Factors and DNA Motifs Involved in Ig Class Switch

描述（由申请人提供）：免疫球蛋白（IG）同种型转换通过环出和缺失过程发生，该过程集中于位于恒定区上游的转换区，C δ除外。关于开关复合（SR）的机制知之甚少。我们最近设计了一种基于质粒的SR瞬时转染试验。使用这种试验，我们表明有不同的开关活动，介导mu-> γ 3，mu-> α mu-> β和mu-> γ 1开关。在这里报告的初步结果中，我们表明开关质粒测定是AID依赖性的。 这一观察结果加强了这样的结论，即开关质粒测定忠实地重演生理SR。这些研究表明，有S区的开关活动的同种型特异性识别。然而，基本上没有什么是已知的开关活动如何识别S区。目的I是为了测试是否可以在体内证实与开关基板所获得的结果。我们将使用靶向同源重组将内源性Sgamma 3区域与Sgamma 1序列交换。这个实验将使我们能够确定是否Sgamma 1特异性开关因子的情况下，将导致无法进行SR时，S？1序列存在于S区。这样的结果将证实在生理环境中存在同种型特异性转换因子。本申请中的目的II集中于定义S区的分子识别的性质。使用质粒检测，我们计划操纵的大小和序列的S区划定SR的最小目标。我们还将创建合成开关区域，在特定的残基突变，我们将定位的位置，在S DNA的双链断裂。总之，这些研究将使我们能够映射功能重组基序（FRM）位于串联重复序列。在第三个目标中，我们将使用目标II中定义的FRM，并询问DNA结合蛋白是否与这些基序相互作用，并使用凝胶位移和竞争结合分析介导SR。如果FRM与先前描述的SNIP和SNAP识别基序重叠，那么我们将询问SNIP和/或SNAP是否在功能上参与SR。我们还将探讨FRM结合蛋白的参与，通过调节其表达水平，并确定重组是否因此受到影响。

DESCRIPTION (provided by applicant): Immunoglobulin (Ig) isotype switching occurs by a looping-out and deletion process which is focused on switch regions located upstream of the constant regions, with the exception of Cdelta. Little is known regarding the mechanism of switch recombination (SR). We recently devised a plasmid-based transient transfection assay for SR. Using this assay we showed that there are distinct switching activities which mediate mu->gamma3, mu->alpha mu->epsilon and mu->gamma1 switching. In preliminary results reported here we show that the switch plasmid assay is AID dependent. This observation strengthens the conclusion that the switch plasmid assay faithfully recapitulates physiological SR. These studies suggest that there is isotype specific recognition of S regions by switching activities. However, essentially nothing is known regarding how switching activities identify S regions. Aim I is designed to test whether the results obtained with the switch substrates can be confirmed in vivo. We will exchange the endogenous Sgamma3 region with Sgamma1 sequence using targeted homologous recombination. This experiment will allow us to determine whether the absence of the Sgamma1 specific switching factor will lead to an inability of the Sgamma3 endogenous locus to undergo SR when S?1 sequence is present in the S region. Such an outcome would confirm the existence of isotype specific switching factors in a physiological setting. Aim II in this application is focused on defining the nature of molecular recognition of S regions. Using the plasmid assay we plan to manipulate the size and sequence of the S regions to delineate a minimal target for SR. We will also create synthetic switch regions which are mutated at specific residues and we will locate the position of double strand breaks in the S DNA. Taken together, these studies will allow us to map functional recombination motifs (FRMs) which are located in the tandem repeats. In the third Aim we will use the FRMs defined in Aim II and ask whether DNA binding proteins interact with these motifs and mediate SR using gel shift and competition binding analyses. If the FRMs overlap with previously described SNIP and SNAP recognition motifs then we will ask whether SNIP and/or SNAP are functionally involved in SR. We will also explore the involvement of the FRM binding proteins by modulating their expression levels and determining whether recombination is affected as a consequence.