Mechanisms of Gene Silencing

发育和疾病进展等复杂的过程是由基因表达调控的。这个 大多数基因的表达是通过染色质包装来控制的。异染色质蛋白1(HP1)是 染色质包装和基因所需的进化保守的非组蛋白染色体蛋白 表情。HP1在染色体上呈不均匀分布，集中在着丝粒内。 异染色质，也定位于端粒和常染色质部位。这项提议的长期目标是 目的是确定HP1在各种染色质环境中调节基因的机制。HP1展品 沿着染色体“传播”并使基因沉默的特性。在具体目标1中，我们提出了HP1 果蝇的系留实验和遗传筛选以确定沉默染色质的机制 正在扩散。最近，HP1被认为与异染色质和常染色质基因的激活有关。 在具体目标2中，我们建议确定HP1靶向所需的序列。此外，我们还将 确定HP1在异染色质基因表达中的作用 染色质环境。从果蝇研究中获得的HP1功能的知识很重要 对乳腺癌进展的影响。我们已经证明，HP1Hsa是HP1家族的三个成员之一 在人类中，在转移性乳腺癌细胞系和转移性临床样本中下调。 HP1Hsct水平的调节改变了乳腺癌细胞的侵袭潜力。因此，我们假设 HP1Hsct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HP1Hsa水平下调后哪些基因被错误调控，并确定直接靶点 通过染色质免疫沉淀获得基因。HP1Hsa在这些基因上的定位和作用机制 将由拆卸和替换策略决定，其中内源性HP1Hsa被替换为 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 HP1蛋白调控基因的机制及分子调控途径的确定 转移。 与公共卫生相关：这里描述的研究将确定基因和分子途径 参与调节乳腺癌的侵袭/转移。由这些基因编码的蛋白质产物可能 代表了转移进展的新标记物和治疗的靶点。

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, one of three HP1 family members in humans, is down-regulated in metastatic breast cancer cell lines and metastatic clinical samples. Modulation of HP1Hsct levels alters the invasive potential of breast cancer cells. Therefore, we hypothesize that HP1Hsct 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.