An in vitro approach to test the possible role of candidate factors in the transcriptional regulation of the RET proto-oncogene.
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 autonomic 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 HOX11L1. 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.
Bachetti
Tiziana
T
Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy.
Borghini
Silvia
S
Ravazzolo
Roberto
R
Ceccherini
Isabella
I
D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D066298:In Vitro Techniques; D058610:Isobutyrates; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009432:Neural Crest / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D015513:Oncogene Proteins / Q000096:biosynthesis; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D002087:Butyrates; D017931:DNA Primers; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D058610:Isobutyrates; D008156:Luciferases; D009841:Oligonucleotides; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret; D014157:Transcription Factors; C065500:ASCL1 protein, human; C000715470:MAS1 protein, human; C109220:NBPhox protein; C491061:PHOX2A protein, human; C491534:TLX2 protein, human; C489494:TLX3 protein, human; C020380:isobutyric acid