Human hypothalamic neuronal epigenomics and risk for obesity

将体重调节的遗传风险信息转化为分子机制可能会有 对干预和治疗产生重大影响。我们正在寻求识别基因变异和它们的 通过直接影响下丘脑影响肥胖的分子机制，因为下丘脑是大脑的中枢 这调节了能量的动态平衡，现在有相当多的证据表明遗传影响会影响这一点。 大脑区域。然而，大多数与这种常见的慢性疾病相关的遗传基因座 种群位于基因组的非编码区，这表明它们对能量动态平衡有影响 是通过对Regulome的改变来体现的。因此，准确定位人类的因果变异并将 它们与其大脑下游靶点的关系给研究带来了组织获取的挑战，因为 表观遗传控制是特定于物种、组织和背景的。克服有限的人体组织的障碍 Access，我们开发了一种生成人类诱导多能干细胞(IPSC)的强大协议- 衍生的神经元培养，概括了下丘脑弓状核神经元的许多特征 核，包括通过将这个体外模型与体内在下丘脑中起关键作用的事件进行比较 发展。我们将使用这个人体模型和最先进的高通量分析来绘制目前 人类下丘脑神经元在发育的3个阶段(早期， 中期和晚期)和在实验的肥胖条件下。接下来，为了准确地定位功能 通过下丘脑表观基因组影响体重调节的BMI基因座变异 调控，我们将通过检测来确定那些影响染色质可及性和/或靶基因表达的因素 在圣安东尼奥墨西哥裔美国家庭受试者产生的100个IPSC来源的神经细胞系中 研究。同时具有这两种特性的GWA型变异体具有很高的因果关系，对人体有明显的影响 通过染色质结构变化的质量指数。将对其中的一组进行因果关系确定 使用基因组编辑技术(如CRISPR/Cas9)产生等基因人类神经细胞的变体 仅在单个基因座上存在基因型差异的品系。外显子特异性靶基因表达的变化和 染色质状态将在三个发育阶段和每种肥胖条件下进行评估。 与遗传易感性相关的表观遗传机制的发现将转化遗传风险信息和 确定遗传性和饮食诱导性肥胖易感性背后的潜在潜在因素。

Translating information on genetic risk for body weight regulation into molecular mechanisms can have a significant impact on intervention and therapies. We are seeking to identify genetic variation and their molecular mechanisms that influences obesity through direct effects on the hypothalamus as it is the brain hub that regulates energy homeostasis and there is now considerable evidence for genetic influence to impact this brain region. However, the majority of genetic loci associated with this common, chronic disorder in the general population are located in noncoding regions of the genome, suggesting their influence on energy homeostasis is manifested through changes to the regulome. Thus, pinpointing the causal human variants and connecting them to their downstream targets in brain presents challenges of tissue access for study because much epigenetic control is species-, tissue- and context-specific. To overcome the barrier of limited human tissue access, we have developed a robust protocol for generating human induced pluripotent stem cell (iPSC)- derived neuronal cultures that recapitulate many of the features of hypothalamic neurons from the arcuate nucleus, including by benchmarking this in vitro model to in vivo events that are pivotal in hypothalamic development. We will use this human model and state of the art high throughput assays to map the currently uncharted regulatory landscape of the human hypothalamic neurons across 3 stages in development (early, mid, and late) and under experimental obesogenic conditions. Next, in order to precisely pinpoint the functional variants in BMI GWAS loci that have influence on body weight regulation through hypothalamic epigenomic regulation, we will identify those that influence chromatin accessibility and/or target gene expression by assay in 100 iPSC-derived neuronal lines generated from subjects of the San Antonio Mexican American Family Studies. GWAS variants with both properties have high potential to be causal and manifest effects on body mass index through changes in chromatin structure. Causal determination will be made for a set of these variants using genome editing techniques such as CRISPR/Cas9 to generate isogenic human neuronal cell lines that differ by genotype only at the single locus. Changes in exon-specific target gene expression and chromatin status will be assessed across the 3 developmental stages and under each obesogenic condition. Discovery of epigenetic mechanisms connected to genetic liability will translate the genetic risk information and identify potential underlying factors behind both heritable and diet-induced obesity susceptibility.