An in vitro approach to test the possible role of candidate factors in the transcriptional regulation of the RET proto-oncogene

Tiziana Bachetti, Silvia Borghini, Roberto Ravazzolo, Isabella Ceccherini

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Neural crest cells arise from the epithelium of the dorsal neural tube and migrate to various districts giving origin, among others, to sympathetic, parasympathetic, and enteric ganglia. It has been shown that the transcription factors HOX11L1, HOX11L2, MASH1, PHOX2A, and PHOX2B are all necessary, to various extents, to the correct development of the autonomie nervous system. To investigate their possible role in the transcriptional regulation of the RET proto-oncogene, a gene playing a crucial role in correct intestinal innervation, we undertook a specific in vitro experimental strategy. Two neuroblastoma cell lines (SK-N-MC and SK-N-BE) were cotransfected with each transcription factor expressing plasmids and sequential deletion constructs of the 5′ c-RET flanking region cloned upstream of the Luciferase reporter gene. Here we show that HOX11L1 enhances the activity of the c-RET promoter in SK-N-MC cell line by stimulating a region between -166 bp and -35 bp. Gel shift assays performed with oligonucleotides spanning this promoter sequence showed a change of the SP1 interaction with its binding sites, consequent to transfection with HOX11LL While HOX11L2 showed no effect in both the cell lines, we have observed PHOX2A, PHOX2B, and MASH1 triggering a reproducible increase in the Luciferase activity in SK-N-BE cell line. A sequence responsible of the PHOX2A-dependent activation has been identified, while PHOX2B seems to act indirectly, as no physical binding has been demonstrated on c-RET promoter.

Original languageEnglish
Pages (from-to)137-149
Number of pages13
JournalGene Expression
Volume12
Issue number3
Publication statusPublished - 2004

Fingerprint

Proto-Oncogenes
Cell Line
Luciferases
Transcription Factors
Parasympathetic Ganglia
Neural Tube
Neural Crest
Neuroblastoma
Reporter Genes
Oligonucleotides
Nervous System
Transfection
Plasmids
Epithelium
Gels
Binding Sites
In Vitro Techniques
Genes

Keywords

  • Autonomic nervous system development
  • Homeobox genes
  • Reporter gene assay
  • RET proto-oncogene
  • Transcriptional regulation

ASJC Scopus subject areas

  • Genetics

Cite this

An in vitro approach to test the possible role of candidate factors in the transcriptional regulation of the RET proto-oncogene. / Bachetti, Tiziana; Borghini, Silvia; Ravazzolo, Roberto; Ceccherini, Isabella.

In: Gene Expression, Vol. 12, No. 3, 2004, p. 137-149.

Research output: Contribution to journalArticle

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