背景:配对框1(PAX1)是一种转录因子,对于咽囊衍生组织的发育至关重要,包括胸腺.PAX1突变在严重联合免疫缺陷(SCID)患者中被鉴定为耳面宫颈综合征2型(OTFCS2)。然而,尽管PAX1在胚胎发育和疾病中的关键作用,对其分子作用模式的详细见解严重不足。
方法:通过荧光素酶报告基因实验研究了PAX1和SCID相关突变体对Wnt信号通路的抑制作用,qRT-PCR和原位杂交在HEK293FT,HCT116细胞和斑马鱼胚胎,分别。进行了免疫共沉淀(co-IP)和蛋白质印迹测定以鉴定PAX1在Wnt信号通路中的作用的分子机制。基于hESC的内胚层分化,流式细胞术,高通量测序数据分析,和qRT-PCR分析用于确定PAX1在内胚层分化过程中的作用。
结果:这里,我们发现PAX1在脊椎动物细胞中抑制经典的Wnt信号通路。机械上,PAX1与SUMOE3连接酶PIASy竞争结合TCF7L2,从而扰乱TCF7L2SUMO化水平,进一步降低其转录活性和蛋白质稳定性。此外,我们发现PAX1在hESC衍生的定形和前肠/咽内胚层细胞中起双重作用,产生胸腺上皮,通过抑制Wnt信号传导。重要的是,我们的数据显示,在SCID患者中发现的PAX1突变显著损害了PAX1对Wnt信号传导的抑制能力.
结论:我们的研究提出了PAX1在规范Wnt信号传导和内胚层分化调节中的一种新的分子作用模式,从而为PAX1相关SCID的分子基础提供见解,提供更好的理解PAX1在胚胎发生中的行为。
BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (
SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.
METHODS: The repressing roles of PAX1 and
SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1\'s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.
RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in
SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.
CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated
SCID, offering better understanding of the behavior of PAX1 in embryogenesis.