FORMATION, STRUCTURE AND FUNCTION IN HETEROCHROMATIN

长期目标是确定 以异色形式包装基因会导致稳定性 失活。这种“关”式的监管显然具有功能性。 在许多表观遗传系统中的重要性，并似乎延伸到 同源异型基因的调节，其开/关模式对 所有高等生物体的正常发育。许多染色体 稳定沉默所需的蛋白质高度保守，它们的 功能障碍可能会导致疾病状态。果蝇 将使用黑素记录仪，因为它易于监控和 修改位置效果杂色(PEV)。一个P元素携带一个 可见标记(HSP70-白色)和特性良好的测试基因(a 标记型HSP26)驻留在中心周围时表现为PEV 异染色质、端粒和带状细胞内的某些部位 4号染色体区域.着丝粒周围异染色质中的沉默 4号染色体对HP1(异染色质)突变敏感 蛋白质1)，一种定位于这些区域的蛋白质。这些五花八门的 转基因显示染色质结构发生变化。我们会带着 在屏幕上恢复出两行详细的结构分析 在28摄氏度，HS-2和HS-5，显示完全沉默 25℃的转基因(在着丝粒周围的异染色质中)。 我们将绘制核小体阵列图，并查看可及性的限制， 核酸酶消化速度和DNA修饰来确定 初级染色质是否发生了变化 纤维，或在更高级别的包装中，或在两者中，区分 这样的模型实现了沉默。 第二个最近发现的异染色质特异蛋白，HP， 将通过分子和基因技术进行分析。甲醛 染色质的交联和免疫沉淀将是 用来分析组蛋白乙酰化的模式并绘制 HP1和HP2与沉默转基因的相关性。的影响 一种染色体蛋白的突变与另一种意愿的关联 进行细胞学和生化测定，以建立 相互作用的层次，以及对染色质结构的影响。 HP1相互作用的作图位置可能表明DNA序列 核异染色质形成；这些将在P-元素中进行测试 构造。将生成转基因功能图，用于 4号染色体，它似乎由散布的常染色质组成 和异色结构域。将此映射与一个序列关联 带有序列图的图应该提供对染色体的洞察 组织，帮助定义 异染色质和常染色质，并可能导致 识别这些域之间的边界。

The long-term objective is to determine the mechanism by which the packaging of a gene in a heterochromatic form leads to stable inactivation. This type of "off" regulation is clearly of functional importance in many epigenetic systems, and appears to extend to the regulation of the homeotic loci, whose on/off pattern is critical for normal development in all higher organisms. Many of the chromosomal proteins required for stable silencing are high conserved, and their misfunction can contribute to a disease state. Drosophila melanogaster will be used because of the ease of monitoring and modifying position effect variegation (PEV). A P-element carrying a visible marker (hsp70-white) and a well-characterized test gene (a marked form of hsp26) exhibits PEV when resident in the pericentric heterochromatin, the telomeres, and some sites within the banded region of chromosome 4. The silencing in pericentric heterochromatin and chromosome 4 is sensitive to mutations in HP1 (heterochromatin protein 1), a protein localized to these regions. These variegating transgenes show alterations in chromatin structure. We will carry out a detailed structural analysis in two lines recovered in a screen at 28 degreesC, HS-2 and HS-5, that show complete silencing of the transgenes (which are in pericentric heterochromatin) at 25 degreesC. We will map the nucleosome array and look at limits of accessibility, rates of nuclease digestion, and DNA modification to determine whether or not alterations have occurred in the primary chromatin fiber, or in higher order packaging, or in both, distinguishing among such models for achieving silencing. A second recently identified heterochromatin-specific protein, HP2, will be analyzed by molecular and genetic techniques. Formaldehyde crosslinking of chromatin followed by immunoprecipitation will be used to analyze the pattern of histone acetylation and map the association of HP1 and HP2 with the silent transgenes. The effect of mutations in one chromosomal protein on the association of other will be determined cytologically and biochemically to establish the hierarchy of interactions, and the impact on chromatin structure. Mapping sites of HP1 interaction may indicate DNA sequences that nucleate heterochromatin formation; these will be tested in P-element constructs. A map of transgene function will be generated for chromosome 4, which appears to be made up of interspersed euchromatic and heterochromatic domains. Correlating this map with a sequence map with a sequence map should provide insights into chromosome organization, helping to define the differences between heterochromatin and euchromatin and potentially leading to identification of boundaries between these domains.