Adipogenic Transcriptional Regulation Mediated by PARP7 Through NAD+ Sensing Mechanism [RNA-Seq]

The process of differentiating from a preadipocyte into a mature, fat storing, adipocyte (adipogenesis) is a complex and tightly regulated process vital to human health. The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined. Here we have identified a mono(ADP-ribosyl)transferase (MART), PARP7, that plays a pivotal role in adipogenesis. ADP-ribosylation – the process whereby specific members of the poly(ADP-ribosyl)polymerase (PARP) family facilitate the covalent transfer of ADP-ribose moieties from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery. Herein we have found that PARP7 is required for modulation of the adipogenic transcriptional program during adipogenesis, and that the loss of PARP7 results in a decrease of the adipogenic process. Contrary to previous studies, the ability to modulate the adipogenic transcriptional program is independent of PARP7 catalytic activity. However, the catalytic activity of PARP7 plays a vital role in regulating protein stability, in a time and space specific manner, which is required for modulation of the adipogenic transcriptional program. This study has found a novel pathway which utilizes NAD+ compartmentalization to stabilize PARP7 at a specific time during the adipogenic process allowing for the regulation of adipogenic genes, through modulation of the well-known adipogenic transcription factor C/EBPß, in a time sensitive manner. Therefore, acting as another check and balance to the adipogenic differentiation process. Overall design: 3T3-L1 cells were seeded in 6-well plates and treated as described above. The cells and tissues were collected, and total RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer's protocols. Total RNA was reverse transcribed using oligo (dT) primers and MMLV reverse transcriptase (Promega) to generate cDNA. The cDNA samples were subjected to qPCR using gene-specific primers, as described below. For the reverse transcription quantitative real-time PCR (RT-qPCR) analyses, “relative expression” was determined in comparison to a value of the control sample.

从前脂肪细胞分化为成熟储脂脂肪细胞的过程（即脂肪生成，adipogenesis）是一个复杂且受严格调控的过程，对人体健康至关重要。目前调控脂肪生成的分子机制尚未完全阐明，尽管学界已明确其信号通路与调控通路的关键环节。本研究鉴定出一种单ADP核糖基转移酶（mono(ADP-ribosyl)transferase, MART）PARP7，其在脂肪生成过程中发挥关键作用。ADP核糖基化修饰——即多聚ADP核糖聚合酶（poly(ADP-ribosyl)polymerase, PARP）家族的特定成员介导NAD+上的ADP核糖基团共价转移至底物蛋白的过程——已被证实可调控脂肪生成调控机制的多个组分。本研究发现，PARP7是脂肪生成过程中调控脂肪生成转录程序所必需的，且PARP7的缺失会削弱脂肪生成过程。与既往研究相悖的是，PARP7调控脂肪生成转录程序的能力并不依赖于其催化活性。然而，PARP7的催化活性以时空特异性的方式在调控蛋白质稳定性方面发挥关键作用，而这一过程是调控脂肪生成转录程序所必需的。本研究揭示了一条全新的通路：该通路利用NAD+区室化作用在脂肪生成过程的特定时段稳定PARP7，进而通过以时间敏感性方式调控经典脂肪生成转录因子C/EBPβ来调控脂肪生成相关基因，因此可作为脂肪生成分化过程的又一制衡机制。总体实验设计：将3T3-L1细胞接种于6孔板中，按前述方法处理。收集细胞与组织，依照生产商说明书使用Trizol试剂（Invitrogen）提取总RNA。使用寡聚(dT)引物与MMLV反转录酶（Promega）将总RNA反转录为cDNA。依照下述方法，使用基因特异性引物对cDNA样本进行qPCR检测。对于反转录定量实时PCR（RT-qPCR）分析，"相对表达量"以对照样本的数值为基准进行计算。