The reducing equivalent NADPH dictates histone acetylation via direct inactivation of HDAC3

NADPH has been long well-recognized as a key cofactor for antioxidant defense and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We found that reduction of cellular NADPH levels by silencing malic enzyme (ME) or G6PD impairs global histone acetylation and transcription in both adipocytes and tumor cells. These effects can be reversed by supplementation of exogenous NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically,NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator Ncor2 (SMRT) or Ncor1, thereby impairs HDAC3 activation. Interestingly, it appears that NADPH and Ins(1,4,5,6)P4 bind to the same domains on HDAC3, and NADPH has relatively higher affinity towards HDAC3. Thus, while Ins(1,4,5,6)P4 acts as an ‘intermolecular glue’, NADPH may function as a HDAC3-Ncor assembly inhibitor. Collectively, our findings uncovered a previous unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3. Briefly, 3T3-L1 preadipocytes cells were transfected with control, ME1, or ME2 siRNAs as indicated and two days later induced to differentiate into adipocytes. After another 7 days, cells were washed in PBS buffer and subsequently cross-linked with 1% formaldehyde solution at room temperature (RT) for 15 min. After washing in cold PBS, cross-linking reaction was stopped by the addition of 0.125M glycine with gently shaking for 5min. Cells were then centrifuged and lysed in 300 μL SDS lysis buffer (50mM Tris-HCl pH 8.0, 10mM EDTA pH 8.0 , 1% SDS, protease inhibitors) for 15 minutes on ice. Cell lysates were sonicated to generate DNA fragments approximately 200-1000 bp and subjected to immunoprecipitation with indicated antibodies against Ac-H2B and Ac-H3. Immunoprecipitations were carried out overnight, samples were then washed with low salt buffer, high salt buffer, LiCl salt buffer and TE buffer sequentially. Histone-DNA complexes were eluted using elution buffer (1%SDS, 100 mM NaHCO3), incubated at 65ºC for overnight in the presence of 0.54 M NaCl. The next day, DNase-free RNase(10 mg/ml)was added into samples and incubated for 1 hour at 37℃.Samples were then incubated with Proteinase K for another 1 hour at 55ºC.Finally, DNA were purified using Qiaex II beads (Qiagen) and analyzed by quantitative PCR (qPCR). For ChIP-seq analysis, briefly, 3T3-L1 cells were treated with siRNA for 2 days and confluent cells were induced to differentiate into adipocytes. Cell were cross-linked, and the cell lysates were sonicated to generate DNA fragments and subjected to immunoprecipitation using Ac-H2B, Ac-H3 or HDAC3 antibodies as described above. Bound DNA fragments were eluted for ChIP-seq library construction and sequencing.

烟酰胺腺嘌呤二核苷酸磷酸（NADPH）长期以来被公认为抗氧化防御与还原性生物合成的关键辅因子。本研究报道了NADPH不依赖代谢途径的全新功能：调控表观遗传状态与转录。我们发现，通过沉默苹果酸酶（ME）或葡萄糖-6-磷酸脱氢酶（G6PD）降低细胞内NADPH水平，会损伤脂肪细胞与肿瘤细胞的全局组蛋白乙酰化水平及转录活性。该效应可通过补充外源性NADPH或抑制组蛋白去乙酰化酶3（HDAC3）逆转。机制层面，NADPH可直接与HDAC3结合，阻断HDAC3与其辅激活因子核受体辅阻遏因子2（SMRT，又称Ncor2）或核受体辅阻遏因子1（Ncor1）的结合，从而削弱HDAC3的激活活性。有趣的是，肌醇四磷酸（Ins(1,4,5,6)P4）与NADPH结合于HDAC3的同一结构域，且NADPH对HDAC3的亲和力相对更高。因此，尽管Ins(1,4,5,6)P4作为“分子胶水”发挥作用，NADPH或可作为HDAC3-Ncor复合物组装抑制剂。综上，本研究发现了NADPH此前未被报道的、不依赖代谢途径的全新功能：通过作为HDAC3的内源性抑制剂，调控表观遗传改变与基因表达。

简要而言，按实验指示将3T3-L1前脂肪细胞转染对照、ME1或ME2小干扰RNA（siRNA），两天后诱导其分化为脂肪细胞。再培养7天后，用磷酸盐缓冲液（PBS）洗涤细胞，随后于室温（RT）用1%甲醛溶液交联15分钟。用预冷PBS洗涤后，加入0.125M甘氨酸终止交联反应，轻柔摇晃5分钟。离心收集细胞，于冰上用300 μL SDS裂解缓冲液（50mM Tris-HCl pH 8.0、10mM EDTA pH 8.0、1% SDS、蛋白酶抑制剂）裂解15分钟。将细胞裂解液超声破碎，得到约200-1000 bp的DNA片段，随后使用针对乙酰化组蛋白H2B（Ac-H2B）与乙酰化组蛋白H3（Ac-H3）的指定抗体进行免疫沉淀。免疫沉淀反应于过夜进行，随后依次用低盐缓冲液、高盐缓冲液、氯化锂缓冲液及TE缓冲液洗涤样品。使用洗脱缓冲液（1% SDS、100 mM NaHCO3）洗脱组蛋白-DNA复合物，于65℃下在0.54 M NaCl存在的条件下孵育过夜。次日，向样品中加入10 mg/ml无DNase核糖核酸酶（DNase-free RNase），于37℃孵育1小时。随后加入蛋白酶K，于55℃继续孵育1小时。最终使用Qiaex II磁珠（Qiagen）纯化DNA，并通过定量聚合酶链式反应（qPCR）进行分析。

对于染色质免疫共沉淀测序（ChIP-seq）分析，简要流程如下：将3T3-L1细胞用siRNA处理2天，待细胞汇合后诱导其分化为脂肪细胞。按前述方法进行细胞交联、裂解液超声破碎得到DNA片段，并使用针对Ac-H2B、Ac-H3或HDAC3的抗体进行免疫沉淀。洗脱结合的DNA片段用于ChIP-seq文库构建与测序。