Transkripciona regulacija ekspresije humanog SOX14 gena

Abstract

Ekspresija Sox 14 gena detektovana je tokom različitih faza razvića nervnog sistema. U literaturi je poznato da je ekspresija Sox 14 gena miša i pileta dozno zavisna od signalnog molekula Sonic hedgehog (SHH), kao i to da je opšti transkripcioni faktor NF-Y važan za transkripcionu regulaciju humanog SOX14 gena. U ovoj tezi su po prvi put analizirani molekulami mehanizmi uključeni u regulaciju ekspresije humanog SOX14 gena, kao i uticaj FOXA2 i SHH signalnog molekula na njegovu ekspresiju. Metodom elongacije reverznog oligonukleotida odreden je start transkripcije ovog gena, koji se nalazi 251 nukleotid uzvodno od ATG kodona. Korišćenjem promotor-reporterskih konstrukata definisan je minimalni promotorski region SOX14 gena (-470/+201) koji sadrži regulatome elemente neophodne za bazalnu transkripcionu aktivnost u HepG2 ćelijama. Takođe, delecionom analizom optimalnog promotora identifikovan je pozitivni regulatomi region, na poziciji -783/-470. U okviru ovog regulatomog regiona in silico analizom je otkriveno potencijalno vezivno mesto za FOXA2 transkripcioni faktor koje je evolutivno očuvano kod sisara, kako po poziciji tako i po nukleotidnom sastavu. Rezultati prezentovani u ovom radu pokazali su da je tkivno specifični FOXA2 transkripcioni faktor važan za regulaciju ekspresije SOX14 gena u U87MG ćelijama, kao i to da je upravo ovaj transkripcioni faktor odgovoran za posredovanje uticaj a SHH signalnog puta na ekspresiju SOX14 gena u HepG2 ćelijama. Identifikacijom Sox 14 gena kod koze (Capra hircus), pacova (Rattus norvegicus) i govečeta (Bos taurus) dopunjena je baza podataka deponovanih sekvenci za Soxl4 gen kičmenjaka. Uporedna analiza SOX14 ortologa pokazala je izuzetno visok stepen evolutivne očuvanosti ovog proteina. Uporednim mapiranjem SOXB gena kod različitih kičmenjaka izvršena je dopuna predloženog modela evolucije ove grape gena. Ova teza predstavlja prvu funkcionalnu karakterizaciju regulatomog regiona SOX14 gena čoveka, kao i defmisanje uloge transkripcionog faktora FOXA2 koji je uključen u signalni put kojim SHH ostvaraje uticaj na ekspresiju SOX14 gena.

Soxl4 gene expression is detected during different stages of neural development. It was previously shown that expression of Soxl4 gene in spinal cord explants of chick and mouse is regulated by Sonic hedgehog (SHH) in a dose-dependent manner, and that NF-Y transcription factor has important role in the transcriptional regulation of human SOX14 gene. For the first time, we have performed analysis of the molecular mechanisms underlying the transcriptional regulation of SOX14 gene expression, and demonstrated the role of FOXA2 and SHH in up- regulation of SOX14 gene expression. Using primer extension, we have identified the transcription start point of SOX14 gene, placed 251 nt upstream of the ATG codon. Using promoter reporter constructs, we have determined the minimal SOX14 gene promoter region (-470/+201) that confers the basal promoter activity in HepG2 cells. Deletion analysis of the optimal promoter region (-1123/+201) revealed cis regulatory region (-783/-470) that is necessary for SOX14 promoter activity. Within this regulatory region, in silico analysis revealed the presence of consensus binding site for FOXA2 transcription factor which is conserved in both position and sequence among all analyzed mammalian orthologues. The presented results demonstrated that tissue specific transcription factor FOXA2 has important role in regulation of SOX14 gene expression in U87MG cells and also that this transcription factor participates in SHH signaling pathway leading to SOX14 gene activation in HepG2 cells. In this thesis, for the first time, we have identified Soxl4 gene in goat (Capra hircus), cow (Bos taurus) and rat (Rattus norvegicus). Comparative analysis of SOX14 protein in vertebrates revealed remarkable evolutionary conservation. Mapping data presented in this thesis, has not only supported the proposed model of group B Sox genes evolution, but has also revealed additional events that occurred during evolution of those genes in vertebrates. This work represents the first insight into functional characterization of the human SOX14 gene regulatory region as well as signaling pathway implicated in SHH regulation of SOX14 gene expression.