从化疗脱发揭示Shh基因表达调控的新机制

The regulation of Shh gene expression in space and time is an important issue in developmental biology. In our previous work, we have shown that chemotherapy drugs damage normal tissue including avian feather, mouse hair and human hair via down-regulation of Shh gene expression. We further showed that (1) chemo-drugs inhibit Shh gene expression in limb bud development and in cancer cells; (2) phosphoproteomics analysis revealed ERK signaling is activated by chemo-drugs which then down-regulates Shh gene expression. The transcription factor involved could be FOS; however, the exact mechanism remains unclear. We hypothesize that chemo-drugs inhibit Shh gene expression via the ERK-Fos signaling axis. Here we propose to (1) identify the upstream signaling events and key transcription factors that inhibit Shh gene expression; (2) identify the tissue-specific enhancers that mediate Shh gene expression in skin/hair follicles through ATAC-seq, CHIP-seq and sequence homologous analysis of human, mouse, and chicken genomes, and contruct enhancer reporter mice to confirm the results; (3) test the functional relevance of the identified cis- and trans- factors in the side effects of chemotherapy, and hair loss in particular via dCas9 interference of enhancer function, and CRISPR/Cas9-mediated enhancer editing. This study will elucidate novel regulatory mechanisms of Shh gene expression, which will be relevant to hair development, its damage responses and protection.

Shh基因的表达调控是发育生物学中广受关注的问题。前期工作表明，化疗药物引起鸡羽毛发育损伤，小鼠和人的脱发等，Shh基因表达的下调起到了关键作用。进一步：（1）化疗药物还影响肢芽发育和肿瘤细胞中Shh基因的表达；（2）磷酸化蛋白质组分析表明，化疗药物活化ERK信号并由此抑制Shh基因表达；其中的转录因子可能是Fos。我们猜想：化疗药物通过ERK-Fos信号轴抑制Shh基因表达。本计划将（1）明确化疗抑制Shh基因表达的信号通路和关键转录因子；（2）通过ATAC-seq/CHIP-seq及分析人、鸡、小鼠的序列同源性，构建增强子报告基因小鼠，明确调控毛囊Shh基因表达的组织特异增强子；（3）通过dCas9干扰增强子功能，及CRISPR/Cas9方法构建增强子编辑小鼠，验证相关顺/反调控元件对化疗脱发和组织损伤的影响。本研究将揭示Shh基因表达调控的新机制，对毛发发育及损伤防护有一定意义。

本研究的目标是阐述化疗脱发的原理，其中化疗药物是如何抑制SHH基因表达。已知SHH基因表达可能包含远端增强子的作用，而且化疗药物引起的转录因子的变化也不清楚。因此，本研究需要阐述影响SHH基因表达的顺式元件和反式因子两个方面。应用小鼠毛发和临床化疗患者毛发样品，发现化疗引起毛囊中SHH基因表达下降，其中包括MAPK/ERK2信号通路的激活（Haslam et al., J Invest Dermatol 2021）；进一步，应用细胞培养模型，阐述了EGR1/AP1-SHH信号轴的作用，以及相应的化疗特异增强子（Zhang et al., J Genet Genomics 2022）。最后，结合ATAC-seq/CHIP-seq及构建LacZ报告基因小鼠，发现介导毛囊中SHH基因表达的特异增强子，以及EGR1-SHH信号轴在化疗脱发中的功能意义（结果整理中）。本研究基本阐明了化疗脱发中SHH基因表达调控的原理，很好的完成了预期目标，并为毛发再生修复提供了新的可能（提交专利申请一份）。