title: The TLX2 homeobox gene is a transcriptional target of PHOX2B in neural-crest-derived cells.
abstract: The TLX2 (HOX11L1, Ncx, Enx) and PHOX2B genes encode transcription factors crucial in the development of neural-crest-derived cells, leading to ANS (autonomic nervous system) specific neuronal lineages. Moreover, they share a similar expression pattern and are both involved in downstream steps of BMP (bone morphogenetic protein) signalling. In an attempt to reconstruct the gene network sustaining the correct development of the ANS, we have undertaken an in vitro experimental strategy to identify direct upstream regulators of the TLX2 gene. After characterizing a sequence displaying enhancer property in its 5' flanking region, we confirmed the functional link between the human PHOX2B and TLX2 genes. Transient transfections and electrophoretic-mobility-shift assays suggested that PHOX2B is able to bind the cell-specific element in the 5' regulatory region of the TLX2 gene, determining its transactivation in neuroblastoma cells. Such interaction was also confirmed in vivo by means of chromatin immunoprecipitation assay and, in addition, up-regulation of endogenous TLX2 mRNA level was demonstrated following PHOX2B over-expression, by quantitative real-time PCR. Finally, PHOX2B proteins carrying mutations responsible for CCHS (congenital central hypoventilation syndrome) development showed a severe impairment in activating TLX2 expression, both in vitro and in vivo. Taken together, these results support the PHOX2B-TLX2 promoter interaction, suggesting a physiological role in the transcription-factor cascade underlying the differentiation of neuronal lineages of the ANS during human embryogenesis.
authors.affiliation: Laboratorio di Genetica Molecolare, Istituto Gerolamo Gaslini, 16148 Genova, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Borghini
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bachetti
authors.forename: Monica
authors.initials: M
authors.lastname: Fava
authors.forename: Marco
authors.initials: M
authors.lastname: Di Duca
authors.forename: Francesca
authors.initials: F
authors.lastname: Cargnin
authors.forename: Diego
authors.initials: D
authors.lastname: Fornasari
authors.forename: Roberto
authors.initials: R
authors.lastname: Ravazzolo
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceccherini
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D005801:Genes, Homeobox / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007040:Hypoventilation / Q000151:congenital / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009432:Neural Crest / Q000166:cytology* / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; C109220:NBPhox protein; C491534:TLX2 protein, human; D014157:Transcription Factors