Molecular basis of Replication origin activation/silecing during tissue-specific

描述（由申请人提供）：组织特异性基因调控过程中复制起始激活/沉默的分子基础。了解基因表达的远程调控对于理解哺乳动物基因组的功能组织以及细胞分化和发育至关重要。这也有助于改进转基因载体的设计，更好地了解染色体缺失、插入和易位对邻近基因表达的影响。我们最近的研究表明，在B细胞发育过程中，免疫球蛋白重链（Igh）基因表达的调控伴随着复制起源活性的长期变化。多个起源的活性在数百个碱基大小的基因组区域中被修改。这是在Igh基因座激活过程中最早检测到的修饰之一。阐明细胞分化过程中起源调控的机制将有助于阐明组织特异性基因表达模式是如何在增殖细胞中建立和维持的。初步结果表明，先前表征的远程调控元件，即Igh内含子增强子的缺失，减少了各种发育调控启动子的基因表达，但不会阻止发育调控复制起点的激活。同一基因组区域对其他B细胞特异性事件（如D-J重组）的可及性也没有显著影响。这表明起源活性和位点可及性的长期调控至少部分独立于增强子元件。在早期S期活跃的起源簇可以表征在细胞分化过程中易于转录调控或重组的基因组区域。在各种B细胞特异性基因位点上识别这样的簇将使我们能够识别哺乳动物染色体的功能单位。初步结果还表明，发育调控因子，如Pax5，直接或间接地参与了Igh位点大部分的起源调控。我们将研究原代野生型和Pax5 -/- pro-B细胞（AIM I），以及Pax5重组的pro-B细胞（AIM II），以及Pax5条件失活的pro-B细胞（AIM II），以阐明基因表达的发育调控与不同基因位点的起源激活之间的关系。我们还将开始剖析Pax5或其他发育调节因子诱导这些变化的途径（AIM III）。

DESCRIPTION (provided by applicant): Molecular basis of replication origin activation/silencing during tissue-specific gene regulation. Understanding long-range regulation of gene expression is of paramount importance to comprehend the functional organization of mammalian genomes, as well as cell differentiation and development. This could also help to improve the design of transgenic vectors and to better appreciate the effect of chromosomal deletions, insertions, and translocations on the expression of adjacent genes. We have recently shown that regulation of gene expression in the immunoglobulin heavy chain (Igh) locus is accompanied, during B cell development, by long range changes in replication origin activity. The activity of multiple origins is modified across genomic regions that are hundreds of kilobases in size. This is one of the earliest modifications detectable during the activation of the Igh locus. Elucidating the mechanisms involved in origin regulation during cell differentiation will help to clarify how tissue-specific patterns of gene expression are established and maintained in proliferating cells. Preliminary results indicate that the deletion of a previously characterized long range regulatory element, the Igh intronic enhancer, reduces gene expression from various developmentally regulated promoters but does not prevent the activation of the developmentally regulated origins of replication. The accessibility of the same genomic region to other B cell-specific events (e.g. D-J recombination) is also not significantly affected. This suggests that the long range regulation of origin activity and locus accessibility are at least partially independent from enhancer elements. Clusters of origins active in early S phase could characterize genomic regions that become accessible to transcriptional regulation or recombination during cell differentiation. The identification of such clusters at various B cell-specific gene loci will allow us to identify functional units of mammalian chromosomes. Preliminary results also indicate that developmental regulators, such as Pax5, are directly or indirectly involved in origin regulation across large portions of the Igh locus. We will examine primary wild-type and Pax5 -/- pro-B cells (AIM I), as well as Pax5 reconstituted pro-B cells (AIM II), and pro-B cells in which Pax5 has been conditionally inactivated (AIM II), to clarify the relationship between developmental regulation of gene expression and origin activation at various gene loci. We will also begin to dissect the pathway by which Pax5 or other developmental regulators induce these changes (AIM III).