Abstract
Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives. Our previous study (Mayanil, C. S. K., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) J. Biol. Chem. 275, 23259-23266) reveals that overexpression of Pax3 in a human medulloblastoma cell line, DAOY, resulted in an up-regulation in alpha-2,8-polysialyltransferase (STX) gene expression and an increase in polysialic acid on neural cell adhesion molecule. This finding suggests that STX might be a previously undescribed downstream target of Pax3. Because Pax3 is important in diverse cellular functions during development, we are interested in the identification of additional downstream targets of Pax3. We utilized oligonucleotide arrays and RNA isolated from stable Pax3 transfectants to identify potential target genes. A total of 270 genes were altered in the Pax3 transfectants as compared with the vector control and parental cell line. An independent analysis by cDNA expression array and real-time quantitative polymerase chain reaction of several genes confirmed the changes observed by the oligonucleotide microarray data. Of the genes that displayed significant changes in expression, several contain paired and homeodomain binding motifs of Pax3 in their promoter regions. Using promoter-luciferase reporter transfection assays and electromobility shift assays, we showed at least one previously undescribed downstream target, STX, to be a biological downstream target of Pax3. Thus we report several previously undescribed candidate genes to be potential downstream targets of Pax3.
Highlights
Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives
Using oligonucleotide arrays and RNA from stable Pax3transfected DAOY cells to identify potential Pax3 downstream targets, a total of 270 genes were altered by Pax3 transfection
Real-time quantitative RTPCR and cDNA expression array analysis confirmed the changes of RhoGDI, MyoD, Drebrin, Tenascin, and glutathione S-transferase
Summary
Stable transfection of mouse Pax in the human medulloblastomaderived cell line, DAOY, was done as described earlier [16]. Each 50 l of RT-PCR reaction (PerkinElmer Life Sciences) volume included 25 ng of total RNA, 5 l of 10ϫ TaqMan buffer A (composed of 500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM passive reference dye, pH 8.3, at room temperature), 10 l of 25 mM MgCl2, 1.5 l of each dNTP (10 mM dATP, dCTP, dGTP, and 20 mM dUTP), 0.5 l of forward and reverse primers (10 M), 1 l of the corresponding TaqMan probe (5000 nM), 0.25 l of AmpliTaq Gold supplied at 5 units/l, and 0.25 l of murine leukemia virus reverse transcriptase. The gels were dried and subjected to PhosphorImager analysis to view the shifted bands
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