# Diet and temperature interactively impact brown adipose tissue gene regulation controlled by DNA methylation

## TL;DR

The study shows how diet and temperature together affect gene regulation in brown fat through DNA methylation, with obesity limiting cold adaptation.

## Contribution

The first evidence that epigenetic cold responses in brown adipose tissue differ based on metabolic state.

## Key findings

- Obese mice show impaired cold adaptation and reduced thermogenic gene activation.
- Obesity limits epigenetic remodeling in BAT, with fewer DMEGs and increased gene-body methylation.
- Cold-induced pathways in obesity shift toward stress and diabetogenic signaling.

## Abstract

Controlling brown adipose tissue (BAT) plasticity offers potential for novel obesity therapies. DNA methylation is closely linked to thermogenic and metabolic pathways and thereby influences BAT function. How metabolic state and cold exposure interact to shape methylation-dependent BAT gene regulation was investigated.

Five-week-old mice were fed either chow for 11 weeks (lean) or high-fat diet for 22 weeks to induce obesity (DIO), after which cold exposure was applied for seven days. BAT transcriptomes (RNAseq) and methylomes (RRBS) were generated, and differentially methylated and expressed genes (DMEGs) showing metabolic state–dependent cold responses were identified. Pathway enrichment, epigenetic regulator screening, and transcription factor (TF) motif analyses were performed. DNA methylation was experimentally modulated in vitro to validate selected gene expression responses.

A total of 1,364 differentially expressed genes (DEGs) were uniquely affected by the interaction of metabolic state and cold, with most downregulated in DIO mice. Sixty-five DMEGs (4 % of DEGs) showed metabolic state–specific responses to cold. In DIO mice, DMEGs were enriched in pathways associated with mitochondrial dysfunction, altered lipid metabolism, neuroendocrine signaling, and stress responses. Several epigenetic regulators, including Tet2, Dnmt3a, and Apobec1, exhibited metabolic state- and cold-dependent expression, and TF-motif analyses highlighted roles for AhrArnt and Foxn1. In vitro assays confirmed that DNA methylation influences expression of thermogenic genes.

These findings provide the first evidence that the epigenetic cold response of BAT differs by metabolic condition. BAT remodeling is shaped by coordinated transcriptional and epigenetic mechanisms integrating environmental and metabolic cues.

Mice were housed under cold exposure (8 °C) or thermoneutrality (30 °C) and fed either chow (lean) or high-fat diet (HFD; diet-induced obese (DIO)). RNA sequencing (RNAseq) and reduced representation bisulfite sequencing (RRBS) was performed on brown adipose tissue (BAT) which provide the fundament for the identification of differentially methylated positions (DMP) and regions (DMR) as well as differentially expressed genes (DEG) in three models: COLDlean, comparing 8° versus 30 °C mice on chow diet; COLDDIO, comparing 8° versus 30 °C mice on HFD diet and ΔCOLD, comparing COLDlean versus COLDDIO. Differentially methylated and expressed genes (DMEGs) were identified for all comparisons, based on DMPs within DMRs that significantly correlated with differentially expressed genes (DEGs; the heatmap shows a representative example). DMEGs were further characterized. First, using a published single cell dataset (Shamsi et al., 2023, https://doi.org/10.1038/s42003-023-05140-2), the distribution of DMEGs across cell types was analyzed. For further characterization, DMEGS were categorized into similarly (among COLDlean and COLDDIO) and uniquely (COLDlean, COLDDIOand ΔCOLD) regulated. Pathway enrichment analyses were performed and to identify differentially regulated epigenetic regulators among the DMEGs, the EpiFactor Database (https://epifactors.autosome.org) was used. A TF-binding site motive enrichment analyses (https://jaspar.elixir.no)was applied to add information of most enriched Transcription Factors (TF). Finally, to gain functional insights of DNA methylation changes on selected DMEG candidates, cell cultures of immortalized brown adipocytes and primary brown adipocytes were treated with 5′aza-2′-deoxycytidine (demethylation) or S-adenosylmethionine (upregulation of methylation), following gene expression analyses. The figure was created with BioRender.Image 1

•Obese mice show impaired cold adaptation, with weight loss and persistent hypothermia compared to lean controls.•Cold activates BAT thermogenic genes, but transcriptional responses are quantitatively reduced in obesity.•Epigenetic remodeling of BAT is limited in obesity, with fewer DMEGs and increased gene-body methylation.•Obesity redirects cold-induced pathways toward stress and diabetogenic signaling and away from browning.•Core methylation regulators respond to cold, but adaptive epigenetic reprogramming is constrained in obesity.

Obese mice show impaired cold adaptation, with weight loss and persistent hypothermia compared to lean controls.

Cold activates BAT thermogenic genes, but transcriptional responses are quantitatively reduced in obesity.

Epigenetic remodeling of BAT is limited in obesity, with fewer DMEGs and increased gene-body methylation.

Obesity redirects cold-induced pathways toward stress and diabetogenic signaling and away from browning.

Core methylation regulators respond to cold, but adaptive epigenetic reprogramming is constrained in obesity.