DNA Replication Initiation Sites in Mammalian Cells

描述（由申请人提供）：我们将确定表观遗传修饰对免疫球蛋白重链（Igh）基因座内复制起始激活和沉默的影响。我们已经确定了整个Igh基因座中许多复制起始位点的位置，并确定了它们在B细胞发育过程中如何变化。我们现在将专注于修改小鼠ES和proB细胞中的Igh基因座，以了解与活性和非活性染色质结构域相关的染色质修饰如何影响基因座内DNA复制起始结构域的激活。在这些研究中，我们对胚胎干细胞进行了改造，使其在Igh基因座的两个位置上含有一个可交换的盒体。利用这些胚胎干细胞，我们建议靶向这些位点，特异性蛋白质修饰染色质结构并具有组蛋白修饰活性。我们已经证明，我们可以将转录激活子连接到我们插入到Igh区域的Gal4靶点上，从而导致组蛋白修饰和先前沉默基因的激活。我们还将通过插入包含频繁识别位点的更大的靶向序列来修改位点。我们将确定表观遗传修饰对复制起始、复制叉进程的方向和速率以及在S期Igh和侧翼区域复制的时间的影响。我们将使用我们最近开发的两种新方法：复制DNA的单分子分析（SMARD）和高分辨率定时阵列（HRTA）分析。高阶染色体结构在调节复制中的作用也将被研究。我们将使用带有Igh基因座的胚胎干细胞，通过盒式交换修饰，产生嵌合小鼠和纯合敲入小鼠，以确定Igh基因座表观遗传变化对B细胞发育的影响。我们将利用我们已经获得的关于B系正常发育不同阶段的复制起始和分叉方向以及Igh位点的核组织的详细信息来描述这些变化。

DESCRIPTION (provided by applicant): We will determine the effects of epigenetic modifications on replication origin activation and silencing within the immunoglobulin heavy chain (Igh) locus. We have established the location of many replication initiation sites throughout the Igh locus and have determined how they change during B cell development. We will now focus on modifying the Igh locus in murine ES and proB cells to understand how chromatin modifications associated with active and inactive chromatin domains affect the activation of initiation domains of DNA replication within the locus. For these studies, we have engineered ES cells to contain an exchangeable cassette at two sites within the Igh locus. Using these ES cells, we propose to target to these sites, specific proteins that modify chromatin structure and have histone modifying activities. We already have demonstrated that we can tether a transcriptional activator to Gal4 target sites that we have inserted in the Igh region, resulting in histone modifications and the activation of a previously silent gene. We will also modify the locus by inserting much larger targeting sequences containing frequent recognition sites. We will determine the effects of epigenetic modifications on replication initiation, the direction and rate of replication fork progression and on the time in S phase during which the Igh and flanking regions replicate. We will use two novel approaches that we have recently developed: single molecule analysis of replicated DNA (SMARD) and high-resolution timing array (HRTA) analysis. The role of higher order chromosome structure in regulating replication will also be investigated. We will use the ES cells with the Igh locus, modified by cassette exchange, to produce chimeric mice and homozygous knock-in mice to determine the effect on B cell development of epigenetic changes in the Igh locus. We will characterize these changes using the detailed information we have already obtained about replication initiation and fork direction, and nuclear organization of the Igh locus at different stages of normal development in the B lineage.