Chromatin Structure in Drosophila Telomeres

端粒是染色体末端的核蛋白结构，其需要完全复制线性DNA并将天然染色体末端与双链染色体断裂区分开来以进行DNA修复。在果蝇中，染色体末端通过三种逆转录转座子HeT-A、TAHRE和TART的靶向转座来维持。插入端粒中的亚末端端粒相关序列（TAS）和末端反转录转座子阵列之间或TAS内的转基因被抑制和多样化。这种杂色，被称为端粒位置效应（TPE），似乎是由于抑制诱导TAS和激活启动转录的反转录转座子的相互作用。以前的研究已经确定了TPE发生的遗传条件和抑制TPE的条件。本项目的目的是确定染色质结构的变化相关的活动和抑制TPE。通过微球菌核酸酶和DNA酶1消化监测核小体阵列，并通过染色质免疫沉淀法测定蛋白质含量。迄今为止，TAS和反转录转座子阵列之间的核小体阵列没有差异，但这些DNA结构域之间的蛋白质含量不同。TAS优先结合与异染色质相关的染色质蛋白，而反转录转座子优先结合与活性染色质相关的蛋白。 插入端粒亚末端端粒相关序列（TAS）和末端反转录转座子阵列之间的转基因被抑制和杂色。这种杂色，称为端粒位置效应，TPE，似乎是由于抑制诱导TAS和激活启动HeT-A转录的相互作用。端粒转基因因此提供了这种相互作用的测定。这些转基因提供了一种手段来研究控制HeT-A转录和转座，从而端粒延长。我们已经发现，当转基因在TAS中或附近时，转基因活性被抑制，并且表达随着同源端粒上以及其他端粒上的TAS序列的变化而变化。这些TAS阵列之一的缺陷或缺失增加转基因活性，并且通过延伸，我们推断反转录转座子转录。

Telomeres are nucleoprotein structures at chromosome ends that are required to completely replicate the linear DNA and to distinguish the natural chromosome end from a double strand chromosome break for purposes of DNA repair. In Drosophila, chromosome ends are maintained by the targeted transposition of three retrotransposons, HeT-A, TAHRE and TART. A transgene inserted in the telomere between the subterminal telomere associated sequence (TAS) and the terminal retrotransposon array, or within TAS, is repressed and variegates. This variegation, termed telomeric position effect (TPE), appears to be due to an interaction of repression induced by TAS and activation initiated by transcription of the retrotransposons. Previous studies have identified genetic conditions in which TPE occurs and conditions in which it is suppressed. The purpose of this project is to identify changes in chromatin structure associated active and suppressed TPE. Nucleosome arrays are monitored by Micrococcal nuclease and DNase 1 digestion, and protein content assayed by chromatin immunoprecipitation. To date, no differences in the nucleosome array have been identified between TAS and the retrotransposon array, but protein content varies between these DNA domains. TAS preferentially binds chromatin proteins associated with heterochromatin, while the retrotransposons preferentially bind proteins associated with active chromatin. A transgene inserted into the telomere between the subterminal telomere associated sequence (TAS) and the terminal retrotransposon array is repressed and variegates. This variegation, termed telomeric position effect, TPE, appears to be due to an interaction of repression induced by TAS and activation initiated by HeT-A transcription. A telomeric transgene thus provides an assay for this interaction. These transgenes provide a means to investigate the control of HeT-A transcription and transposition, and thus telomere elongation. We have found that when the transgene is in or near TAS, transgene activity is repressed, and expression varies with changes in TAS sequences on the homologous telomere, as well as at other telomeres. Defects in, or deletions of, one of these TAS arrays increase transgene activity, and by extension we infer retrotransposon transcription.