Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress

Authors: TAYLOR, BETHANY - Baylor College Of Medicine; STEINTHAL, LOIC - Children'S Nutrition Research Center (CNRC); DIAS, MICHELLE - Baylor College Of Medicine; YALAMANCHILI, HARI - Children'S Nutrition Research Center (CNRC); OCHSNER, SCOTT - Baylor College Of Medicine; ZAPATA, GLADYS - Baylor College Of Medicine; MEHTA, NITESH - Children'S Nutrition Research Center (CNRC); MCKENNA, NEIL - Baylor College Of Medicine; YOUNG, NICOLAS - Baylor College Of Medicine; NUOTIO-ANTAR, ALLI - Children'S Nutrition Research Center (CNRC)

Submitted to: Epigenetics and Chromatin
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2024
Publication Date: 4/27/2024
Citation: Taylor, B.C., Steinthal, L.H., Dias, M., Yalamanchili, H.K., Ochsner, S.A., Zapata, G.E., Mehta, N.R., McKenna, N.J., Young, N.L., Nuotio-Antar, A.M. 2024. Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress. Epigenetics and Chromatin. 17:12. https://doi.org/10.1186/s13072-024-00536-8.
DOI: https://doi.org/10.1186/s13072-024-00536-8

Interpretive Summary: The U.S. and worldwide obesity and type 2 diabetes epidemics represent grave public health concerns. Increasing body heat production to burn energy has been suggested as a potential therapeutic target for obesity and type 2 diabetes mellitus. Brown fat plays an important role in the regulation of body temperature through the production of heat. Brown fat increases heat production in colder ambient temperature conditions and decreases heat production as ambient temperatures become warmer. We wanted to know what factors in brown fat regulate heat production in response to cold. Therefore, we exposed mice to three different chronic housing temperatures: 1.) thermoneutral, which simulates the human comfort zone, 2.) mild cold (normal room temperature) or 3.) severe cold and examined changes in epigenetic factors, which regulate gene expression, to determine what, if any changes occurred. We discovered that DNA methylation is reduced in brown fat under cold housing conditions, indicating a permissive state with regard to gene expression. However, histone modifications showed both "on" and "off" signals with regard to gene expression. Taken together, our data provide new insights into the regulation of heat production by brown fat and yield additional unexplored targets for the prevention and treatment of obesity and type 2 diabetes mellitus.

Technical Abstract: Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Our preliminary analyses of RNA-Seq data uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. Motivated to understand how BAT function is regulated epigenetically, we developed a novel method for the first-ever unbiased top-down proteomic quantitation of histone modifications in BAT and validated our results with a multi-omic approach. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C) and BAT was analyzed for DNA methylation and histone modifications. Methylation of promoters and intragenic regions in genomic DNA decrease in response to chronic cold exposure. Integration of DNA methylation and RNA expression datasets suggest a role for epigenetic modification of DNA in regulation of gene expression in response to cold. In response to cold housing, we observe increased bulk acetylation of histones H3.2 and H4, increased histone H3.2 proteoforms with di- and trimethylation of lysine 9 (K9me2 and K9me3), and increased histone H4 proteoforms with acetylation of lysine 16 (K16ac) in BAT. Our results reveal global epigenetically-regulated transcriptional "on" and "off" signals in murine BAT in response to varying degrees of chronic cold stimuli and establish a novel methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Additionally, we make histone PTM and proteoform quantitation, RNA splicing, RRBS, and transcriptional footprint datasets available as a resource for future research.