Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany.
PLoS One. 2018 Aug 2;13(8):e0200964. doi: 10.1371/journal.pone.0200964. eCollection 2018.
The TBX18 transcription factor is a crucial developmental regulator of several organ systems in mice, and loss of its transcriptional repression activity causes dilative nephropathies in humans. The molecular complexes with which TBX18 regulates transcription are poorly understood prompting us to use an unbiased proteomic approach to search for protein interaction partners. Using overexpressed dual tagged TBX18 as bait, we identified by tandem purification and subsequent LC-MS analysis TBX18 binding proteins in 293 cells. Clustering of functional annotations of the identified proteins revealed a highly significant enrichment of transcriptional cofactors and homeobox transcription factors. Using nuclear recruitment assays as well as GST pull-downs, we validated CBFB, GAR1, IKZF2, NCOA5, SBNO2 and CHD7 binding to the T-box of TBX18 in vitro. From these transcriptional cofactors, CBFB, CHD7 and IKZF2 enhanced the transcriptional repression of TBX18, while NCOA5 and SBNO2 dose-dependently relieved it. All tested homeobox transcription factors interacted with the T-box of TBX18 in pull-down assays, with members of the Pbx and Prrx subfamilies showing coexpression with Tbx18 in the developing ureter of the mouse. In summary, we identified and characterized new TBX18 binding partners that may influence the transcriptional activity of TBX18 in vivo.
TBX18 转录因子是小鼠几种器官系统发育的关键调节因子，其转录抑制活性的丧失会导致人类扩张性肾病。与 TBX18 调节转录的分子复合物知之甚少，这促使我们使用无偏 proteomic 方法来寻找蛋白相互作用伙伴。我们使用过表达的双标签 TBX18 作为诱饵，通过串联纯化和随后的 LC-MS 分析，在 293 细胞中鉴定到 TBX18 结合蛋白。鉴定到的蛋白质的功能注释聚类显示转录共因子和同源盒转录因子的高度显著富集。使用核募集测定和 GST 下拉实验，我们验证了 CBFB、GAR1、IKZF2、NCOA5、SBNO2 和 CHD7 在体外与 TBX18 的 T 盒结合。在这些转录共因子中，CBFB、CHD7 和 IKZF2 增强了 TBX18 的转录抑制，而 NCOA5 和 SBNO2 则剂量依赖性地缓解了抑制。所有测试的同源盒转录因子在 pull-down 实验中与 TBX18 的 T 盒相互作用，Pbx 和 Prrx 亚家族的成员在小鼠输尿管发育过程中与 Tbx18 共表达。总之，我们鉴定并表征了新的 TBX18 结合伙伴，这些伙伴可能影响 TBX18 在体内的转录活性。
