Mechanisms of Gene Silencing

基因沉默的机制

• 批准号: 7146494
• 负责人: Lori L Wallrath
• 金额: $ 29.87万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7146494
• 关键词: Drosophilidae; alternatives to animals in research; biochemistry; chromatin; cytology; gene induction /repression; genetic promoter element; genetic regulation; genetic transcription; heterochromatin; immunofluorescence technique; immunoprecipitation; in situ hybridization; molecular genetics; molecular site; northern blottings; transfection; western blottings

DESCRIPTION (provided by applicant): Complex processes such as development and disease progression are regulated by gene expression. The majority of gene expression is controlled through chromatin packaging. Heterochromatin Protein 1 (HP1) is an evolutionary conserved non-histone chromosomal protein required for chromatin packaging and gene expression. HP1 shows a non-uniform distribution on chromosomes, being enriched within centric heterochromatin and also localizing to telomeres and euchromatic sites. The long-term goal of this proposal is to determine the mechanisms of gene regulation by HP1 in a variety of chromatin contexts. HP1 exhibits the property of "spreading" along the chromosome and silencing genes. In Specific Aim 1, we propose HP1 tethering experiments and genetic screens in Drosophila to determine the mechanism of silent chromatin spreading. Recently, HP1 has been linked to the activation of both heterochromatin and euchromatic genes. In Specific Aim 2, we propose to identify the sequences required for HP1 targeting. In addition, we will determine the role of HP1 in the expression of heterochromatic genes that normally reside in a repressive chromatin environment. Knowledge of HP1 function obtained from studies in Drosophila has important implications for breast cancer progression. We have shown that HP1Hsa, 1 of 3 HP1 family members in humans, is down-regulated in metastatic breast cancer cell lines and metastatic clinical samples. Modulation of HP1Hsa levels alters the invasive potential of breast cancer cells. Therefore, we hypothesize that HP1Hsa functions to regulate genes required for invasion and metastasis. In Specific Aim 3, we will determine which genes are mis-regulated following knock-down of HP1Hsa levels and identify direct target genes by chromatin immunoprecipitation. The mechanism of HP1Hsa localization and function at such genes will be determined by a knock-down and replacement strategy where endogenous HP1Hsa is exchanged for mutant forms lacking specific functions. Collectively, these studies will expand our knowledge about the mechanisms of gene regulation by HP1 proteins and identify the molecular pathways that regulate metastasis. Relevance to Public Heath: Studies described here will identify the genes and molecular pathways involved in regulating breast cancer invasion/metastasis. Protein products encoded by such genes could represent new markers for metastatic progression and targets for therapy.

描述（由申请人提供）：诸如发育和疾病进展之类的复杂过程受基因表达调节。大多数基因表达都是通过染色质包装来控制的。异染色质蛋白1（HP1）是一种进化保守的非固定染色体蛋白，需要染色质包装和基因表达。 HP1在染色体上显示出不均匀的分布，富集在中心异染色质中，并且位于端粒和正念位点。该提案的长期目标是确定HP1在多种染色质环境中通过HP1调节的机制。 HP1沿染色体和沉默基因表现出“扩散”的特性。在特定的目标1中，我们提出了果蝇中的HP1束缚实验和遗传筛选，以确定无声染色质扩散的机理。最近，HP1已与异染色质和正式基因的激活联系在一起。在特定目标2中，我们建议确定HP1靶向所需的序列。此外，我们将确定HP1通常驻留在抑制性染色质环境中的杂色基因表达中的作用。从果蝇研究中获得的HP1功能的知识对乳腺癌的进展具有重要意义。我们已经表明，在人类中3个HP1家族成员中有1个HP1HSA在转移性乳腺癌细胞系和转移性临床样品中被下调。 HP1HSA水平的调节改变了乳腺癌细胞的侵入性潜力。因此，我们假设HP1HSA功能可以调节侵袭和转移所需的基因。在特定的目标3中，我们将确定在HP1HSA水平敲低的基因并通过染色质免疫沉淀识别直接靶基因后，哪些基因被错误调节。 HP1HSA定位和此类基因功能的机制将取决于内源性HP1HSA的敲低和替代策略，以交换缺乏特定功能的突变体形式。总的来说，这些研究将扩大我们对HP1蛋白基因调节机制的了解，并确定调节转移的分子途径。与公共卫生的相关性：此处描述的研究将确定与调节乳腺癌侵袭/转移有关的基因和分子途径。由这种基因编码的蛋白质产物可以代表转移性进展的新标记和治疗靶标。