SOX3/Sox3 gene represents one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Despite the mounting evidence that SOX3 is the key player in early developmental gene regulation, little is known about the transcriptional regulation of the Sox3 gene itself. In this thesis, for the first time, we have performed analysis of the molecular mechanisms underlying the transcriptional regulation of SOX3 gene expression. Using primer extension, we have identified the transcription start point of this gene, 252 nt upstream of the ATG codone. Using promoter–reporter constructs, we have determined the minimal SOX3 promoter region (-219/+67) that confers the basal promoter activity in NT2/D1 cells. Deletion analysis of the minimal promoter revealed two cis regulatory regions (-219/-100 and +27/+67) that are necessary for SOX3 promoter activity. Also, in this study we have identified two regions, -427/-219 and +67/+286, that harbor positive cis regulatory elements necessary for both, optimal and retinoic acid (RA)-inducible promoter activity of the SOX3 gene. By additional functional analysis we have narrowed down the region required for RA induction of this gene to fragment -427/-293. Comparative analysis of the promoter sequences of SOX3 orthologues showed that binding sites for transcription factors Sp1, USF, NF-Y and CREB , as well as, TATA box are conserved in both position and sequence among all analysed mammalian orthologues. Data presented in this thesis, for the first time, suggest that transcription factors Sp1, USF1, NF-Y, CREB and MAZ function as transcriptional activators of the SOX3 gene expression. On the other hand, the presented results indicate that ZBP-89 transcription factor inhibits the response of SOX3 promoter upon retinoic acid induction. In this study, we have performed the first characterization of the human SOX3 promoter and identified transcription factors involved in regulation of its expression. Results presented in this thesis suggest that transcriptional regulation of the SOX3 gene depends on the combined action of distinct regulatory modules within the promoter region of this gene. minimalni promotor, SOX3, Sp1, USF1, NF-Y, CREB, MAZ, ZBP-89 minimal promoter, SOX3, Sp1, USF1, NF-Y, CREB, MAZ, ZBP-89

Characterization of the basal promoter of the human SOX3 gene and identification of the regulatory elements responsible for the SOX3 gene induction by retinoic acid

[N. Kovačević Grujičić]

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Биологија - молекуларна биологија / Bbiology - molecular biology

SOX3/Sox3 gen je jedan od najranijih neuralnih markera kod kičmenjaka, koji determiniše neuronalni tip ćelijske diferencijacije. Iako je SOX3 gen ključni regulator ranih faza embrionalnog razvića, do skora je u literaturi postojalo malo podataka o mehanizmima regulacije aktivnosti ovog gena. U ovoj tezi su, po prvi put, analizirani molekularni mehanizmi uključeni u transkripcionu regulaciju ekspresije SOX3 gena. Metodom elongacije reverznog oligonukleotida određen je start transkripcije ovog gena, 252 nukleotida uzvodno od ATG kodona. Funkcionalnom analizom definisan je minimalni promotorski region (-219/+67) neophodan za bazalnu transkripcionu aktivnost SOX3 gena u NT2/D1 ćelijama. Delecionom analizom minimalnog promotora identifikovana su dva cis regulatorna regiona, -219/-100 i +27/+67, koja su neophodna za aktivnost SOX3 promotora. Takođe, identifikovana su i dva regiona (-427/-219 i +67/+286) koji sadrže pozitivne cis regulatorne elemente neophodne, kako za optimalnu aktivnost, tako i za indukciju SOX3 promotora retinoičnom kiselinom. Dodatna funkcionalna analiza je ukazala da je fragment -427/-293 neophodan za inducibilnost promotora ovog gena. Takođe, poređenje sekvenci je pokazalo da su mesta za vezivanje Sp1, USF, NF-Y i CREB proteina, kao i TATA boks, očuvani i po nukleotidnom sastavu i po poziciji u minimalnim promotorskim regionima kod svih analiziranih SOX3 ortologa sisara. Funkcionalna analiza je po prvi put pokazala da su transkripcioni faktori Sp1, USF1, NF-Y, CREB i MAZ transkripcioni aktivatori ekspresije SOX3 gena. S druge strane, rezultati prikazani u ovoj tezi su ukazali da transkripcioni faktor ZBP-89 inhibira odgovor SOX3 promotora na indukciju retinoičnom kiselinom. Ova teza predstavlja prvu funkcionalnu karakterizaciju promotorskog regiona SOX3 gena čoveka, kao i identifikaciju transkripcionih faktora uključenih u regulaciju ekspresije ovog gena. Posebno treba istaći da rezultati prikazani u ovoj tezi ukazuju na kompleksnu, modularnu prirodu cis regulatornih elemenata odgovornih za transkripcionu regulaciju SOX3 gena.

SOX3/Sox3 gene represents one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Despite the mounting evidence that SOX3 is the key player in early developmental gene regulation, little is known about the transcriptional regulation of the Sox3 gene itself. In this thesis, for the first time, we have performed analysis of the molecular mechanisms underlying the transcriptional regulation of SOX3 gene expression. Using primer extension, we have identified the transcription start point of this gene, 252 nt upstream of the ATG codone. Using promoter–reporter constructs, we have determined the minimal SOX3 promoter region (-219/+67) that confers the basal promoter activity in NT2/D1 cells. Deletion analysis of the minimal promoter revealed two cis regulatory regions (-219/-100 and +27/+67) that are necessary for SOX3 promoter activity. Also, in this study we have identified two regions, -427/-219 and +67/+286, that harbor positive cis regulatory elements necessary for both, optimal and retinoic acid (RA)-inducible promoter activity of the SOX3 gene. By additional functional analysis we have narrowed down the region required for RA induction of this gene to fragment -427/-293. Comparative analysis of the promoter sequences of SOX3 orthologues showed that binding sites for transcription factors Sp1, USF, NF-Y and CREB , as well as, TATA box are conserved in both position and sequence among all analysed mammalian orthologues. Data presented in this thesis, for the first time, suggest that transcription factors Sp1, USF1, NF-Y, CREB and MAZ function as transcriptional activators of the SOX3 gene expression. On the other hand, the presented results indicate that ZBP-89 transcription factor inhibits the response of SOX3 promoter upon retinoic acid induction. In this study, we have performed the first characterization of the human SOX3 promoter and identified transcription factors involved in regulation of its expression. Results presented in this thesis suggest that transcriptional regulation of the SOX3 gene depends on the combined action of distinct regulatory modules within the promoter region of this gene.

minimalni promotor, SOX3, Sp1, USF1, NF-Y, CREB, MAZ, ZBP-89

minimal promoter, SOX3, Sp1, USF1, NF-Y, CREB, MAZ, ZBP-89

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