Regulação pré- e pós-transcricional do gene Scratch2 durante a neurogênese no tubo neural posterior.

Abstract

Goes, CP. Pre-and Post-transcriptional Regulation of cScratch2 during neurogenesis in the posterior neural tube. [Doctoral thesis (Systems Biology)]. Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo; 2019. Scratch2 is a transcription factor expressed in early post-mitotic neural cells that has a conserved role in survival, differentiation and migration. Scrt2 is expressed in a very restricted population of cells in the inner layer of the intermediate zone in the chick embryo, and this pattern remains conserved in vertebrates. The mechanisms that control its expression remain unknown and could contribute towards to our understanding of gene regulation during neural differentiation. Here we investigate the pre (enhancers and transcription factors) and posttranscriptional (miRNAs) regulatory mechanisms involved in Scrt2 regulation. We performed an in silico analysis and identified 1) a potential conserved cis-element (E1) in both chicken and mouse genome that could be involved in Scrt2 expression; 2) Ascl1, Brn2 and Sox2 as putative upstream TFs in the gene cascade, and 3) binding sites in the Scrt2 3'UTR for miR-125b, -200b, -429, -211 and -204. We tested the biological function of the candidate regulatory region E1 and 3'UTR through electroporation in chick embryos. Modulation of E1driven transcription via TFs was tested in luciferase reporter assay in HEK293T cells. We also performed Chromosome Conformation Capture (3C) to verify interactions between E1 and cScrt2 promoter and CRISPR/Cas9 methodology to edit E1 and miRNAs target sites in cScrt2 3'UTR. Our results indicate that E1 is a neural enhancer, driving Scrt2 expression, possibly in the equatorial neural tube domain. The overexpression of selected TFs in the neural tube increased cScr2 expression in the dorsal portion of the neural tube and decreased the size of some differentiated cells subpopulations in the neural tube. Moreover, the CRISPR-mediated edition of miRNAs target sites increased Scrt2 expression, suggesting a possible role for miR-125b and -200b in post-transcriptional regulation of cScrt2. In conclusion, we propose that Scrt2 expression domain is generated by the conjunction of pre and post-transcriptional regulatory mechanisms. In this scenario, E1 could modulate cScrt2 transcription through Ascl1 and Brn2 transcription factors. After transcription, the levels of cScrt2 transcripts would be further delimited by the binding of miR-125b and -200b in the 3'UTR.