The structural role of ING proteins in chromatin remodeling.

描述（由申请人提供）：染色质动力学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对交易因子的可及性程度。与常染色质基因组区域相关的最常见标记之一是赖氨酸4处组蛋白H3的甲基化（H3K4）。H3K4可以是单甲基化，二甲基化或三甲基化，三甲基化的物种（H3K4Me3）与活性基因相关。生长抑制剂3,4和5 （ING3， ING4和ING5）是肿瘤抑制因子，在调节细胞生长，增殖，应激反应和衰老中起重要作用。最近，我们小组发现ING蛋白的PHD指可以识别甲基化的H3K4。此外，虽然ING4和ING5优先与H3K4Me3相互作用，但ING3的PHD指似乎更强烈地与H3K4Me2组蛋白尾部结合。然而，目前尚不清楚这些非常相似的PHD结合域如何区分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，确定PHD手指如何靶向INGS， ING4和INGS到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指与组蛋白肽的特定相互作用。为了研究PHD手指结构域识别Me2和Me3组蛋白标记的分子基础，我们将ING4和ING5与H3K4Me3肽共结晶，ING3与H3K4Me3和H3K4Me2肽共结晶。一旦我们分析了结构数据，我们将利用这些信息在ING3、ING4和INGS的PHD结构域中制造特定的突变，以测试哪些残基对与Me2和Me3肽的相互作用最重要。我们将使用核磁共振和色氨酸荧光滴定组蛋白肽来测量这些突变对肽结合的影响，以确定它们的解离常数（Kds）。这些突变的功能意义也将通过染色质免疫沉淀和荧光显微镜在体内进行测试。这些数据将为区分ING3、ING4和INGS博士结构域非常相似的组蛋白标记的分子机制提供关键的结构见解，并对有效读取组蛋白代码所需的机制基础有更深入的了解。

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.