The Role of Transcription in Replication Blockage at Common Fragile Sites

我之前的研究表明，包含非常大的转录活性基因的基因组区域与受干扰复制的首选位点之间存在关系，在中期染色体中，常见脆弱位点（CFSs）的非凝聚染色质。CFSs是预定的染色体断裂区域，与癌症的致病作用有关。本研究旨在研究CFSs的转录、复制和染色体凝聚的相互依赖性。通过比较三个长基因是否表达的不同细胞类型，我获得了一些初步结果，确实表明这些基因的转录参与了CFS的形成。我现在将研究RNA Pol II与DNA聚合酶的共定位是否有利于cfs。同时，大型CFS基因的复制时间分析结果将与活性/非活性转录和CFS形成进行比较。通过过度表达正转录延伸因子，我将研究这些大基因的异常缓慢转录是否可能是观察到的cfs复制延迟的潜在原因。为了测试在染色质重塑中转录和复制的干扰偶联的参与，我还计划分析cfs中的组蛋白修饰。该项目的综合结果有望(i)为活细胞中不同过程的相互交织提供新的见解，例如Pol II转录延伸，复制和染色体凝聚；（II）更好地理解CFS的形成。

My previous studies suggested a relation between genomic regions containing very large transcriptionally active genes and preferred sites of disturbed replication, seen in metaphase chromosomes as uncondensed chromatin at common fragile sites (CFSs). CFSs are predetermined chromosomal breakage regions and have been implicated to have a causative role in cancer. The present proposal is aimed to study the interdependence of transcription, replication and chromosome condensation at CFSs. Some first results that I obtained by comparing different cell types in which three long genes are expressed or not, show indeed the involvement of transcription of these genes in CFS formation. I will now investigate if the colocalization of RNA Pol II with DNA polymerase is favoured at CFSs. In parallel, results of replication timing analysis across large CFS genes will be compared to active/inactive transcription and to CFS formation. By overexpressing positive transcription elongation factors I will investigate whether an abnormally slow transcription of these large genes could be a potential cause of the observed delayed replication at CFSs. To test for an involvement of perturbed coupling of transcription and replication in chromatin remodeling, I also plan to analyze histone modifications within CFSs. The combined results of this project are expected to (i) give new insights into the intertwining of distinct processes, such as Pol II transcription elongation, replication and chromosome condensation, in living cells and (ii) to better understand CFS formation.