z in mouse or human cells reduced binding of metabolic genes that was associated with reduced posttranslational histone modifications including acetylation and succinylation. The relevance, of which, is exemplified by fatty-induced increase in chromatin-bound ACAA2 and differential modulation of gene expression, which is abrogated by a nuclear localization signal (NLS) mutation. In this proposal, we will focus on investigating the nuclear role of 2 enzymes, representatives of the two pathways that oxidize glucose and fatty acids; including, OGDH, which converts aKG into suc-CoA, and ACAA2, which converts 3-ketoacyl-CoA into acetyl-CoA and acyl-CoA in the last reaction of the b-oxidation spiral, respectively. We hypothesize that, 1- The nucleus harbors mitochondrial enzymes of the TCA cycle and b-oxidation spiral that are specifically localized to H2A.Z-bound chromatin at the TSS of select genes. 2- In accordance, this renders specific genes directly responsive to either glucose or fatty acids, via the local production of acetyl-CoA, suc-CoA, and the production/consumption of aKG, which are required for histone modifications necessary for transcriptional activation or repression. 3- Perturbations of the nuclear concentrations of these genes results in substrate-dependent modulation of histone marks and transcription, at select promoters, which influences the development of cardiac hypertrophy and failure. Thus, our aims are to:1- Identify the chromatin-association sites of ACAA2 and OGDH, the underlying histone marks, and their regulation by diet in the normal and hypertrophied hearts. 2- Determine the roles of nuclear ACAA2 and OGDH in regulating histone modifications and gene transcription during myocyte hypertrophy. 3- Determine the roles of nuclear ACAA2 and OGDH in the development of cardiac hypertrophy and failure in mice.
The current dogma is that metabolic oxidative enzymes and substrate oxidation are specifically confined to the mitochondria. However, our recent findings challenge this belief, as we not only show the presence of metabolic oxidative enzymes in the nucleus, but also their association with H2A.Z-bound chromatin, in the heart. These enzymes are expected to locally produce acetyl- CoA, a-ketoglutarate, and succinyl-CoA, among other acyl-CoAs, which are substrates for histones and DNA modifications, thus regulating transcription. As proof-of-principle, we show that acetyl-CoA acyltransferase 2 associates with the transcription start sites of select genes and renders them specifically responsive to palmitate. This would explain how the regulation of gene transcription is directly and specifically regulated by metabolism. In addition, it would demonstrate, for the first time, how the type of substrate/diet can directly impact the transcriptional phenotype and, thus, a cell?s or organ?s health.
