Nakadai, T. | Search bioRxiv

bioRxiv2021

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, and transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we have uncovered negative biochemical crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.

biochemistry↗

bioRxiv2018

A non-canonical-PPARγ/RXRα-binding sequence regulates leptin expression in response to changes in adipose tissue mass

Leptin expression decreases after fat loss and is increased when obesity develops and its proper quantitative regulation is essential for the homeostatic control of fat mass. We previously reported that a distant leptin enhancer (LE1), 16kb upstream from the transcription start site (TSS), confers fat-specific expression in a BAC transgenic reporter mouse (BACTG). However this and the other elements that we identified do not account for the quantitative changes in leptin expression that accompany alterations of adipose mass. In this report, we used ATAC-seq to identify a 17bp non-canonical-PPAR{gamma}/RXR-binding site leptin regulatory element 1 (LepRE1) within LE1, and show that it is necessary for the fat-regulated quantitative control of reporter (luciferase) expression. While BACTG reporter mice with mutations in this sequence still show fat-specific expression, luciferase is no longer decreased after food restriction and weight loss. Similarly the increased expression of leptin reporter associated with obesity in ob/ob mice is impaired. A functionally analogous LepRE1 site is also found in a second, redundant DNA regulatory element 13kb downstream of the TSS. These data uncouple the mechanisms conferring qualitative and quantitative expression of the leptin gene and further suggest that factor(s) that bind to LepRE1 quantitatively control leptin expression and might be components of a lipid sensing system in adipocytes.\n\nSignificanceLeptin gene expression is highly correlated with the lipid content of individual fat cells suggesting that it is regulated by a \"fat sensing\" signal transduction pathway. This study is thus analogous to studies that led to the identification of a cholesterol-sensing pathway by studying the regulation of the LDL receptor gene by intracellular cholesterol. Several lines of investigation have suggested that, in addition to adipocytes, liver, neurons and other cell types can also sense changes in lipid content though the molecular mechanisms are unknown. The data here provide a critical first step toward elucidating the components of this system, which would be of great importance. These studies also identify a previously underappreciated role of PPAR{gamma}/RXR complex to regulate leptin expression.

physiology↗