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Updated: Oct 4, 2026
Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
Published on: March 27, 2020
Microarray analysis detects novel Pax3 downstream target genes
C S Mayanil1, D George, L Freilich
1Pediatric Brain Tumor Research Program, Department of Neurological Surgery, Northwestern University Medical School and Children's Memorial Institute of Education and Research, Chicago, Illinois 60614, USA.
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.
Insights
Paired box 3 (Pax3) transcription factor regulates gene expression. This study identified new downstream targets of Pax3, including alpha-2,8-polysialyltransferase (STX), crucial for neural development.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Pax3 is a crucial transcription factor for embryonic development, including neural tube and neural crest formation.
- Previous research indicated Pax3's role in up-regulating alpha-2,8-polysialyltransferase (STX) gene expression and polysialic acid levels.
Purpose of the Study:
- To identify novel downstream target genes of the Pax3 transcription factor.
- To further investigate the regulatory relationship between Pax3 and STX.
Main Methods:
- Utilized oligonucleotide arrays and RNA from stable Pax3 transfectants to screen for gene expression changes.
- Confirmed array data using cDNA expression arrays and real-time quantitative polymerase chain reaction.
- Employed promoter-luciferase reporter assays and electromobility shift assays to validate STX as a direct target.
Main Results:
- A total of 270 genes showed altered expression in Pax3 transfectants compared to controls.
- Several identified genes possess potential Pax3 binding motifs in their promoter regions.
- Confirmed STX as a direct downstream target of Pax3 through functional assays.
Conclusions:
- Pax3 regulates a significant number of genes involved in cellular functions.
- STX is a validated downstream target of Pax3, expanding the understanding of its regulatory network.
- Several novel candidate genes are proposed as potential downstream targets of Pax3 for future research.
