KRAB關聯蛋白-1(KAP1)在B細胞分化過程中所扮演角色

Abstract

KRAB-associated protein 1 (KAP1)在細胞中作為染色質調控因子，透過和核小體去乙醯酶 (NuRD)複合體及H3K9組蛋白甲基轉移酶等組蛋白修飾因子協同作用來使得染色質轉變為較緊密的異染色質，進而抑制基因表達。過去的研究文獻中指出KAP1會調控第17型輔助T細胞(Th17)的發育，以及影響調節型T細胞的功能。在B細胞中，KAP1缺失則被發現會造成邊緣區B細胞(Marginal zone B cell)及濾泡型B細胞(Follicular B cell)數目減少，說明KAP1參與在B細胞早期發育過程中。然而，KAP1在漿細胞分化中所扮演的角色目前仍未釐清。在本研究中，我們以Trim28f/f IgHCγ1-Cre小鼠為模式，探討KAP1基因(Trim28)在生發中心B細胞(Germinal center B cell)中被剔除後對漿細胞分化造成之影響。我們發現在T-dependent抗原刺激下，KAP1剔除後分化出的漿細胞減少，同時伴隨著較少的生發中心B細胞。下一步欲探討的問題是KAP1對漿細胞生產免疫球蛋白(Immunoglobulin, Ig)的功能是否有影響。結果顯示在初級及次級T-dependent免疫反應中，Trim28基因剔除後之漿細胞產生的IgG數量皆顯著降低。然而在in vitro實驗中，則可觀察到生發中心B細胞及漿細胞在刺激過後之複製率及細胞凋亡率並無差異，說明其並非造成漿細胞及生發中心B細胞數量降低之原因。漿細胞分化過程中的主要調控因子為B lymphocyte-induced maturation protein-1 (Blimp-1)，我們發現到在KAP1剔除之CD138+漿細胞中的Blimp-1 蛋白質及RNA皆下降，說明KAP1對Blimp-1的表現有重要影響，進而可影響到漿細胞之分化及功能。在蛋白質交互作用檢測(proximity ligation assay, PLA)及免疫共沈澱實驗(co-immunoprecipitation)中顯示，KAP1和Blimp-1主要在漿細胞核中結合，說明KAP1可能透過和Blimp-1結合並在細胞核中調控基因表現。為探討KAP1是否會調控Prdm1 (Blimp-1基因)表現，我們進行染色質免疫沈澱定量聚合酶鏈鎖反應實驗(ChIP-qPCR)，卻發現在漿細胞中KAP1並未結合至已知作用於Prdm1之基因位置，然而此結果不能排除KAP1藉由結合至遠端之基因位置進行調控或是有更多蛋白質參與調控的可能性。綜合而言，本研究中發現到KAP1可能在漿細胞中透過和Blimp-1結合來調控Blimp-1本身的表現，並影響漿細胞之分化過程，而KAP1在漿細胞中調控基因的機制還需要更進一步的研究探討。

KRAB-associated protein 1 (KAP1) is a chromatin remodeling regulator that cooperates with histone modifiers, such as Nucleosome Remodeling Deacetylase (NuRD) complex or H3K9 methyltransferase SETDB1 to form heterochromatin, thus repress the gene expression. Recent reports also indicated that KAP1 mainly functions as a transcriptional activator to regulate T-helper 17 (Th17) cell development and the function of regulatory T cells. KAP1 also regulates early B cell development through maintaining the balance of homeostasis of marginal zone and follicular B cells. However, the role of KAP1 in plasma cell differentiation is still unknown. Hence, in the study, we deleted KAP1 gene, Trim28, in germinal center (GC) B cells by crossing Trim28f/f mice with IghCγ1-Cre mice. We found that GC-specific depletion of KAP1 impairs plasma cell generation accompanying with reduced GC B cells in vivo and in vitro. We next sought to investigate the function of KAP1 in Ig secretion. We found a significant reduction of IgG production in GC-specific Trim28 deletion mice after primary and secondary T-dependent immunizations. The Trim28-depleted plasma cells and GC B cells showed similar proliferation and apoptotic rates with the control cells in vitro. However, both protein and RNA levels of Blimp-1, which is the master regulator of plasma cell differentiation, are downregulated in KAP1 depleted CD138+ plasma cells, which implies that KAP1 is important for Blimp-1 expression and thus leads to the differentiation of plasma cells. The proximity ligation assay (PLA) and Co-IP experiments also provided the evidences that KAP1 interacts with Blimp-1 in the nucleus, suggesting that KAP1 may involve in the regulation of genes by associating with Blimp-1 in plasma cells. To further identified whether KAP1 regulates Prdm1 expression, we performed chromatin immunoprecipitation-qPCR (ChIP-qPCR), and revealed that KAP1 did not bind to the intron and promoter region of Prdm1 in plasma cells, but we couldn’t exclude the possibility that KAP1 may regulate Prdm1 expression through a binding sequence that was far from the Prdm1 gene or the interaction with other protein. The detailed mechanisms of how KAP1 regulate gene expression in plasma cells need further studies. Taken together, our results suggest that KAP1 associates with Blimp-1 and may involve in the regulation of Blimp-1 and thus promotes plasma cell differentiation.