FORMATION, STRUCTURE AND FUNCTION IN HETEROCHROMATIN

这项建议的长期目标是确定机制 通过将基因包装在异染色质中导致稳定的 失活。这种“关”式的监管显然具有功能性。 在染色体印迹中很重要，并可能延伸到对 同源异型基因，其开启/关闭模式对正常发育至关重要- 存在于所有高等生物体中。整个过程中都会用到果蝇 因为便于操作和位置监控而学习 效应杂化，一种出现的表观遗传下调 以反映测试基因的包装。我们已经确定了一个 异染色质相关蛋白，HP1，在该基因中起作用 失活；HP1基因突变导致位置效应抑制 果蝇的花纹。我们的第一个目标是识别 使用生化和遗传方法与HP1相互作用。互动 将使用(A)果蝇的基因筛查来寻找蛋白质；(B) 与抗HP1抗体共沉淀；和(C)在酵母中进行筛选， 需要蛋白质-蛋白质相互作用才能获得肯定的结果(字段 和宋，1989，自然340,245-246)。在基因筛查中，误解 突变，包括HP1的温度敏感等位基因，正在 寻找的，以及不互补的第二位点突变。互动 鉴定出的蛋白质将被生化和遗传鉴定。 以确定它们在异染色质形成中的作用。 在研究人类猪型流感的同时，我们正进行有关 插入的转基因的核小体和更高级别的包装 利用P元件介导的异染色质转化。两种不同的 正在使用的基因是hsp26，一种热休克基因，其5‘上游 调节区通常预置在可接近染色质结构中 在基因激活之前，以及控制下的CAT报告基因 糖皮质激素反应元件，一种可能是 其5‘上游调控区域被包装在核小体中的那些 数组，并且必须重新建模才能激活。我们将描述 包装这些基因时的染色质结构 异染色质，与它们的常染色质包装相比，在 以了解有关失活过程的更多信息。拥有如此特定的 在异染色质环境中测试基因也将在 评估上述染色体蛋白的作用。这些 研究应该使我们更接近于理解自然和 高阶染色质结构的调节作用。

The long-term objective of this proposal is to determine the mechanism by which the packaging of a gene in heterochromatin leads to stable inactivation. This type of "off" regulation is clearly of functional important in chromosome imprinting, and may extend to the regulation of the homeotic loci, whose on/off pattern is critical for normal develop- ment in all higher organisms. Drosophila will be used throughout this study because of the ease of manipulation and monitoring of Position effect variegation, one type of epigenetic down-regulation that appears to reflect the packaging of the test gene. We have identified a heterochromatin-associated protein, HP1, which plays a role in this gene inactivation; mutations in HP1 result in suppression of position effect variegation in Drosophila. Our first aim is to identify proteins which interact with HP1, using biochemical and genetic approaches. Interacting proteins will be sought using (a) a genetic screen in Drosophila; (b) coprecipitation with anti-HP1 antibodies; and (c) a screen in yeast that requires protein-protein interactions to score a positive result (Fields and Song, 1989, Nature 340, 245-246). In the genetic screen, missense mutations, including temperature-sensitive alleles of HP1, are being sought, as well as noncomplementing second-site mutations. Interacting proteins identified will be characterized biochemically and genetically to determine their role in heterochromatin formation. In parallel with the study of HP1, we are carrying out studies of the nucleosomal and higher order packaging of transgenes inserted into heterochromatin using P-element mediated transformation. Two different genes are being used, hsp26, a heat shock gene whose 5' upstream regulatory region is normally preset in an accessible chromatin structure prior to gene activation, and a CAT reporter gene under the control of glucocorticoid response elements, a gene likely to be characteristic of those whose 5' upstream regulatory region is packaged in a nucleosomal array and must be remodeled for activation. We will characterize the chromatin structure of these genes when they are packaged in heterochromatin, in comparison to their packaging in euchromatin, in order to learn more about the inactivation process. Having such specific test genes in a heterochromatin environment will also be invaluable in assessing the role of the chromosomal proteins identified above. These studies should move us much closer to an understanding of the nature and regulatory role of higher order chromatin structure.