H2A.Z-dependent and -independent recruitment of metabolic enzymes to chromatin required for histone modifications

Abstract

13H2A.Z plays a fundamental role in the regulation of transcription and epigenetics, however, the mechanisms that 14 underlie its functions are not fully understood. Using rapid chromatin immunoprecipitation-mass spectrometry, 15 we uncovered the association of H2A.Z-bound chromatin with an array of tricarboxylic acid cycle and beta-16 oxidation enzymes in the mouse heart. Recombinant green florescence fusion proteins combined with mutations 17 of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), 18 oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and 19 chromatin binding in both rodent and human cells. Conclusively, chromatin immunoprecipitation-deep 20 sequencing, confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start 21 sites. Finally, human H2A.Z-deficient HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, with 22 the exception of pyruvate dehydrogenase, accompanied with reduced posttranslational histone modifications. 23Thus, the data show that metabolic enzymes are recruited to active promoters for potential site-directed 24 epigenetic modifications. 45Generally, organisms respond to metabolic cues by exacting a change in gene transcription that influences their 46 development and growth, or homeostasis. These signals include ATP:ADP:AMP and NAD + :NADH ratios, and 47 the availability of metabolites that are involved in histone and DNA modifications -e.g. acetyl-CoA (ac-CoA), a-48 ketoglutarate (aKG), and succinyl-CoA (suc-CoA), not discounting other acyl-CoAs 9 . In the case of acetyl-CoA, 49we know that during substrate abundance, citrate is exported from the mitochondria and into the cytosol and 50 nucleus, where ATP citrate lyase (ACLY) converts it into acetyl-CoA, which is a substrate for histone acetylation 51 10 . Alternatively, during substrate shortage, acetate is imported from the circulation and converted into acetyl- 4CoA by acyl-CoA synthetase short chain family member 2 (ACSS2) 11 . As for the other CoA-linked metabolites 53 and aKG, the mechanism for nuclear delivery is less well-established. Moreover, the question of how substrates 54 modulate the expression of specific target genes remains a challenge. 55The current dogma is that metabolic oxidative enzymes and substrate oxidation are largely confined to the 56 mitochondria. However, recent findings, including our own, challenge this belief. In specific, Sutendra et al, 57reported the presence of all subunits of the pyruvate dehydrogenase (PDH) complex in human sperm, and 58 normal and cancerous lung epithelial cells 12 , while Nagaraj et al reported it in the human 4/8-cell stage zygote 59 13 , and show that it is required for generating acetyl-CoA for histone acetylation. Likewise, oxoglutarate 60 dehydrogenase (OGDH) 14 and isocitrate dehydrogenase (IDH2) 15 have also been shown to partly localize to 61 the nucleus, as these findings are further supported by a proteomics study th...