Brown adipose tissue histone proteoform analysis reveals an epigenetic response to cold stress

Regulation of the thermogenic response by brown adipose tissue (BAT) is an important mechanism in the basic biology and treatment of obesity and diabetes. Consensus transcriptional regulatory analysis of a publicly-available RNA-seq dataset uncovered a large number of nodes representing epigenetic modifiers that were altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic activation of BAT results in epigenetic modifications of DNA and histones that affect the tissue function. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C). Reduced representation bisulfite sequencing (RRBS) revealed a marked decrease in methylation of promoters and intragenic regions in BAT genomic DNA in response to varying degrees of chronic cold exposure. Integration of our RRBS and the publicly available RNA-Seq dataset suggested a role for epigenetic modification of DNA in gene regulation in response to cold. To analyze histone modifications, we developed a robust method for the isolation and quantitation of histone proteoforms, which enabled the first comparison of histone H3.2 and H4 proteoforms in BAT. We report distinct on/off histone signals that are observed with histone H3.2 in BAT, but not in the liver, when mice are acclimated to severe cold in comparison with mild cold. Specifically, we observe a larger percentage of histone H3.2 with the K9 dimethylation mark coupled with K36 mono- or di-methylation and K23 acetylation, suggesting strong repression of previously active chromatin. Taken together, our results provide novel findings supporting global epigenetic modification in murine BAT in response to varying degrees of chronic cold stimuli and establish a methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response. Overall design: To investigate the effect of housing temperature on DNA methylation in brown adipose tissue (BAT), male C57BL/6J mice were housed at thermoneutral (TN, 28oC) or standard room temperatures (RT, 22oC) beginning at weaning (3 weeks of age). At 8 weeks of age, a cohort of RT-housed mice were housed under severe cold (SC, 8oC) for two weeks. BAT was dissected when mice were 10 weeks old. n=4 per TN, RT, or SC-housed group.