Transcriptional Control of Obesity

虽然有超过20个神经元表达的基因与体重的调节有关，但这些基因响应能量平衡变化而表达的转录和转录后机制仍不清楚。 我实验室的最新证据表明，NhIh 2转录因子在控制体重的下丘脑基因的调节中起着关键作用。 NhIh 2基因敲除小鼠（N2 KO）显示成年期肥胖，NhIh 2在控制食欲的下丘脑区域表达，包括下丘脑的弓状核（ARC）。 在双标记原位杂交研究中，我们发现几乎所有的前阿黑皮素（POMC）神经元在ARC表达NhIh 2。 在这些神经元中，POMC前体蛋白被加工成α-黑素细胞刺激激素（alphaMSH），一种调节食物摄入和能量消耗的肽。 在比较N2 KO和正常小鼠中POMC mRNA和肽水平的研究中，我们发现，虽然两组之间的POMC mRNA水平相同，但两种加工形式的POMC，β-内啡肽（β-END）和促肾上腺皮质激素（ACTH）的水平显着降低，而N2 KO动物中的α MSH水平适度降低。 这些发现表明，N2 KO动物可能具有减少POMC前体加工的缺陷。 事实上，在N2 KO动物中，将POMC切割成生物活性肽激素原转化酶I（PC 1）和II（PC 2）所需的两种加工酶的mRNA水平降低了80%。 因此，PC 1和PC 2可能是NhIh 2转录因子的直接基因调控靶点，并且完全加工的神经肽的减少导致N2 KO动物的肥胖。 此外，我们预测NhIh 2是瘦素信号通路的直接靶点，因为它在含有瘦素受体的POMC神经元中表达，并且在其启动子中含有STAT基序。基于这些初步研究结果，我们提出，正能量平衡增加NhIh 2的表达，这反过来又增加了PC 1和PC 2基因的转录速率。 为了验证这一假设，我们将在前两个特定目标中询问NhIh 2、PC 1和PC 2是否响应于正能量平衡而增加，以及PC 1和PC 2基因的N2 KO是否在N2 KO动物中丢失。 在具体目标3中，我们将切换到NhIh 2启动子的分子研究，以确定AP 1和STAT 3转录因子是否可以结合并反式激活NhIh 2基因。 然后，我们将在具体目标4中询问NhIh 2转录因子是否与活化的STAT 3合作结合并反式激活PC 1启动子。 所提出的实验使用分子和动物为基础的方法来问一个基本的问题，转录和转录后机制控制体重。

Although over 20 neuronally-expressed genes have been implicated in the regulation of body weight, the transcriptional and post-transcriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Recent evidence from my laboratory suggests that the NhIh2 transcription factor plays a key role in the regulation of hypothalamic genes controlling body weight. NhIh2 knockout mice (N2KO) display adult-onset obesity and NhIh2 is expressed in the hypothalamic regions that control appetite-including the arcuate ncuelus (ARC) of the hypothalamus. In dual-label in situ hybridization studies, we found that nearly all pro- opiomelanocortin (POMC) neurons in the ARC expressed NhIh2. In these neurons, the POMC precursor protein is processed to alpha- melanocyte stimulating hormone (alphaMSH), a peptide that regulates food intake and energy expenditure. In studies comparing POMC mRNA and peptide levels in N2KO and normal mice, we found that while POMC mRNA levels were identical between the two groups, levels of two processed forms of POMC, beta-endorphin (betaEND) and adrenocorticotropin hormone (ACTH) were dramatically reduced, while levels of alphaMSH were modestly reduced in the N2KO animals. These findings suggest that N2KO animals could have a defect that reduces processing of the POMC precursor. Indeed, mRNA levels for two of the processing enzymes necessary for cleaving POMC into bioactive peptides prohormone convertases I (PC1) and II (PC2) were reduced up to 80 percent in N2KO animals. Thus, it is likely that PC1 and PC2 are direct gene regulatory targets of the NhIh2 transcription factor and that a reduction in fully processed neuropeptides contributes to obesity in the N2KO animals. In addition, we predict that NhIh2 is a direct target of leptin-signaling pathways, as it is expressed in the leptin-receptor containing POMC neurons and contains STAT motifs in its promoter. Based on these preliminary findings, we propose that positive energy balance increases the expression of NhIh2, which in turn increases the transcription rate of the PC1 and PC2 genes. To test this hypothesis, we will ask in the first two specific aims, if NhIh2, PC1 and PC2 are increased in response to a positive energy balance and if the of the PC1 and PC2 genes is lost in N2KO animals. In specific aim 3, we will switch to a molecular investigation of the NhIh2 promoter to determine if AP1 and STAT3 transcription factors can bind to and transactivate the NhIh2 gene. We will then ask in specific aim 4 if the NhIh2 transcription factor, in cooperation with activated STAT3, binds to and transactivates the PC1 promoter. The proposed experiments use both molecular and animal-based approaches to ask a fundamental question about transcriptional and post- transcriptional mechanisms controlling body weight.