The transcription factor Zfp281 serves redundantly with Zfp148 to support CD4+ T cell development and functions [Thymus Visium]

The transcription factor Gata3 is essential both for CD4+ T cell development in the thymus and for the differentiation of Th2 cells, which mediate responses against extracellular parasites and contribute to asthma and allergies. Here we report that the transcription factor Zfp281, in part redundantly with its paralog Zfp148, cooperates with Gata3 to promote CD4+ T cell development and Th2 cell differentiation. Disruption of both factors in T cells resulted in reduced numbers of spleen CD4+ T cells. Additionally, we show that Zfp281 interacts with Gata3, and that Zfp281 binds to Th2 cytokine loci in Th2 cells. Finally, we found that Zfp148 and Zfp281 promote Th2 cytokine expression. During airway inflammation, Zfp148/281-deficient CD4+ T cells failed to express Th2 cytokines, despite normal expression of Gata3. Altogether our data identify Zfp148 and Zfp281 as essential for enabling Gata3 functions in both CD4+ T cell development and Th2 function. Tissue sections from FFPE fixed thymi from C57BL/6 and tdKO mice were first destained and decrosslinked according to the 10X Demonstrated Protocol CG000520. Probe hybridization and all subsequent steps were performed as per 10X User guide for Visium CytAssist Spatial Gene Expression (CG000495). In brief, probe hybridization was performed overnight on the destained and decrosslinked tissue sections followed by probe ligation, and CytAssist enabled RNA digestion and tissue removal. Subsequently, probe extension was carried out and the ligation product was eluted, and a pre-amplification step performed. A subset of the pre-amplification product was used for library construction. Quality and concentration of the resulting sequencing library was checked using Bioanalyzer instrument (Agilent 2100).转录因子Gata3对于胸腺内CD4+ T细胞的发育以及Th2细胞的分化均不可或缺；Th2细胞可介导针对胞外寄生虫的免疫应答，并参与哮喘与过敏反应的发生发展。本研究报道，转录因子Zfp281与其旁系同源因子Zfp148存在部分功能冗余，可与Gata3协同促进CD4+ T细胞发育与Th2细胞分化。在T细胞中同时敲除这两个因子，会导致脾脏CD4+ T细胞数量显著降低。此外，本研究证实Zfp281可与Gata3发生相互作用，且Zfp281能够结合Th2细胞中的Th2细胞因子基因座。最后，我们发现Zfp148与Zfp281可正向调控Th2细胞因子的表达。在气道炎症过程中，尽管Gata3的表达水平正常，但Zfp148/281缺陷型CD4+ T细胞无法正常表达Th2细胞因子。综上，本研究数据表明Zfp148与Zfp281是Gata3在CD4+ T细胞发育及Th2细胞功能中发挥正常功能所必需的调控因子。 取自C57BL/6小鼠与tdKO小鼠的福尔马林固定石蜡包埋（FFPE）胸腺组织切片，首先按照10X官方示范方案CG000520完成脱蜡与解交联处理。探针杂交及后续所有步骤均参照10X Visium CytAssist空间基因表达用户指南（CG000495）执行。简言之，经脱蜡、解交联处理的组织切片先进行过夜探针杂交，随后完成探针连接反应；借助CytAssist系统进行RNA酶消化与组织移除操作，继而开展探针延伸反应，洗脱连接产物并进行预扩增步骤。取部分预扩增产物用于测序文库构建。采用安捷伦2100生物分析仪（Agilent 2100）对最终获得的测序文库的质量与浓度进行检测。