Role of Zfp318 in the functional development of B cells

 描述（由申请人提供）：该修订的R21申请的主要重点是定义锌指家族成员Zfp 318在B细胞发育和功能中的功能。以前，我们证明了Zfp 318在B细胞成熟过程中表达，并且在成熟的幼稚卵泡B细胞中显示出最高水平的表达。Zfp 318具有2个锌指结构域和核定位基序：它是一类具有RNA和/或DNA结合潜力的蛋白质。 我们已经创建了Zfp 318条件性缺失等位基因，并在本申请中包括的数据和出版的手稿中显示，缺乏该蛋白质的B细胞不能产生IgD。在幼稚成熟和成熟B细胞中，IgD通过Ighm/Ighd基因座的选择性剪接与IgM协同表达。缺乏Zfp 318的B细胞继续表达VDJ-IgM，但失去产生VDJ-IgD产物的能力。自第一次提交以来产生的新数据表明，Zfp 318不起改变Ighm/Ighd hnRNA的选择性剪接的作用，而是通过阻断IgM编码外显子末端的转录终止位点来产生整个hnRNA。在不存在Zfp 318的情况下，几乎所有Ighm转录物在编码IgD的外显子之前终止。令人惊讶的是，Ighm/Ighd基因是B细胞中唯一显示出这种改变的表达谱的基因座，这意味着Zfp 318已经进化为单独控制该事件。 本应用程序部分的实验计划侧重于实现三个特定目标。一是 确定是否存在对B细胞的功能性后果和由Zfp 318的缺失产生的免疫应答。IgD和Zfp 318自原始脊椎动物发育以来就共同进化，我们的Zfp 318缺陷动物将为我们提供一个独特的Vantage，以确定这些蛋白质对免疫应答的重要性。其次，我们将讨论Zfp 318的功能。它能和DNA结合还是和RNA结合Zfp 318仅调节Ighm/Ighd表达的事实表明该蛋白具有非常高水平的序列/结构特异性。最后，我们将解决如何Zfp 318基因的表达调控整合到B细胞的发育和激活。从我们以前的研究中我们知道Zfp 318的表达受到Mef 2c的显著影响，Mef 2c是一种已知调节B细胞发育和活化的转录因子。Zfp 318如何适应这个表达和功能网络是最后一个问题的主要目标。

 DESCRIPTION (provided by applicant): The primary focus of this revised R21 application is on defining the functions(s) of the zinc finger family member Zfp318 in the development and function of B cells. Previously we demonstrated that Zfp318 is expressed during B cell maturation and shows the highest level of expression in mature, naïve follicular B cells. Zfp318 possesses 2 zinc finger domains as well as nuclear localization motifs: it is in a class of protein with RNA and/or DNA binding potential. We have created a Zfp318 conditional deletion allele and show in data included in this application and a manuscript in press that B cells lacking the protein fail to produce IgD. IgD, in naïve maturing and mature B cells, is coordinately expressed with IgM via alternative splicing of the Ighm/Ighd locus. B cells lacking Zfp318 continue to express VDJ-IgM but lose the ability to generate the VDJ-IgD product. New data generated since the first submission demonstrates that Zfp318 does not function to alter the alternative splicing of the Ighm/Ighd hnRNA, but instead allows for the production of this entire hnRNA by blocking the transcription termination site at the end of the IgM- encoding exons. In the absence of Zfp318, virtually all of the Ighm transcripts terminate prior to the IgD-encoding exons. Amazingly, the Ighm/Ighd gene is the only locus in B cells that shows such altered expression profiles which means that Zfp318 has evolved to singularly control this event. The experimental plan of this application section is focused upon accomplishing three specific goals. First, we will determine if there are functional consequences to the B cell and the immune response created by the absence of Zfp318. IgD and Zfp318 have co-evolved since the development of primitive vertebrates, our Zfp318 deficient animal will provide us a unique vantage to ascertain the importance of these proteins to the immune response. Secondly we will address how Zfp318 functions. Does it bind to DNA, does it bind to RNA? The fact Zfp318 only modulates Ighm/Ighd expression indicates the protein possesses a very high level of sequence/structure specificity. And finally we will address how the regulation of the expression of the Zfp318 gene is integrated into B cell development and activation. We know from our previous studies that the expression of Zfp318 is dramatically influenced by Mef2c, a transcription factor known to modulate B cell development and activation. How Zfp318 fits into this network of expression and function is the primary goal of this last question.