Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses. Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of BCL6, which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells. Overall design: ChIP-Seq analyses using naïve FO B cells,with rabbit IgG, anti-IRF8, anti-PU.1, anti-H3K27Ac. RNA-Seq analyses using total RNA from FACS-purified FO B cell of WT and DKO mice.

干扰素调节因子（Interferon Regulatory Factor, IRF）与Ets家族转录因子，可调控一系列参与免疫细胞发育与功能的基因的表达。然而，由于家族成员间存在功能冗余，且对多种细胞谱系具有广泛调控作用，目前学界对各家族成员的分子调控机制的认知仍较为有限。本研究通过Mb1-Cre系统在B细胞谱系中条件性敲除floxed Irf8与Spi1（后者编码PU.1蛋白），构建双基因敲除小鼠（DKO小鼠），结果显示该小鼠的滤泡型与生发中心（GC）B细胞发育存在严重缺陷。DKO小鼠的抗体类别转换重组与抗体亲和力成熟过程亦出现明显受损。通过RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq）分析，我们发现滤泡型与活化B细胞中存在IRF8与PU.1的特异性靶基因。DKO小鼠的B细胞中，维持滤泡型B细胞身份与生发中心发育所必需的靶基因表达水平显著下调。此外，本研究证实，对生发中心B细胞发育至关重要的BCL6基因的体内表达依赖于IRF8与PU.1，这为二者在生发中心B细胞发育中的关键作用提供了分子机制层面的合理解释。整体实验设计：采用初始滤泡型B细胞（naïve FO B cells）进行ChIP-seq分析，所用抗体包括兔IgG、抗IRF8、抗PU.1及抗H3K27Ac；同时提取经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化的野生型（WT）与DKO小鼠的滤泡型B细胞总RNA，开展RNA-seq分析。