Intermediary Metabolism, Histone Acetylation, and Transcriptional Regulation

描述（由申请人提供）：我们实验室的长期目标是了解中间代谢如何影响组蛋白乙酰化和转录调控，并利用这些信息开发癌症治疗的新策略。组蛋白乙酰化是一种 影响染色质结构并调节多种细胞功能的动态修饰，如基因表达、DNA修复和细胞增殖。组蛋白乙酰转移酶（HAT）和组蛋白脱乙酰酶（HDAC）平衡作用的扰动改变了参与细胞生长的基因的表达模式，导致肿瘤发生。另一方面，许多HDAC抑制剂重新激活在癌症中沉默的多个基因的转录，从而具有抗癌活性。组蛋白乙酰化依赖于中间代谢，用于在核胞质隔室中提供乙酰辅酶A。在芽殖酵母中，乙酰辅酶A通过糖酵解途径产生。我们以前的研究表明，通过糖酵解途径合成乙酰辅酶A，随后组蛋白乙酰化，在磷脂酶C（PLC 1）突变的细胞中显着减少。由于核胞质乙酰辅酶A也用于脂肪酸的从头合成，组蛋白乙酰化和脂肪酸的合成竞争相同的乙酰辅酶A库。脂肪酸从头合成中的第一个和限速反应是乙酰辅酶A羧化形成丙二酰辅酶A，由乙酰辅酶A羧化酶（ACC）催化。我们已经表明，ACC在酵母细胞中的减弱表达导致组蛋白乙酰化增加和转录调控改变，暗示ACC作为中间代谢和组蛋白乙酰化之间的调节联系。基于我们的初步数据，这一提议的中心假设是葡萄糖摄取、糖酵解和ACC的调节影响乙酰辅酶A的稳态，从而影响组蛋白乙酰化和转录调节。由于酵母细胞中的直系同源物Snf 1和哺乳动物细胞中的AMP活化蛋白激酶（AMPK）磷酸化并抑制ACC，因此本提案的目的1解决了Snf 1和AMPK是否分别调节酵母和哺乳动物细胞中的组蛋白乙酰化。我们还将确定是否ACC的抑制或AMPK在人乳腺癌MCF 7细胞中的激活导致组蛋白的乙酰化增加，改变转录调控，并增加细胞凋亡。在目的2中，我们将测试的假设，组蛋白低乙酰化在plc 1细胞是由于葡萄糖转运和代谢的缺陷所造成的失败，降解转录抑制因子Mth 1的泛素-蛋白酶体途径。本提案中提出的方法是基于先前未被充分认识的中间代谢和组蛋白乙酰化之间的关系，并将测试一种新的概念，用于开发癌症治疗，旨在将乙酰辅酶A从脂质合成重定向到组蛋白乙酰化，从而改变转录调控并诱导细胞凋亡。此外，该项目将为本科生和研究生提供一个良好的研究环境，因为他们准备在生物医学研究的职业生涯。

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand how intermediary metabolism affects histone acetylation and transcriptional regulation and to utilize this information to develop novel strategies for cancer treatment. Histone acetylation is a dynamic modification that affects chromatin structure and regulates diverse cellular functions, such as gene expression, DNA repair, and cell proliferation. Perturbation of the balanced action of histone acetyltransferases (HATs) and histone deacetylases (HDACs) alters the expression pattern of genes involved in cellular growth, resulting in tumorigenesis. On the other hand, many HDAC inhibitors reactivate the transcription of multiple genes that are silenced in cancer and thus possess anti-cancer activity. Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment. In budding yeast, acetyl-CoA is generated by the glycolytic pathway. Our previous studies revealed that the synthesis of acetyl-CoA via the glycolytic pathway, and subsequently histone acetylation, are significantly reduced in cells with mutation of phospholipase C (PLC1). Since nucleocytosolic acetyl-CoA is also used for de novo synthesis of fatty acids, histone acetylation and the synthesis of fatty acids compete for the same acetyl-CoA pool. The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC). We have shown that attenuated expression of ACC in yeast cells results in increased histone acetylation and altered transcriptional regulation, implicating ACC as a regulatory link between intermediary metabolism and histone acetylation. The central hypothesis of this proposal, based on our preliminary data, is that regulation of glucose uptake, glycolysis, and ACC affect homeostasis of acetyl-CoA and consequently histone acetylation and transcriptional regulation. Since orthologs Snf1 in yeast cells and AMP-activated protein kinase (AMPK) in mammalian cells phosphorylate and inhibit ACC, the Aim 1 of this proposal addresses whether Snf1 and AMPK regulate histone acetylation in yeast and mammalian cells, respectively. We will also determine whether the inhibition of ACC or the activation of AMPK in human breast cancer MCF7 cells results in increased acetylation of histones, altered transcriptional regulation, and increased apoptosis. In Aim 2, we will test the hypothesis that the histone hypoacetylation in the plc1 cells is due to the defect in glucose transport and metabolism caused by the failure to degrade transcriptional repressor Mth1 by the ubiquitin-proteasome pathway. The approach presented in this proposal is based on the previously underappreciated relationship between intermediary metabolism and histone acetylation and will test a novel concept for the development of cancer therapy aimed at redirecting acetyl-CoA from lipid synthesis to histone acetylation, thus altering transcriptional regulation and inducing apoptosis. In addition, this project will provide an excellent research environment for undergraduate and graduate students, as they prepare for careers in biomedical research.