The structural role of ING proteins in chromatin remodeling.

ING 蛋白在染色质重塑中的结构作用。

• 批准号: 7405637
• 负责人: Karen Champagne Glass
• 金额: $ 4.96万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405637
• 关键词: Affinity; Aging; Apoptosis; Binding; Binding Sites; Biological Assay; Cells; Chromatin; Complex; DNA; DNA Repair; DNA Structure; Data; Discrimination; Disease; Dissociation; Enzymes; Epigenetic Process; Family; Fingers; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Genomics; Growth; Growth Inhibitors; Histone Code; Histone Deacetylase; Histone H3; Histones; ING1 gene; In Vitro; Investigation; Link; Lysine; Malignant Neoplasms; Measures; Methylation; Modification; Molecular; Mono-S; Mutate; Mutation; NMR Spectroscopy; Nuclear; Nuclear Magnetic Resonance; PHD Finger; Peptides; Physiological; Play; Process; Protein Family; Proteins; Reading; Regulation; Resolution; Role; Specificity; Structure; Tail; Techniques; Testing; Thinking; Transact; Tryptophan; Tumor Suppressor Proteins; X-Ray Crystallography; base; biological adaptation to stress; cell growth regulation; chromatin immunoprecipitation; chromatin remodeling; histone acetyltransferase; in vivo; insight; member; mutant; novel; research study; senescence; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): Chromatin dynamics regulate diverse nuclear processes that influence cellular viability and tumorigenesis. Different histone modifications are linked to discrete chromatin states and are thought to regulate the extent of accessibility of DNA to transacting factors. One of the most common marks associated with euchromatic genomic regions is methylation of histone H3 at lysine 4 (H3K4). H3K4 can be mono-, di- or tri-methylated, with the tri-methylated species (H3K4Me3) associated with active genes. Inhibitor of growth 3, 4 and 5 (ING3, ING4, and ING5) are tumor suppressors that play a major role in the regulation of cell growth, proliferation, stress responses and aging. Recently, our group found that the PHD fingers of ING proteins recognize methylated H3K4. Furthermore, while ING4 and ING5 interact preferentially with H3K4Me3, the PHD finger of ING3 appears to bind more strongly to the H3K4Me2 histone tail. However, it is unknown how these very similar PHD binding domains differentiate between the Me2 and Me3 histone marks. The objective of this proposal is to determine how binding of PHD fingers targets INGS, ING4 and INGS to the H3K4me2 and H3K4me3 histone tails by characterizing the binding sites of these PHD domains using structural and functional techniques. This will allow us to compare specific interactions of the ING3, ING4 and ING5 PHD fingers with histone peptides at the atomic level. To investigate the molecular basis for the recognition of the Me2 and Me3 histone marks by the PHD finger domains we will co-crystallize ING4 and ING5 with the H3K4Me3 peptide, and ING3 with both the H3K4Me3 and H3K4Me2 peptides. Once we have analyzed the structural data we will use this information to make specific mutations in the PHD domains of ING3, ING4 and INGS to test which residues are most important for the interactions with the Me2 and Me3 peptides. We will measure the effect of these mutations on peptide binding using NMR and tryptophan fluorescence by titrating in histone peptides to determine their dissociation constants (Kds). The functional significance of these mutations will also be tested in vivo by chromatin immunoprecipitation and fluorescence microscopy. These data will provide critical structural insights into the molecular mechanism used to discriminate between very similar histone marks by the PHD domains of ING3, ING4 and INGS, and impart a greater understanding of the mechanistic basis required to effectively read the histone code. ING3, ING4 and INGS are tumor suppressor proteins involved in controlling the way DNA is packaged within our cells. Understanding how higher order DNA structures effect in gene expression is important as loss of this regulation may underlie many disease processes, particularly cancer.

描述（由申请人提供）：染色质动态调节影响细胞活力和肿瘤发生的多种核过程。不同的组蛋白修饰与离散的染色质状态有关，并被认为调节DNA对反式作用因子的可及性程度。与常染色质基因组区域相关的最常见标记之一是组蛋白H3在赖氨酸4（H3K4）处的甲基化。H3K4可以是单甲基化、二甲基化或三甲基化的，其中三甲基化的种类（H3K4Me3）与活性基因相关。生长抑制剂3、4和5（ING3、ING4和ING5）是在调节细胞生长、增殖、应激反应和衰老中起主要作用的肿瘤抑制剂。最近，我们的小组发现ING蛋白的PHD指识别甲基化的H3K4。此外，虽然ING4和ING5优先与H3K4Me3相互作用，但ING3的PHD指似乎更强烈地结合H3K4Me2组蛋白尾部。然而，目前还不清楚这些非常相似的PHD结合结构域如何区分Me2和Me3组蛋白标记。本提案的目的是通过使用结构和功能技术表征这些PHD结构域的结合位点来确定PHD指如何将INGS、ING4和INGS靶向至H3K4me2和H3K4me3组蛋白尾部。这将使我们能够在原子水平上比较ING3、ING4和ING5 PHD指与组蛋白肽的特异性相互作用。为了研究PHD指结构域识别Me2和Me3组蛋白标记的分子基础，我们将使ING4和ING5与H3K4Me3肽共结晶，并使ING3与H3K4Me3和H3K4Me2肽共结晶。一旦我们分析了结构数据，我们将使用这些信息在ING3、ING4和INGS的PHD结构域中进行特定突变，以测试哪些残基对于与Me2和Me3肽的相互作用最重要。我们将使用NMR和色氨酸荧光通过滴定组蛋白肽来测量这些突变对肽结合的影响，以确定其解离常数（Kds）。还将通过染色质免疫沉淀和荧光显微镜在体内测试这些突变的功能意义。这些数据将提供关键的结构见解的分子机制，用于区分非常相似的组蛋白标记的PHD结构域的ING3，ING4和INGS，并赋予更好的理解所需的机制基础，有效地读取组蛋白代码。 ING3，ING4和INGS是肿瘤抑制蛋白，参与控制DNA在我们细胞内的包装方式。了解高阶DNA结构如何影响基因表达是重要的，因为这种调节的丧失可能是许多疾病过程的基础，特别是癌症。