Functional Definition of MYC Acetylation

MYC是一种转录因子，靶向基因组的很大一部分(~15%)，在正常细胞中受到严格调控，对大多数动物的正常发育和动态平衡是必不可少的。去调控的MYC表达(例如，过度表达)在小鼠中是致癌的，并与人类的大多数类型的癌症有关。MYC的解除调控可能通过改变c-myc基因本身(例如，易位和扩增)或通过异常激活上游信号通路来激活c-myc基因的表达和/或通过翻译后修饰(PTM)增加MYC蛋白的稳定性。然而，MYC的PTMS的特征很差，它们在MYC的多种细胞和分子活动中的作用仍然不清楚，也存在争议。直到最近，只有少数残基的磷酸化和O-糖基化是报道的影响蛋白酶体泛化和降解MYC的PTMS。我们实验室最近发现了MYC的新的PTMS，并发现MYC在几个赖氨酸(K)残基上被具有组蛋白乙酰转移酶(HAT)活性的转录共激活物(即GCN5和p300)乙酰化。本研究的总体目标是提供确凿的证据，证明哺乳动物细胞中天然的MYC是通过乙酰化来调节的，并确定特定残基上MYC乙酰化的细胞和分子功能。 目的：利用乙酰化位点特异性抗体(MYC-K148Ac、MYC-K157Ac、MYC-K323Ac)在哺乳动物细胞中建立和鉴定天然MYC的乙酰化，目前尚未证实内源性MYC在任何细胞类型中发生乙酰化。我将使用能识别人类MYC乙酰化残基K148、K157和K323的新抗体来分析几个未转化和转化/癌细胞系中这些不同部位的MYC乙酰化情况。 目的：为确定MYC乙酰化残基K148(149)和K157(158)的功能，检测MYC乙酰化残基K148和K157(与小鼠MYC K149和K158同源的人类坐标)在MYC诱导的细胞增殖、转化和凋亡中的作用。 目的3：建立和确定MYC乙酰化在靶基因调控中的作用(S)。通过对初步实验(如Nrf1)和目标2中确定的受R158(或R149)替换影响的MYC靶基因进行分析，以探讨K158(或K149)位乙酰化在调节MYC转录功能中的可能作用。将使用各种方法，包括染色质免疫沉淀(ChIP)分析、RNA干扰(RNAi)和报告基因分析。

MYC is a transcription factor that targets a large fraction (~15%) of the genome and is tightly regulated in normal cells and essential for normal development and homeostasis in most animals. Deregulated MYC expression (e.g., overexpression) is tumorigenic in mice and associated with most types of cancer in humans. MYC deregulation may occur via alterations of the c-myc gene itself (e.g., translocation and amplification) or via aberrant activation of upstream signaling pathways that activate c-myc gene expression and/or increase MYC protein stability via post-translational modifications (PTMs). The PTMs of MYC are, however, poorly characterized and their roles in the multiple cellular and molecular activities of MYC are still poorly defined and controversial. Until recently phosphorylation and O-glycosylation of a few residues were the only PTMs reported, which affect MYC ubiqitination and degradation by the proteasome. Our lab recently uncovered novel PTMs of MYC and found that MYC is acetylated at several lysine (K) residues by transcription coactivators with histone acetyltransferase (HAT) activity (i.e., GCN5 and p300). The general objective of my research is to provide definitive evidence that native MYC in mammalian cells is regulated via acetylation and to define the cellular and molecular functions of MYC acetylation at specific residues. AIM 1: To establish and characterize acetylation of native MYC in mammalian cells using acetylated site-specific antibodies (MYC-K148Ac, MYC-K157Ac, MYC-K323Ac) Acetylation of endogenous MYC has yet to be demonstrated in any cell type. I will use new antibodies that recognize specifically human MYC acetylated residues K148, K157, and K323 to profile acetylation of MYC at these different sites in several non-transformed and transformed/cancer cell lines. AIM 2: To identify functions of MYC acetylated residues K148(149) and K157(158) I will test the role of MYC acetylated residues K148 and K157 (human coordinates homologous to mouse MYC K149 and K158) in MYC-induced cell proliferation, transformation and apoptosis. AIM 3: To establish and define the role(s) of MYC acetylation in target gene regulation Selected MYC target genes affected by the R158 (or R149) substitution identified in preliminary experiments (e.g. Nrf1) and in Aim 2 will be analyzed to tes the possible role of acetylation at K158 (or K149) in regulating MYC transcription functions. Various methods, including chromatin immunoprecipitation (ChIP) assays, RNA interference (RNAi), and reporter gene assays will be used.