Identify transcriptional regulators of Mc4r in the hypothalamus [scRNA-Seq]

Human MC4R mutations can increase or decrease obesity risk. Other than intracellular signaling, MC4R function is also influenced by its levels. However, the genetic programs that govern MC4R transcription in the brain remain largely unknown. Moreover, it is unclear whether human genetic variants exist that affect Mc4r expression and obesity risk. Here, we identify the homeodomain transcription factor Otp as one regulator of Mc4r expression in a specific subset of hypothalamic neurons. Selective loss of Otp in these neurons during development or adulthood results in reduced Mc4r expression and excessive body weight gain. Moreover, OTP interacts with upstream regulatory sequences of Mc4r to modulate its transcription. To investigate the changes of PVH neuron populations and mRNA expressions in OtpQ153R mice, we carried out single-nucleus RNA sequencing (snRNA-seq) in the PVH of Sim1-Cre; Sun1-GFP and Sim1-Cre; Sun-GFP; Otpfl/+ and Sim1-Cre; Sun1-GFP; OtpQ153R/+ mice. Following PVH collection at four weeks old, we used fluorescence-activated cell sorting (FACS) to enrich GFP-positive nuclei from seven Sim1-Cre; Sun1-GFP mice and four Sim1-Cre; Sun-GFP; Otpfl/+ mice and six Sim1-Cre; Sun1-GFP; OtpQ153R/+ mice.人类MC4R基因突变可升高或降低肥胖患病风险。除细胞内信号转导外，MC4R的功能还受其表达水平的调控。然而，调控大脑内MC4R转录的遗传程序仍不完全明确。此外，目前尚不清楚是否存在影响MC4R表达与肥胖风险的人类遗传变异体。本研究鉴定出同源域转录因子Otp是下丘脑神经元特定亚群中MC4R表达的调控因子之一。在发育阶段或成年期，这些神经元中Otp的特异性缺失会导致MC4R表达降低以及体重过度增加。此外，OTP可与MC4R的上游调控序列结合以调控其转录。为探究OtpQ153R小鼠的PVH（室旁下丘脑核，paraventricular hypothalamic nucleus）神经元群体与mRNA表达变化，我们对Sim1-Cre; Sun1-GFP、Sim1-Cre; Sun-GFP; Otpfl/+以及Sim1-Cre; Sun1-GFP; OtpQ153R/+小鼠的PVH脑组织开展了单细胞核RNA测序（snRNA-seq）。于小鼠4周龄时采集PVH脑组织后，我们通过荧光激活细胞分选（FACS）分别富集了7只Sim1-Cre; Sun1-GFP小鼠、4只Sim1-Cre; Sun-GFP; Otpfl/+小鼠以及6只Sim1-Cre; Sun1-GFP; OtpQ153R/+小鼠的GFP阳性细胞核。