Identification of target genes of the transcription factors Tbx1, Pax3, and Hoxa3 by microarray analysis
Lipner, Shari R.
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DiGeorge Syndrome/Velo-Cardio-Facial Syndrome (DGS/VCFS) is the most common microdeletion syndrome in humans having a frequency of 1/4,000 live births. Most patients exhibit craniofacial abnormalities, heart defects, velopharyngeal insufficiency, defective cellular immunity, and hypocalcemia, as well as learning disabilities. While the phenotype is variable, 83% of patients with DGS/VCFS have a 1.5 to 3 Mb hemizygous deletion on chromosome 22q11. Since the 1.5 Mb region contains at least 24 genes, it was unclear at the inception of this project which gene or genes is responsible for the phenotypes seen in DGS/VCFS patients. While the genes, Pax3 and Hoxa3 do not map to this region, mice that are null for either of these genes have a phenotypic spectrum that closely resembles that of DGS/VCFS patients. We hypothesized that if a gene from the 22q11 region is altered in expression in the Pax3-/- and/or Hoxa3-/- mice, then the gene would be a likely candidate for causing the phenotypes seen in DGS/VCFS patients.;For this purpose, we compared gene expression profiles between wild type and Pax3 or Hoxa3 deficient embryos using cDNA microarrays that included 19/24 22q11 genes as well as 8946 other cDNAs. None of the genes in 22q11 exhibited significant differences in expression between wild type and Pax3 null embryos or between wild type and Hoxa3 null embryos.;Comparison of the gene expression changes occurring in Pax3 and Hoxa3 mutant embryos revealed that the mRNA for dopachrome tautomerase (Dct), an enzyme responsible for pigment synthesis in melanocytes, is downregulated in both mutants. While Pax3 is known to play an important role in melanocyte development, Hoxa3 had not previously been shown to be involved in this pathway. To further explore the role of Hoxa3 in melanocyte development, we investigated expression of Hoxa3 in melanocyte cell lines. The results showed that Hoxa3 is expressed in melanoblasts, but not in melanocytes, and that its expression strongly decreases as melanoblasts differentiate into melanocytes. Furthermore, we found that Hoxa3 is expressed in a very high proportion of human and mouse melanomas.;Inspection of the Dct promoter revealed 8 potential binding sites for Hoxa3. Transient transfection experiments showed that Hoxa3 repressed, rather than activated, the Dct promoter. Repression of the promoter was observed even when Hoxa3 was co-transfected with two transcription factors, Mitf and Sox 10, which were shown previously to activate the Dct promoter. We also found that Hoxa3 can activate the Mitf promoter. Therefore, we concluded that Hoxa3 is involved in two pathways controlling Dct expression; one in which the Dct promoter is repressed directly by Hoxa3 and the other in which the Dct promoter is activated through Hoxa3 mediated positive regulation of Mitf synthesis. (Abstract shortened by UMI.).