Regulation of one carbon metabolism and epigenetics by SIRT5

依赖NAD的sirtuin家族脱酰酶调节细胞和生物体内平衡的不同方面。 其中，SIRT5仍然是一个有点神秘的蛋白质。SIRT5主要是一种线粒体sirtuin 具有非典型的催化性能，从赖氨酸中去除琥珀酸基、丙二酸基和戊二酸基 它的底物蛋白质。尽管SIRT5是针对这些翻译后基因的主要细胞活动 修饰(PTM)，细胞和整个动物中的SIRT5缺乏只会引起非常轻微的表型， 尤其是在基本条件下。因此，SIRT5及其靶标PTMS的生理功能仍然存在 有点神秘。使用shRNA和基于CRISPR/Cas9的方法，我们发现sirtuin SIRT5是 对特定癌症类型的生存至关重要，包括黑色素瘤和尤文肉瘤(EWS)。整体 转录组分析表明SIRT5影响与生存和应激相关的基因的表达 反应包括黑色素瘤系基因MITF和FOXO3a转录因子。通过代谢物 通过分析，我们发现了SIRT5缺失导致的一个碳代谢(1 Cm)的缺陷。1厘米 由一系列复杂的生化反应组成，产生通用的甲基供体S- 腺苷蛋氨酸(SAM)，组蛋白和DNA甲基化的共同底物。在黑色素瘤和EWS细胞中 缺乏SIRT5，H3K4me3水平降低，通过用SIRT5小分子处理而概括的一种表型 我们最近开发的分子降解剂。值得注意的是，SAM重组完全拯救了 黑色素瘤中与SIRT5缺失相关的致死性。长期目标是阐明SIRT5的作用 来维持细胞内环境的稳定。这项建议的目的是阐明SIRT5在 调控1 cM和核基因表达。中心假说是SIRT5调节 1 cm的酶促进SAM的生成、H3K4的三甲基化和适当的基因表达。其基本原理是 1 cm是细胞SAM和其他关键细胞代谢物的来源，因此表征了 1 cm调控的新机制具有重要的生物学意义。这项工作将在 三个具体目标的背景。首先，我们将机械地阐明SIRT5在调节1 cM中的作用。 使用代谢物跟踪、生物化学和其他补充方法，最初专注于 Mthfd1酶作为SIRT5的候选靶标。第二，H3K4me3在调节H3K4me3的作用 SIRT5对基因表达的影响将利用定向芯片和全局芯片-SEQ来阐明。第三，政府的作用 SIRT5在调节H3K4me3水平和1C应激反应中的存活率将表现为正常 细胞和老鼠。这一应用是创新的，因为目前还没有文献将SIRT5与1 cm联系起来，而且大多数 文献中描述的SIRT5缺失的表型非常有限。这项工作意义重大， 由于阐明SIRT5在调节1 cm中的功能将识别1 CM生物学的新方面，以及 潜在地提供了新的治疗机会。

NAD+-dependent sirtuin-family deacylases regulate diverse aspects of cellular and organismal homeostasis. Among these, SIRT5 has remained a somewhat enigmatic protein. SIRT5 is a primarily mitochondrial sirtuin that possesses atypical catalytic properties, removing succinyl, malonyl, and glutaryl groups from lysines on its substrate proteins. Although SIRT5 is the dominant cellular activity that targets these post-translational modifications (PTMs), SIRT5 deficiency in cells and whole animals provokes only very mild phenotypes, particularly under basal conditions. Thus, physiologic functions of SIRT5 and its target PTMs have remained somewhat mysterious. Using shRNA and CRISPR/Cas9-based approaches, we find that the sirtuin SIRT5 is critical for survival of specific cancer types, including melanoma and Ewing sarcoma (EWS). Whole transcriptome profiling demonstrates that SIRT5 affects expression of genes integral to survival and stress responses, including the melanoma lineage gene MITF and the FOXO3A transcription factor. Via metabolite profiling, we have identified defects in one carbon metabolism (1CM) conferred by SIRT5 depletion. 1CM consists of a complex set of biochemical reactions required for generation of the universal methyl donor S- adenosyl methionine (SAM), the co-substrate for histone and DNA methylation. In melanoma and EWS cells depleted for SIRT5, H3K4me3 levels are reduced, a phenotype recapitulated by treatment with a SIRT5 small molecule degrader that we recently developed. Remarkably, SAM reconstitution completely rescues the lethality associated with SIRT5 depletion in melanoma. The long-term goal is to elucidate the roles of SIRT5 in maintaining cellular homeostasis. The objective of this proposal is to elucidate roles for SIRT5 in regulating 1CM and nuclear gene expression. The central hypothesis is that SIRT5 regulates activities of 1CM enzymes to promote SAM generation, H3K4 trimethylation and proper gene expression. The rationale is that 1CM is the source of cellular SAM and other key cellular metabolites, and hence characterization of new mechanisms of 1CM regulation is of fundamental biological importance. The work will take place in the context of three Specific Aims. First, roles of SIRT5 in regulating 1CM will be elucidated mechanistically, using metabolite tracing, biochemistry, and other complementary approaches, focusing initially on the MTHFD1L enzyme as a candidate SIRT5 target. Second, the role of H3K4me3 in mediating the effects of SIRT5 on gene expression will be elucidated, using directed ChIP and global ChIP-seq. Third, the role of SIRT5 in regulating H3K4me3 levels and survival in response to 1C stress will be characterized in normal cells and mice. The application is innovative, in that no literature currently links SIRT5 to 1CM, and most phenotypes of SIRT5 depletion described in the literature are remarkably modest. The work is significant, as elucidation of SIRT5 functions in regulating 1CM will identify new aspects of 1CM biology, as well as potentially illuminating new therapeutic opportunities.