The Hypothalamic Epigenetic Landscape in Dietary Obesity

Abstract The hypothalamus in the brain plays a pivotal role in controlling energy balance in vertebrates. Nutritional excess through high‐fat diet (HFD) feeding can dysregulate hypothalamic signaling at multiple levels. Yet, it remains largely unknown in what magnitude HFD feeding may impact epigenetics in this brain region. Here, it is shown that HFD feeding can significantly alter hypothalamic epigenetic events, including posttranslational histone modifications, DNA methylation, and chromatin accessibility. The authors comprehensively analyze the chromatin immunoprecipitation‐sequencing (ChIP‐seq), methylated DNA immunoprecipitation‐sequencing (MeDIP‐seq), single nucleus assay for transposase‐accessible chromatin using sequencing (snATAC‐seq), and RNA‐seq data of the hypothalamus of C57 BL/6 mice fed with a chow or HFD for 1 to 6 months. The chromatins are categorized into 6 states using the obtained ChIP‐seq data for H3K4me3, H3K27ac, H3K9me3, H3K27me3, and H3K36me3. A 1‐month HFD feeding dysregulates histone modifications and DNA methylation more pronouncedly than that of 3‐ or 6‐month. Besides, HFD feeding differentially impacts chromatin accessibility in hypothalamic cells. Thus, the epigenetic landscape is dysregulated in the hypothalamus of dietary obesity mice.


Figure S2 .
Figure S2.Summary of the chromosomal distribution of epigenetic marks.a) Circos plots summarizing the chromosomal distribution of epigenetic marks.The outermost circle depicts the ideograms of each chromosome.The second outermost circle represents gene density, with purple and white indicating high and low density, respectively.Dot plots in other circles display the density of epigenetic marks, from outer to inner: H3K4me3, H3K9me3, H3K27ac, H3K27me3, H3K36me3.b) Relative proportion of annotated genomic elements for each histone modification.

Figure S3 .
Figure S3.Hypothalamic chromatin state following diet treatments.Emission probabilities of the 6-states ChromHMM model were calculated from five histone modifications in the hypothalamus of mice fed with chow for 6 months of HFD for 1, 3, or 6 months.Each row represents a chromatin state, and each column corresponds to a histone modification.The emission parameters were generated from ChIP-seq data for H3K4me3, H3K9me3, H3K27me3, H3K27ac, and H3K36me3, and represent the enrichment possibility, with red and white indicating a high and low possibility, respectively.The heatmap to the right of the emission parameters displays the proportion of the whole genome occupied by each state (Genome %).The heatmap to the right of the genome proportion displays the overlap fold enrichment for various external genomic annotations (GeneAnno).TES, transcription end site.The heatmap to the right of GeneAnno shows the fold enrichment for each state for each 200-bp bin position within 2 kb around a set of transcription start sites (TSSs).A red color corresponds to a greater fold enrichment on a column-specific coloring scale.

Figure S4 .
Figure S4.Representative genes showing the pattern of occupancy in an open region, such as Marcks, and a bivalent region, such as Insm2, in the 1-month HFD (a, b), 3-month HFD (c, d), 6-month chow (e, f), and 6-month HFD datasets (g, h).

Figure S5 .
Figure S5.The pattern of histone modification of a representative gene, Nrxn1, in the five experimental groups.

Figure S6 .
Figure S6.The commonly impacted genes inferred by ChIP-seq datasets in the indicated treatment groups.a) Venn diagram showing the number of imputed upregulated genes whose promoter regions were enriched with H3K4me3 and H3K27ac, but not with H3K9me3 or H3K27me3 ChIP-seq peaks in mouse hypothalamus.b) Venn diagram showing the number of imputed down -regulated genes whose promoter regions were not enriched with H3K4me3 and H3K27ac, but with H3K9me3 or H3K27me3 ChIPseq peaks in the hypothalamus.

Figure S7 .
Figure S7.The overlapped genes whose promoter regions were differentially occupied by peaks of histone marks.a) Venn diagrams showing the overlapping of

Figure S8 .
Figure S8.Trend analysis of histone modification profile.The trend of changes of ChIP-seq peaks for the active histone modification mark H3K4me3 and suppressive histone mark H3K9me3 (a), H3K4me3 and H3K27me3 (b), H3K27ac and H3K27me3 (c), as well as H3K27ac and H3K9me3 (d).The numbers of genes impacted in both modifications are shown (a-d).

Figure S9 .
Figure S9.The proportion of profiles for H3K4me3 and H3K27ac (a), as well as H3K9me3 and H3K27me3 (b).

Figure S10 .
Figure S10.The DNA methylation level in each chromatin state.a-d) The DNA methylation levels in each chromatin state, which is the proportion of the peaks in MeDIPseq over the genomic coverage of each state, are shown for the indicated treatment groups.The line in the boxplot represents the median and the boxes represent the first and third quartiles.The whiskers represent data that are within the 1.5× interquartile range.Data beyond the end of the whiskers are outlying points that are plotted individually.The numbers of overlapped regions between chromatin states and MeDIP-seq peak regions are shown at the bottom.

Figure S11 .
Figure S11.Unsupervised clustering of snATAC-seq data of mouse hypothalamus.a) Unsupervised clustering of the snATAC-seq dataset revealed 21 cell types.b) Dot plot showing the inferred marker gene activities in each cell type.Dot size corresponds to the proportion of cells with detected activity of the indicated gene, and dot density corresponds to average gene activity relative to that in all cell types.

Figure
Figure S13.snATAC-seq data analysis to reveal the commonly impacted genes between two cell types.OPC, oligodendrocyte precursor cell.

Figure S14 .
Figure S14.Representative genes impacted by HFD feeding in respective hypothalamic cell type.Red boxes indicate the significantly impacted genes between dietary treatment groups.Dot size corresponds to the proportion of cells with detected activity of the indicated gene and dot density corresponds to average gene activity relative to that in all cell types.OPC, oligodendrocyte precursor cell.

Figure S15 .
Figure S15.Integrated analysis of RNA-seq and ChIP-seq data.a) Dot plot showing significantly enriched GO terms for the up-or down-regulated genes.The color represents the p value, and the dot size represents the gene ratio.BP, biological process; MF, molecular function.b) Enrichment map networks displaying pathways for differentially