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Multi-acetylated histones are a source of carbon for lipid synthesis

ABSTRACT: Histone modifications commonly integrate environmental stimuli to cellular metabolic outputs by affecting gene expression. Many modifications, including some histone acetylation marks, do not always correlate to transcription, thus pointing towards an alternative role of histone modifications as potential metabolic reservoirs. Using an approach that integrates mass spectrometry- based epi-proteomics and metabolomics with stable isotope tracer studies, we demonstrate that elevated lipids in histone acetyltransferase (HAT)-depleted hepatocytes result from carbon atoms flowing from the deacetylation of multi-acetylated histone H4 to fatty acids. Consistent with this, the enhanced lipid synthesis in HAT-depleted hepatocytes is dependent on the activity of histone deacetylases (HDACs) and acetyl-CoA synthetase ACSS2. Furthermore, we show that during diet-induced lipid synthesis there is a reduction of multi-acetylated histone H4 in hepatocytes and in mouse liver. In addition, overexpression of histone acetyltransferases can reverse diet-induced lipogenesis by blocking lipid droplet accumulation and maintaining the levels of multi-acetylated histone H4. This study unveils an additional link between epigenetics and metabolism whereby histone acetylation reservoirs may serve as a carbon source for lipid synthesis.

INSTRUMENT(S): Q Exactive Plus, Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Roberta Noberini

LAB HEAD: Tiziana Bonaldi

PROVIDER: PXD042624 | Pride | 2024-02-14

REPOSITORIES: Pride

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Project description:In this study we focus on the MYST-type acetyltransferase MOF (males-absent-on-the-first, KAT8), which is the effector enzyme of two transcription regulator complexes in Dro-sophila. As part of the male-specific-lethal dosage compensation complex (MSL-DCC, or DCC for short), the enzyme is targeted to transcribed genes on the X chromosome, where it acetylates H4K16 to unfold the chromatin fiber to boost transcription (15-17). In the context of the NSL complex (16) MOF has been reported to acetylate different combina-tions of histone H4 lysines 5, 8, 12 and 16 at promoters in different cells, illustrating the potential of complex subunits to modulate substrate specificity (18-21). Defining the substrate selectivity of KATs in vivo poses several challenges (1). The intrin-sic substrate selectivity of enzymes may be modulated by its molecular environment, be it the complex assembly or the chromatin context. Redundant activities of different en-zymes, indirect effects and the action of lysine deacetylases (KDACs) may occlude the consequences of loss-of-function manipulation. Furthermore, the specificity of the anti-bodies that are commonly used to detect distinct acetylated lysines in histones may be compromised due to low-level off-target effects, interference of modifications next to acetylated lysines, and the tendency of Kac-antibodies to react with relaxed selectivity with oligo-acetylated histone tails (22-25). A more reliable strategy involves the detection of modified histone peptides by mass spectrometry, which unequivocally detects individ-ual modifications and defined combinations of modifications and has a much better dy-namic range than traditional Western blotting (26-30). In a complementary approach one may determine the intrinsic substrate selectivity of defined recombinant enzymes and KAT complexes in biochemical assays (31). We re-cently reconstituted a recombinant MOF-containing core DCC (here termed ‘4-MSL’, since it contains the subunits MOF, MSL1, MSL2 and MSL3 (32)) and now report on a study of its substrate selectivity on recombinant nucleosome arrays. Pilot experiments using traditional Western blotting of diagnostic antibodies suggested that, in addition to the expected H4K16 acetylation, H4K12 was modified. Since this finding disagrees with the prevailing view that the DCC selectively acetylates H4K16, we applied the targeted mass spectrometry described in (26) to our in vitro reactions, focusing on acetylation of the H4 N-terminus. We compared the acetylation reaction of the 4-MSL complex with another MYST enzyme, dTip60 (KAT5), in the context of a trimeric complex, akin to the yeast piccolo NuA4 complex, which is thought to have a more relaxed lysine selectivity on the H4 N-terminus (33-35). We found that during short reaction times, the 4-MSL complex acetylated H4K16 with excellent selectivity, while the dTip60piccolo complex preferentially modified H4K12. To our surprise, we also observed that during longer incubations the 4-MSL complex progres-sively acetylated K12, K8 and K5, leading to a complex mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains bound to the substrate and continues to acetylate more N-terminal H4 lysines in a processive manner. Because the MSL-DCC in vivo contains long, non-coding roX RNA (15,36-38), we ex-plored whether RNA affected the specificity of the reaction. We observed that the addi-tion of RNA to the HAT reaction improves the specificity of the reaction and suppresses the generation of oligo-acetylated forms, possibly by reducing the dwell time of the en-zyme on the nucleosome substrate. The comparison of MOF and dTip60 activities in the context of recombinant HAT com-plexes revealed significant differences in activity and specificity. Our study describes a powerful experimental approach and conceptual framework for the analysis of physiolog-ically relevant components that modulate intrinsic activities.

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Multi-acetylated histones are a source of carbon for lipid synthesis

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