Transkripciona regulacija ekspresije humanog SOX18 gena

Abstract

Humani SOX18 gen pripada familiji SOX gena koji kodiraju DNK-vezujuće proteine koji imaju ulogu transkripcionih faktora i arhitektonskih komponenti hromatina. SOX18 gen ima važnu ulogu u regulaciji vaskularnog razvića, i učestvuje u specifikaciji i diferencijaciji endotelijalnih ćelija, angiogenezi i limfangiogenezi. Mutacije u SOX18 genu kod čoveka su povezane sa sidromom Hipotrihoza-Limfedem-Talengiektazija (eng. Hypotrichosis-Lymphedema-Talengiectasia) čije su karakteristike poremećaji u razviću dlake, vaskularnog i limfnog sistema. Iako do danas ima dosta podataka o ulozi SOX18 gena u procesima vaskularogeneze, angiogeneze i limfangiogeneze, još uvek se malo zna o molekularnim mehanizmima uključenim u regulaciju ekspresije ovog gena. Osnovni ciljevi, istraživanja predstavljenog u ovoj tezi, bili su analiza transkripcione regulacije ekspresije humanog SOX18 gena, kao i analiza uticaja pro-angiogenetskih faktora i inhibitora angiogeneze na ekspresiju ovog gena u endotelijalnim ćelijama. Ispitivanja transkripcione regulacije su obuhvatala analizu uloge određenih transkripcionih faktora u regulaciji aktivnosti SOX18 promotora, kao i u regulaciji endogene ekspresije SOX18 gena. Transkripciona regulacija je ispitivana u dva model sistema: HeLa ćelijama, koje su korišćena kao tumorski model sistem i EA.hy926 ćelijama, koje su korišćene kao endotelijalni model sistem. In silico analizom su identifikovana potencijalna vezivna mesta za različite transkripcione faktore koji mogu biti uključeni u regulaciju ekspresije SOX18 gena. Za dalju funkcionalnu analizu odabrani su transkripcioni faktori Sp3, ZBP-89, NF-Y i EGR1. Na osnovu eksperimenata smanjene elektroforetske pokretljivosti, funkcionalnih/mutacionih analiza, i analiza ekspresije u nativnom kontekstu, pokazano je da su transkripcioni faktori Sp3 i ZBP-89 negativni, a NF-Y i EGR1 pozitivni regulatori transkripcije humanog SOX18 gena. Na ovaj način pokazana je funkcionalna veza između transkripcionih faktora Sp3, ZBP-89, NF-Y i EGR1 i SOX18 gena i omogućeno je bolje razumevanje, dela, transkripcione kontrole ekpresije ovog gena...

Human SOX18 gene belongs to the family of SOX genes that encode DNA-binding proteins, which display properties of both transcription factors and architectural components of chromatin. SOX18 gene plays important role in vascular development, endothelial cell specification and differentiation, angiogenesis and lymphangiogenesis. Mutations in human SOX18 gene are associated with Hypotrichosis-Lymphedema-Talengiectasia syndrome, characterized by defects in hair, vascular and lymphatic development. Despite the mounting evidence that SOX18 gene is an important player in vasculogenesis, angiogenesis and lymphangiogenesis, little is known about molecular mechanisms involved in the regulation of its expression. The aim of this study was to investigate transcriptional regulation of the human SOX18 gene expression, as well as the effecs of pro- and anti-angiogenic factors on SOX18 expression in endothelial cells. Analyses of transcriptional regulation included identification of transcription factors that are involved in regulation of SOX18 promoter activity, as well as in regulation of endogenous SOX18 expression. Two model systems were used: HeLa cells, as a tumor model system, and EA.hy926 cells, as an endothelial model system. Several putative transcription factor binding sites were identified by in silico analysis of the SOX18 promoter sequence. Transcription factors Sp3, ZBP-89, NF-Y and EGR1 were selected for further functional analysis. By in vitro binding assays, functional/mutagenesis assays and analyses of endogenous SOX18, it has been shown that transcription factors Sp3 and ZBP-89 act as negative regulators, while NF-Y and EGR1operate as positive regulators of SOX18 gene expression. These results gave first functional link between Sp3, ZBP-89, NF-Y and EGR1 transcription factors and SOX18 gene, thus providing better understanding of transcriptional regulation of SOX18 gene expression...