Department of Biology, University of Fribourg, Fribourg, Switzerland.
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, United States of America.
PLoS Genet. 2019 Jul 12;15(7):e1008269. doi: 10.1371/journal.pgen.1008269. eCollection 2019 Jul.
Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.
眼组织的发育是由一组称为视网膜决定网络（RDN）的保守转录因子启动的。在果蝇 Drosophila melanogaster 中，锌指转录因子 Glass 直接作用于 RDN 的下游，控制光感受器和非光感受器细胞的身份。在发育过程中，时空基因表达的严格控制是细胞命运决定以及分化组织维持的关键特征。然而，控制玻璃基因表达的分子机制在很大程度上仍然未知。我们在这里确定了控制玻璃基因表达的复杂调控机制。通过将不同的细胞类型特异性和一般增强子与抑制元件结合在一起，将 recapitulate glass 表达的所有信息包含在一个紧凑的 5.2 kb 顺式作用基因组元件中。此外，该基因座的不成熟 RNA 在实际玻璃翻译起始上游包含一个替代的小开放阅读框（smORF），导致小肽而不是三个可能的 Glass 蛋白同工型。基于 CRISPR/Cas9 的诱变表明，smORF 对于功能感光器的形成不是必需的，但能够减弱玻璃表达错误的影响。此外，通过基因组编辑生成消除一个或两个同工型的玻璃基因座显示，只有三种蛋白质中的一种对于功能感光器的形成是至关重要的，而去除另外两种同工型不会导致发育或感光器功能缺陷。我们的研究结果表明，通过靶向玻璃转录本的关键特征进行操纵，眼睛的发育和功能在很大程度上不受影响，这表明存在强烈的选择压力，允许形成一个功能正常的眼睛。
