Integrative single-cell characterization of hypothalamus sex-differential and obesity-associated genes and regulatory elements

Abstract

Over 500 noncoding genomic loci are associated with obesity. The majority of these loci reside near genes that are expressed in the hypothalamus in specific neuronal subpopulations that regulate food intake, hindering the ability to identify and functionally characterize them. Here, we carried out integrative single-cell analysis (RNA/ATAC-seq) on both mouse and human male and female hypothalamus to characterize genes and regulatory elements in specific cell subpopulations. Utilizing both transcriptome and regulome data, we identify over 30 different neuronal and non-neuronal cell subpopulations and a shared core of transcription factors that regulate cell cluster-specific genes between mice and humans. We characterize several sex-specific differentially expressed genes and the regulatory elements that control them in specific cell subpopulations. Overlapping cell-specific scATAC peaks with obesity-associated GWAS variants, identifies potential obesity-associated regulatory elements. Using reporter assays and CRISPR editing, we show that many of these sequences, including the top obesity-associated loci (FTO and MC4R), are functional enhancers whose activity is altered due to the obesity-associated variant and regulate known obesity genes. Combined, our work provides a catalog of genes and regulatory elements in hypothalamus cell subpopulations and uses obesity to showcase how integrative single-cell sequencing can identify functional variants associated with hypothalamus-related phenotypes.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3’ UTR ofFAIM2

“…These results may however not accurately reflect regulation of FAIM2 expression in vivo as this gene is primarily expressed in the brain; furthermore, this miRNA product is a passenger strand that is typically found in lower abundance due to degradation after its complementary guide miRNA is loaded into an Argonaute protein during miRNA processing 68 . More recently, others carrying out global analyses have implicated an enhancer in the region harboring rs7132908 with a luciferase reporter assay and found that, in mouse neuronal hypothalamus cells, the obesity risk A allele significantly decreased enhancer activity with a minimal promoter 69 , consistent with our results.…”