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

描述（由申请方提供）：组织特异性基因调控期间复制起点激活/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，并更好地理解染色体缺失、插入和易位对相邻基因表达的影响。我们最近发现，免疫球蛋白重链（Igh）基因座的基因表达调控是伴随着，在B细胞发育过程中，复制起点活性的长期变化。多个来源的活性在大小为数百个内切酶的基因组区域中被修饰。这是Igh基因座激活过程中最早可检测到的修饰之一。阐明在细胞分化过程中参与起源调控的机制将有助于阐明如何在增殖细胞中建立和维持组织特异性的基因表达模式。初步结果表明，删除以前的特征的远程调控元件，Igh内含子增强子，减少基因表达从各种发育调节启动子，但不阻止激活的发育调节复制起点。相同基因组区域对其他B细胞特异性事件（例如D-J重组）的可及性也不受显著影响。这表明，起源活动和基因座可及性的远程调节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变得易于转录调控或重组的基因组区域。在不同的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，发育调节因子，如Pax 5，直接或间接参与跨越大部分Igh基因座的起源调节。我们将研究原代野生型和Pax 5-/- pro-B细胞（AIM I），以及Pax 5重建的pro-B细胞（AIM II），和其中Pax 5已被条件灭活的pro-B细胞（AIM II），以澄清基因表达的发育调控和各种基因位点的起源激活之间的关系。我们还将开始剖析Pax 5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).