探討修復因子Fancd2在R-loop形成及免疫球蛋白類型轉換的調控機制

Abstract

B 細胞經由輔助型T 細胞 (T helper cell) 活化後會進行體細胞超突變 (somatic hyper-mutation, SHM) 以及免疫球蛋白類型轉換 (class switch recombination, CSR)。 體細胞超突變 (somatic hyper-mutation, SHM) 會在免疫球蛋白重鏈及輕鏈 (immunoglobulin heavy chain and light chain) 的可變區 (variable region) 產生高機率的點突變，增加抗體的多樣性進而對抗原的親和力產生影響，而對抗原親和力較強的抗體會逐漸增加，此為抗體親和力成熟過程。免疫球蛋白類型轉換 (class switch recombination, CSR) 會改變免疫球蛋白重鏈 (immunoglobulin heavy chain) 的恆定區 (constant region) ，但可變區 (variable region) 保持不變，因此抗體對抗原的親和力不會改變，而改變抗體的恆定區 (constant region) 會使B 細胞初始表現的IgM 及IgD 轉換成IgG、IgE 或IgA 等其他不同種類的抗體，而在這個轉換的過程中，在免疫球蛋白重鏈 (immunoglobulin heavy chain) 上的轉換區 (switch region) 會產生R-loop。R-loop 是由RNA:DNA hybrid 及single-stranded DNA 所組成的結構，然而，在細胞中過多的R-loop 累積會造成DNA 雙股斷裂，進而影響基因的穩定性，因此，R-loop 需要被嚴格的調控。在本篇論文中，我們致力於找尋在 免疫球蛋白類型轉換 (class switch recombination, CSR) 中調控R-loop 的因子，其中，我們發現Fancd2 (Fanconi anemia group D2) 可能參與調控免疫球蛋白類型轉換 (class switch recombination, CSR) 中的R-loop 形成過程進而影響抗體的多樣性。

Immunoglobulin rearrangements include somatic hyper-mutation (SHM) and class switch recombination (CSR). Both of them are important for antibody diversification, a process to help fighting against and protecting cells from all types of environmental antigens and threats. The R-loop is a three-stranded structure of nucleic acids consisting of a RNA:DNA hybrid and displaced single-stranded DNA (ssDNA). It is involved in various biological processes, such as recruitment of chromatin regulators, heterochromatin formation, and transcription termination. On the other hand, excess of R-loops formation would cause DNA break, replication fork stalling, as well as stalling and collisions between transcription and replication complexes. All of the above might lead to higher genome instability and evolution rate, thus R-loop formation needs to be strictly constrained and regulated. Here, we focus on searching for key R-loop regulating factors during the process of class switch recombination (CSR) at DNA encoded for the immunoglobulin heavy-chain (IgH), an essential process for B cell maturation. When B cells receive cytokine stimuli, R-loops form at switch region leading to DNA deletion and replacement of downstream DNA with another constant region. In our studies, we first found that Fancd2, a repair factor whose mutations are genetically linked to Fanconi anemia, might play an important regulatory role on R-loop formation, subsequently CSR process and antibody diversification.