Molecular regulation of adipocyte progenitor quiescence and metabolic adaptation to obesity

脂肪组织的扩张是由新的脂肪细胞形成(导致增生性生长)和现有脂肪细胞肥大的组合发生的，它们的相对贡献可能会影响全身代谢健康。脂肪细胞发育失败所致的先天性脂肪营养不良与严重的胰岛素抵抗有关。发育基因的遗传变异引起的脂肪生成的更细微的损害也容易导致胰岛素抵抗和2型糖尿病。这些例子反映了更大的数据体，为代谢健康依赖于功能脂肪细胞群体的假设提供了概念基础。通过利用稳定的同位素示踪剂和高精度的质谱学方法，我们现在也发现了脂肪生成与年龄相关的下降。因此，脂肪祖细胞--新脂肪细胞的细胞来源--必须在成熟的脂肪组织中处于相对静止的状态。尽管脂肪形成的下游驱动因素已经被很好地描述，但在体内，维持脂肪前体细胞静止或其近端向增殖和分化转变的机制仍很不清楚。为了解决这一知识差距，我们利用转录数据以及新分离的初级脂肪细胞前体细胞中可获得的染色质的基因组比例图来模拟导致静止状态的转录因子网络。我们的数据表明，孤儿核受体NR4A1是一个关键的调控节点。体外和体内功能基因组学研究为我们的以NR4A1为中心的假说提供了额外的支持：NR4A1调控着一个转录程序，该程序在脂肪细胞前体细胞中建立了一种有害的代谢静止状态。这些数据还为两个相互关联的具体目标提供了理论基础。在目标1中，我们将检验NR4A1是脂肪细胞前体细胞静止的核心转录调节因子的假设。我们将使用报告分析、CRISPRi和CRISPR/Cas9来功能性地询问关键成脂转录因子上的NR4A1-DNA调控节点。在目标2中，我们将在基因组水平上对脂肪细胞前体细胞中NR4A1相关的染色质调控图景进行无偏见的询问。我们将绘制NR4A1与DNA的相互作用图，并用规范的顺式调节标记对NR4A1结合进行共定位。然后，我们将使用低输入基因组学方法，结合NR4A1功能的获得或丧失，来绘制依赖于NR4A1的染色质调控版图和转录输出。总体而言，通过有针对性的功能基因组学实验和无偏见的基因组规模分析，我们将测试我们的NR4A1模型作为控制祖细胞静止的基因程序的主调节器。确定使常驻脂肪细胞前体细胞静止的基本分子机制有望识别脂肪生成和代谢健康脂肪的分子障碍。

Adipose tissue expansion occurs by a combination of new adipocyte formation (resulting in hyperplastic growth) and hypertrophy of existing adipocytes, the relative contributions of which may impact systemic metabolic health. Congenital lipodystrophy due to failed adipocyte development is associated with severe insulin resistance. More subtle impairments in adipogenesis arising from genetic variants in developmental genes also predispose to insulin resistance and Type 2 Diabetes Mellitus. These examples reflect a larger body of data providing a conceptual basis for the hypothesis that metabolic health is dependent on a functional adipocyte population. By leveraging stable isotope tracers with high precision mass spectrometry methods, we have now also identified an age-dependent decline in adipogenesis. Therefore, adipocyte progenitors—the cellular source of new adipocytes—must reside in a state of relative quiescence in mature adipose tissue. Although downstream drivers of adipogenesis are well described, the mechanisms that maintain quiescence of adipocyte progenitors or their proximal transition to proliferate and differentiate, in vivo, remain largely unknown. To address this knowledge gap, we have leveraged transcriptomic data together with genome scale maps of accessible chromatin in freshly isolated primary adipocyte progenitors to model the network of transcription factors responsible for the quiescent state. Our data implicate the orphan nuclear receptor, NR4A1, as a key regulatory node. In vitro and in vivo functional genomics studies have provided additional support for our NR4A1-centric hypothesis: that NR4A1 regulates a transcriptional program that establishes a metabolically deleterious state of quiescence in adipocyte progenitors. These data also provide rationale for two interrelated Specific Aims. In Aim 1, we will test the hypothesis that NR4A1 is a core transcriptional regulator of adipocyte progenitor quiescence. We will use reporter assays, CRISPRi and CRISPR/Cas9 to functionally interrogate NR4A1-DNA regulatory nodes at key adipogenic transcription factors. In Aim 2, we will perform an unbiased interrogation of the NR4A1-depedent chromatin regulatory landscape in adipocyte progenitor cells at genome scale. We will map NR4A1-DNA interactions and co-localize NR4A1 binding with canonical cis-regulatory marks. We will then use low-input genomics methods coupled with NR4A1 gain or loss of function to map the NR4A1 dependent chromatin regulatory landscape and transcriptional output. Collectively, through targeted functional genomics experiments and unbiased genome scale analyses we will test our model of NR4A1 as a master regulator of a gene program controlling progenitor quiescence. Defining fundamental molecular mechanisms that render resident adipocyte progenitor cells quiescent holds promise to identify molecular barriers to adipogenesis and metabolically healthy fat.