Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 TFs. Mus musculus

Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo, and furthermore, revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4_/_ T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4_/_ mice showed impaired IL- 21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4. Overall design: Affymetrix expression data: Prepare CD4+ T cells from spleen. CD4+ T cells were preactivated, rested, and treated with IL-21 for 1, 6, and 24 hours. ChIP-seq data: Profiling of IRF4 and Stat3 binding with and without IL-21 stimulation in wild type and IRF4 KO mice.

白细胞介素-21（Interleukin-21, IL-21）是一种多效性细胞因子，可诱导由Prdm1编码的转录因子BLIMP1（transcription factor BLIMP1）的表达，而BLIMP1能够调控浆细胞分化与T细胞稳态。本研究鉴定到位于Prdm1基因下游的IL-21应答元件，该元件可结合转录因子STAT3与IRF4，二者对于Prdm1的最佳表达不可或缺。对STAT3与IRF4结合位点的全基因组ChIP测序（ChIP-Seq）分析显示，绝大多数经IL-21诱导结合STAT3的区域在体内同时可结合IRF4；此外，该分析还揭示，在Prdm1基因中至关重要的非经典TTCnnnTAA GAS基序，广泛存在于STAT3的结合位点中。将全基因组表达芯片数据与结合位点数据进行比对后发现，绝大多数受IL-21调控的基因均与STAT3-IRF4联合结合位点相关，而非单纯的STAT3结合位点。相应地，对IRF4敲除（Irf4⁻/⁻）T细胞的ChIP测序分析显示，经IL-21处理后，STAT3的结合水平显著降低；而IRF4敲除小鼠体内IL-21诱导的滤泡辅助T细胞（Follicular helper T cell, Tfh）分化过程也受到损伤。上述结果表明，STAT3与IRF4可广泛协同调控基因表达。

整体实验设计：
Affymetrix表达芯片数据：从脾脏中分离CD4+ T细胞，将CD4+ T细胞进行预激活、静置培养后，分别用IL-21处理1小时、6小时及24小时。
ChIP测序数据：在野生型与IRF4敲除（IRF4 KO）小鼠中，分别检测经IL-21刺激与未刺激状态下IRF4与Stat3的结合情况。