Abstract
Abstract: :
Purpose: To identify human gene targets of LMX1B, mutation of which can lead to glaucoma associated with nail-patella syndrome, and thereby reveal a more defined mechanism for the pathogenesis of this type of glaucoma. Methods: Stable lines of human fetal kidney (293) cells were created in which LMX1B expression was under the control of an ecdysone-inducible promoter. PolyA-enriched RNA samples isolated from control and ponasterone A (an ecdysone analogue) treated cells were converted to cDNA in the presence of either Cy3 or Cy5-labeled nucleotides. Differentially labeled experimental and control cDNAs were simultaneously hybridized to a microarray containing 40,000 sequence-verified human EST clones. Average-linkage hierarchical clustering was performed by the CLUSTER program and the results were displayed with TREEVIEW. Significance Analysis of Microarrays (SAM) was applied to search for genes with statistically significant changes that correlated with LMX1B expression. Results: Ponasterone A administration led to strong expression of LMX1B protein in the stable cell lines and the expression was restricted to the nucleus, as expected. LMX1B protein induction only achieved a modest change of gene expression and no gene was changed more than 3-fold. A small fraction of the genes tested (1,688) was induced or repressed by 1.74-fold or greater in two or more replicated samples. Application of the SAM program to the set of 1,688 genes, using a significance threshold expected to produce fewer than 6 false positives, resulted in a list of 64 cDNA clones with possible statistically significant changes. Conclusion: Gene expression profiling by cDNA microarrays can provide insight into the gene expression responses of 293 cells to LMX1B and lead to the discovery of LMX1B target genes. Quantitative PCR can be carried out to confirm these targets. Administration of a "cofactor" of LMX1B, such as E47/Pan1, which increases the trans-activation of LMX1B protein, may lead to more dramatic changes in gene expression.
Keywords: 417 gene/expression • 605 transcription factors