Abstract
The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.
Highlights
We utilized an established protocol to derive ARC hypothalamic neurons (HN)-like neurons that generate predominantly neurons that express markers such as NPY and POMC (80–95%)[28], and collected cells at three stages of differentiation: pluripotent ESCs, NKX2-1+ hypothalamic progenitors (HPs), and HNs generated from a human ESC line (H9) derived from one female donor
Twelve days were selected as the HP timepoint due to high expression of the neuroprogenitor marker Nestin and the low expression of the neuronal marker Tubulin Beta 3 (TUBB3), while day 27 was chosen as HN timepoint due to high TUBB3 and POMC expression[28]
We grouped the open chromatin regions (OCRs) into three categories (Fig. 2c): (1) OCRs located within promoter regions annotated as “promoter OCRs”; (2) OCRs with direct promoter contacts determined by Capture C, annotated as “promoter-interacting region (PIR)OCRs”; and (3) OCRs that could not be assigned to a gene because they did not fit either criterion, annotated as “non-PIROCRs”. Because they could be annotated to a gene, we considered the sets of 50,952 promoter OCRs and 87,170 PIR-OCRs as putative cis-acting regulatory elements (cREs)
Summary
Given its inaccessibility, studying human hypothalamic gene regulation has proven challenging To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. We integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. The hypothalamus is a critical regulator of many physiological functions, including energy homeostasis, reproduction, sleep, and stress[1] This brain region senses neural and physiological signals, which triggers distinct populations of neurons to release neurotransmitters and peptide neuromodulators to signal the autonomic nervous and endocrine systems[1–3]. Assaying relevant cell types in this regard is critical, as promoter architecture varies across cellular identity and developmental stage[17,24,25]
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