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Transcription of the human 5-Hydroxytryptamine receptor 2B (HTR2B) gene is under the regulatory influence of the transcription factors NFI and RUNX1 in human uveal melanoma

ABSTRACT: Introduction: Uveal melanoma (UM) is the most common type of primary intraocular tumor in the adult population. UM will propagate to the liver as the first metastatic site. Once this organ is invaded, survival becomes a matter of months for the patient as no treatment has proved effectiveness. Among the candidates from the class II gene signature, the serotonin receptor-encoding gene HTR2B appears to be the most discriminating as its expression strongly increases in the tumors of UM patients that will progress toward liver metastases. Our study aims at characterizing the molecular mechanisms that lead to this aberrant expression of HTR2B in metastatic UM cell lines.Materials and Methods: Expression of HTR2B was monitored by microarrays and validated by Western blot in a variety of UM cell lines. A 2 Kbp segment covering both the promoter and 5 '-flanking region of the HTR2B gene was cloned upstream the CAT reporter gene in the plasmid pCATbasic (to yield the -2000/HTR2B construct). Deletion mutants were generated by double digestion of the -2000/HTR2B construct with restriction enzymes and transfected, along with the parental -2000/HTR2B construct, in UM cell lines. The genomic areas of interest were 5’ end-labeled and used as probes in electrophoretic mobility shift assays (EMSAs). DMS methylation footprinting was also used to precisely position the DNA target sites for transcription factors (TFs) that bind the HTR2B regulatory regions.Results and Discussion: Transfection analyses revealed that the upstream regulatory regions of HTR2B promoter is made up of a combination of alternative positive and negative regulatory elements. Repressive regions have an abnormally elevated number of target sites for the TFs NFI. EMSA analyses provided evidence that both the NFI isoforms NFIC and NFIX could interact with the promoter and the upstream negative regulatory element from the HTR2B gene. In addition, the TF RunX1 was shown by DMS methylation interference footprinting to bind a target site from the distal silencer element.Conclusion: This project will help understand better the molecular mechanisms accounting for the abnormal expression of the HTR2B gene in uveal melanoma. In the long term, this study will allow us to identify new potential targets that could help screening patients at risk of progressing to liver metastasis formation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE86915 | GEO | 2016/09/15

SECONDARY ACCESSION(S): PRJNA342951

REPOSITORIES: GEO

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Project description:Î±9b1 is the most recent addition to the integrin family of membrane-bound receptors and consequently remains the one which is the least characterized. To better understand how transcription of the human gene encoding the Î±9 subunit is regulated at the molecular level, we cloned the Î±9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of Î±9 promoter/CAT recombinant plasmids in primary cultured cells and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the Î±9 promoter and positioned the basal Î±9 promoter to within 118 bp from the Î±9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1/Sp3, c-Myb and NFI to the most upstream Î±9 negative regulatory element. The transcription factors Sp1/Sp3 and NFI were found to bind the basal Î±9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Most of all, addition of tenascin-C (TNC), the ligand of Î±9Î²1, to the tissue culture plates prior to cell seeding increased Î±9 transcription whereas it simultaneously decreased expression of the Î±5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the Î±9 and Î±5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing. Primary cultures of human corneal epithelial cells (HCECs; number of replicates: 8), human skin epithelial cells (HSEC; number of replicates: 4), human corneal fibroblast cells (HCFCs; number of replicates: 3), human skin fibroblast cells (HSFCs; number of replicates: 3), human uveal melanocytes (UVM; number of replicates: 3), and various humal uveal melanoma cell lines (T115, T142 and T143; number of replicates: 2-3) were analyzed by gene profiling on microarrays at low culture passages (between passage 2 to 7). HCECs were also cultured on either BSA (number of replicates: 2) or on tenascin-C (number of replicates: 2) and their transcriptome analyzed by gene profiling.

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Transcription of the human 5-Hydroxytryptamine receptor 2B (HTR2B) gene is under the regulatory influence of the transcription factors NFI and RUNX1 in human uveal melanoma

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