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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Brown adipose tissue; DNA methylation; Epigenetics; Gene expression; Histone; Thermogenesis.

Fig. 1

High confidence transcriptional target (HCT) intersection analysis resolves epigenetic writer transcriptional footprints in cold challenge-regulated gene sets. In panels (A-C), nodes that have the strongest (higher odds ratio, OR) and most significant (lower p-value) footprints within the indicated cold challenge-induced gene set are distributed towards the upper right of the plot. Scatterplot showing enrichment of nodes with established roles in thermal regulation among nodes that have the most significant intersections with (A) SC vs TN-induced genes; (B) SC vs RT-induced genes; and (C) RT vs TN-induced genes. (D) HCT intersection p-values for selected epigenetic writers within cold challenge-induced genes are indicated in the form of a heatmap. HCT intersection analysis was carried out as described in the Methods section. White cells represent p > 0.05 intersections. The intensity of the color scheme is proportional to the confidence of the intersection between HCTs for a particular node and genes induced (red, ↑) or repressed (blue, ↓) in each cold challenge contrast. Lower confidence (higher p) intersections are towards the yellow end of the spectrum and higher confidence (lower p) intersections are towards the brick red end of the spectrum. Full numerical data are in Additional file 1. n = 4 per group for RNA-Seq data. TN: thermoneutral. RT: room temperature. SC: severe cold

Fig. 2

Alternative splice variants and reduced representation bisulfite sequencing (RRBS) data reveal changes in brown adipose tissue associated with housing temperature. (A) A comparison of SC vs TN housing RNA-Seq data reveals differential expression of alternate splice variants in BAT. Integrated RNA-Seq and RRBS data for (B) SC vs TN housed mice. (C) SC vs RT housed mice; and (D) RT vs TN housed mice. n = 4 per group for RNA-Seq and RRBS datasets. RRBS data shown are for percent methylation difference >|5|, p_adj < 0.05. DEG: differentially expressed genes. DMP: differentially methylated promoters. DMG: differentially methylated genes. TN: thermoneutral. RT: room temperature. SC: severe cold

Fig. 3

Workflow to obtain histone proteoform data from brown adipose tissue and liver. (A) The general workflow includes tissue homogenization, nuclei isolation, acid extraction, HPLC separation of histone families, and LC–MS/MS data acquisition. HPLC separation of histones shows a clean separation of histone families and H3 variants for (B) BAT and (C) liver. (D) MS1 average spectra of histone H4 extracted from BAT from 40 to 80 min show clear + 16 to + 9 charge states matching the m/z of histone H4. (E) The MS1 spectrum of 11,193.8565 Da species (761.2961 m/z, charge + 15) matches a mass of H4 with N-terminal acetylation + 0 methylations + 3 acetylations + 0 phosphorylations with less than 10 ppm error. (F) The ion map from MS2 fragmentation of species from (E) shows unambiguous localization information for the proteoform H4 < N-acK12acK16acK31ac > from a mass change at less than 10 ppm error. (G) Annotated MS2 spectrum shows most abundant peaks are from the same proteoform, < N-acK12acK16acK31ac > , with less than 10 ppm error

Fig. 4

Housing temperature alters bulk histone acetylation, but not methylation, in BAT and has no effect on bulk histone acetylation or methylation in liver. Percent (A) acetylated (1 + acetylation) and (B) methylated (1 + methylation) histones H3.2 and H4 isolated from liver and BAT. Number of (C) acetyl groups and (D) methyl groups (K4me2 = 2 methyl groups) per histones H3.2 and H4 isolated from liver and BAT. n = 3–5 per group. One-way ANOVA testing was conducted for all histone data from each tissue comparing TN, RT, and SC housing conditions, and p-values are indicated in each plot. TN: thermoneutral. RT: room temperature. SC: severe cold

Fig. 5

Under normal room temperature housing conditions, BAT histone H3.2 and H4 post-translational modifications and proteoforms are distinct from those observed in liver. (A) Histone H3.2 and (B) histone H4 binary PTM combinations in BAT and liver. (C) Dot size references for binary volcano plots (A, B). (D) Histone H3.2 and (E) histone H4 proteoforms from BAT and liver. (F) Dot size references for proteoform volcano plots (D,E). P-values were calculated using Welch’s t-test. Volcano plots show cutoffs of |1.5| for fold change and p < 0.05. Red dots represent both p < 0.05 and fold change >|1.5|; blue dots represent p < 0.05 and fold change <|1.5|; grey dots represent p > 0.05 and fold change >|1.5|; black dots represent p > 0.05 and fold change <|1.5|. n = 3–5 per group. RT: room temperature

Fig. 6

Histone H3.2 K9me2- or K9me3-containing proteoforms increase in brown adipose tissue in response to cold housing. (A) Selected discrete H3.2 PTM abundance at different housing temperatures. (B) Proteoform changes between SC and TN. Dot size corresponds to absolute percentage point change. Welch’s t-test was used for volcano plot data, with cutoffs of |1.5| for fold change and p < 0.05. Red dots represent both p < 0.05 and fold change >|1.5|; blue dots represent p < 0.05 and fold change <|1.5|; grey dots represent p > 0.05 and fold change >|1.5|; black dots represent p > 0.05 and fold change <|1.5|. (C) Ternary (3-PTM) combinations show significant differences with K9 di- and tri-methylation, K23 acetylation, and K36 monomethylation. n = 3–5 per group. One-way ANOVA testing was conducted for all data comparing TN, RT, and SC housing conditions, and p-values are indicated in each plot. pp: percentage point. TN: thermoneutral. RT: room temperature. SC: severe cold

Fig. 7

Housing temperature significantly alters histone H4 K16ac-containing proteoforms in brown adipose tissue. (A) Discrete K16ac PTM marks on histone H4 at different housing temperatures. (B) Histone H4 proteoforms from BAT from SC vs TN housing temperatures. Dot size corresponds to absolute percentage point change. Welch’s t-test was used for volcano plot data, with cutoffs of |1.5| for fold change and p < 0.05. Red dots represent both p < 0.05 and fold change >|1.5|; blue dots represent p < 0.05 and fold change <|1.5|; grey dots represent p > 0.05 and fold change >|1.5|; black dots represent p > 0.05 and fold change <|1.5|. (C) H4 < K12acK16acK20me1K31ac > proteoform abundance at different housing temperatures. (D) H4 < K8acK16acK20me3 > proteoform abundance at different housing temperatures. n = 4–5 per group. One-way ANOVA testing was conducted for all data comparing TN, RT, and SC housing conditions, and p-values are indicated in each plot. pp: percentage point. TN: thermoneutral. RT: room temperature. SC: severe cold