核基质结合因子A对RAG介导的Ig基因V(D)J重组的调控及机制

The diversity of immunoglobulin (Ig) variable region gene is generated from recombination activating enzymes (RAG1 and RAG2)-mediated V(D)J recombination. Presently, numerous molecular regulation mechanisms of recombination remain elusive. Scaffold attachment factor A (SAFA) plays a role in regulation of gene expression. In the preliminary studies, the applicant found that RAG1 proteins interact with SAFA. Over-expression of SAFA enhances the expression of exogenous RAG and RAG-mediated plasmid recombination in 293T cells. Based on these results, the applicant hypothesizes that SAFA plays an important regulatory role in RAG-mediated V(D)J recombination. In this project, the applicant will over-express or knockout SAFA in 293T cells and pre-B cells to determine whether SAFA affects Ig gene V(D)J recombination, RAG’s expression and distribution in nucleus, and recruits Ig gene. The applicant will use SAFA conditional knockout mice to determine whether loss of SAFA affects V(D)J recombination and B cell development. The results will reveal the regulatory role of SAFA in V(D)J recombination. This project will explore a new molecular regulation mechanism of Ig gene recombination, and provide new insights in further exploration of regulation of V(D)J recombination.

免疫球蛋白(Ig)可变区基因的多样性源于重组激活酶（RAG1和RAG2）介导的V(D)J重组。目前许多调控V(D)J重组的分子机理尚未阐明。核基质结合因子A（SAFA）能调控基因表达。申请人前期研究发现RAG1能结合SAFA；在293T细胞中过表达SAFA能增强外源性RAG的表达，促进RAG介导的质粒重组。故假设SAFA对RAG介导的V(D)J重组具有重要的调控作用。该研究拟在293T和前B细胞中过表达或敲除SAFA，探讨SAFA对Ig基因V(D)J重组、RAG蛋白表达及在细胞核中分布的影响，研究SAFA是否募集Ig基因；在小鼠中条件性敲除SAFA，探讨其是否影响小鼠的V(D)J重组和B细胞发育，从而揭示SAFA对Ig基因V(D)J重组的调控及机制。该研究有望揭示新的Ig基因重组的分子调控机制，并为进一步研究V(D)J重组的调控提供新的思路。

免疫球蛋白(Ig)基因可变区的多样性源于重组激活基因(RAG)介导的V(D)J重组。目前调控V(D)J重组的分子机理尚未阐明。核基质结合因子A（SAFA）能调控基因表达等。课题组前期研究发现，SAFA能结合RAG1。本项目进一步验证SAFA与RAG1的相互作用，探讨其对RAG在细胞核中分布的影响；探明SAFA在早期B细胞中结合的基因；通过敲低HEK293T和前B细胞(preB)中的SAFA，探讨其对V(D)J重组的影响。免疫共沉淀实验结果进一步表明，SAFA不与RAG2相互作用，但能与RAG1相互作用。细胞免疫荧光结果显示，在HEK293T细胞中过表达RAG1，RAG1在细胞核中聚集，RAG2抑制RAG1的聚集，而SAFA促进其聚集。利用流式细胞术分选proB细胞，进行染色质免疫共沉淀（CHIP）-seq实验，结果显示，SAFA能够募集重组相关蛋白的基因。敲低HEK293T细胞中的SAFA，RAG介导的质粒重组增强。敲低小鼠preB细胞的SAFA，轻链重组和RAG1表达均增强。以上结果表明，SAFA通过调控重组相关蛋白的表达，以及调控RAG1在细胞核中的分布，进而负调控RAG介导的V(D)J重组。本研究阐明了新的V(D)J重组的调控机制，并为进一步探讨重组的分子调控机制提供了新的研究思路。