Defining STAT5 target genes in thymic precursor cells at pre- and post-commitment stages

STAT5 is critical for inducing lineage specifying transcription factors, establishing effector gene programs, upregulating IL-2Rα (also known as CD25), and supporting cell survival in response to environmental cues in mature T cells. Although it has been shown that IL-7-induced STAT5 signaling is also essential for survival of thymic precursor cells, the exact roles of STAT5A and STAT5B and their target genes that contribute to the developmental program in early thymic development have been unknown. To define STAT5 target genes in these early T-cell progenitors, we have performed CRISPR-Cas9 mediated acute deletion of Stat5a and Stat5b in combination. We first carried out preliminary experiments to determine the stages in which IL-7 could induce maximal STAT5 phosphorylation in thymic double negative (DN) cells (B. Shin, unpublished data). We then focused on genes responding to loss of STAT5 in two different stages: CD25+ Bcl11b- pre-commitment stage, and CD25+ Bcl11b+ post-commitment stage. Of note, to generate these early pro-T cells in OP9-DLL1 coculture, we exploited bone-marrow precursor cells expressing a Bcl2 transgene to minimize survival defects due to loss of STAT5A and STAT5B. Our data show that STAT5 is necessary, in both pre- and post-commitment stages, for optimal expression of the IL-2Rα gene (Il2ra), which is constitutively expressed in normal DN2 and DN3 pro-T cells. This dataset further defines a distinct set of additional genes responding more strongly to loss of STAT5 in pro-T cells. The linked manuscript by R. Spolski et al. and other datasets referenced therein include data documenting the genomic occupancy of STAT5B in corresponding stages. In summary, our data define STAT5-responsive target genes during early T development. Bone-marrow progenitor cells from B6.CRISPR-Cas9;Bcl2;Bcl11b-mCherry/mCherry transgene expressing mice were incubated with IL-7, Flt3-ligand (Flt3l), and SCF for overnight. Three sgRNAs against Stat5a and three sgRNAs against Stat5b were retrovirally introduced, and infected cells were cultured on OP9-DLL1 stromal cells with IL-7 and Flt3l. After 6 days post infection, Lineage marker (Lin)- infection marker+ CD25+ mCherry- (Bcl11b reporter negative, pre-commitment stage) and CD25+mCherry+ (Bcl11b reporter negative, post-commitment stage) cells were FACS sorted and subjected to bulk RNA-seq.

信号转导与转录激活因子5（STAT5）在成熟T细胞中，对于诱导谱系特异性转录因子、建立效应基因程序、上调IL-2受体α链（IL-2Rα，又称CD25），以及响应环境信号支持细胞存活均发挥关键作用。尽管已有研究证实，白细胞介素7（IL-7）介导的STAT5信号通路对胸腺前体细胞的存活同样不可或缺，但STAT5A与STAT5B的确切功能，以及二者在胸腺早期发育程序中所涉及的靶基因，此前仍未明确。
为明确此类早期T细胞祖细胞中的STAT5靶基因，我们联合采用CRISPR-Cas9介导的Stat5a与Stat5b急性敲除策略。我们首先开展预实验，以确定IL-7可诱导胸腺双阴性（DN）细胞达到最大STAT5磷酸化的阶段（B. Shin，未发表数据）。随后我们聚焦于两个不同阶段中响应STAT5缺失的基因：CD25+ Bcl11b- 预定向阶段，以及CD25+ Bcl11b+ 定向后阶段。值得注意的是，为在OP9-DLL1共培养体系中获取此类早期前T细胞，我们利用表达Bcl2转基因的骨髓前体细胞，以最小化因STAT5A与STAT5B缺失导致的存活缺陷。
我们的数据显示，在预定向与定向后两个阶段中，STAT5对于IL-2Rα基因（Il2ra）的最优表达是必需的——该基因在正常DN2与DN3前T细胞中呈组成型表达。本数据集进一步明确了前T细胞中另一组对STAT5缺失响应更为显著的独特基因集合。R. Spolski等人的关联论文及其中引用的其他数据集，包含了对应阶段中STAT5B的基因组结合位点数据。
综上，我们的数据明确了早期T细胞发育过程中响应STAT5的靶基因。实验方法如下：我们将来自B6.CRISPR-Cas9;Bcl2;Bcl11b-mCherry/mCherry转基因小鼠的骨髓祖细胞，与IL-7、Flt3配体（Flt3l）及干细胞因子（SCF）共同孵育过夜。将3条靶向Stat5a的单引导RNA（sgRNA）与3条靶向Stat5b的sgRNA通过逆转录病毒导入细胞，随后将感染细胞在OP9-DLL1基质细胞上，以IL-7与Flt3l进行培养。感染后第6天，通过流式细胞术（FACS）分选谱系标志物（Lin）- 感染标志物+ CD25+ mCherry-（Bcl11b报告基因阴性，预定向阶段）及CD25+ mCherry+（Bcl11b报告基因阳性，定向后阶段）细胞，进行批量RNA测序（bulk RNA-seq）。