Mechanisms of Gene Silencing

描述（由申请人提供）：基因表达调控发育和疾病进展等复杂过程。大多数基因表达是通过染色质包装控制的。异染色质蛋白1（Heterochromatin Protein 1，HP 1）是一种进化上保守的非组蛋白染色体蛋白，是染色质包装和基因表达所必需的。HP 1在染色体上呈非均匀分布，在着丝粒异染色质内富集，也定位于端粒和常染色质位点。这项提案的长期目标是确定HP 1在各种染色质背景下的基因调控机制。HP 1具有沿染色体沿着“扩散”和沉默基因的特性。在具体目标1中，我们提出了在果蝇中进行HP 1系留实验和遗传筛选，以确定沉默染色质扩散的机制。最近，HP 1已被链接到异染色质和常染色质基因的激活。在具体目标2中，我们建议确定HP 1靶向所需的序列。此外，我们将确定HP 1在异染色质基因表达中的作用，这些异染色质基因通常位于抑制性染色质环境中。从果蝇研究中获得的HP 1功能知识对乳腺癌进展具有重要意义。我们已经表明，HP 1Hsa，人类3个HP 1家族成员之一，在转移性乳腺癌细胞系和转移性临床样品中下调。HP 1Hsa水平的调节改变了乳腺癌细胞的侵袭潜力。因此，我们推测HP 1Hsa的功能是调节侵袭和转移所需的基因。在具体目标3中，我们将确定哪些基因在HP 1Hsa水平敲低后被错误调节，并通过染色质免疫沉淀法鉴定直接靶基因。HP 1Hsa在这些基因上的定位和功能的机制将通过敲低和置换策略来确定，其中内源性HP 1Hsa被交换为缺乏特定功能的突变形式。总的来说，这些研究将扩大我们对HP 1蛋白基因调控机制的了解，并确定调节转移的分子途径。与公共卫生的相关性：这里描述的研究将确定参与调节乳腺癌侵袭/转移的基因和分子途径。由这些基因编码的蛋白质产物可能代表转移进展的新标志物和治疗靶点。

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.