Transcriptional regulatory network analysis of retinal pigment epithelial cells during epithelial-mesenchymal transformations
Epithelial-mesenchymal transition (EMT) is a mechanism of tissue remodeling during which cells transition between two behavioral states comprised of populations of either cohesive, epithelial or migrating, mesenchymal cells. EMT occurs as a normal component of many biological and pathobiological processes. The purpose of this study was to elucidate a candidate regulatory network involved in the reciprocal regulation of genes during EMT of Retinal Pigment Epithelial (RPE) cells. We initially selected a set of sixty genes whose expression is altered during EMT. The regulatory regions of the genes were subjected to computational analysis using the Promoter Analysis and Interaction Network Toolset (PAINT), which identified transcription response elements (TREs) statistically overrepresented in the regulatory regions of reciprocally regulated RPE gene clusters. These TREs were then used to construct transcriptional regulatory network models of the two gene clusters. The validity of these models was then tested using RT-PCR to detect differential expression of the corresponding TF mRNAs in both undifferentiated and differentiated ARPE-19 and primary chicken RPE cell cultures. The expression of these transcription factors was tested in cultures of undifferentiated ARPE-19 cells by immunohistochemistry. RNA interference was used to knockdown three of the candidate transcription factors, namely SREBP-1, SMAD3 and Oct-1. Lastly, we determined the expression of these factors in RPE cells of PVR epi-retinal membranes by immunohistochemistry. The computational analysis resulted in the dentification of specific TREs that are candidates for serving as nodes in a transcriptional regulatory network regulating EMT in RPE cells. The models predicted TFs whose differential expression during RPE EMT was successfully verified by RT-PCR analysis, including Oct-1, HNF-1, SMAD3, TFE, Core Binding Factor, GATA-1, IRF, NKX3A, SREBP-1 and LEF-1. Subsequent biochemical analysis identified three TFs, SREBP-1, SMAD3 and Oct-1, which affect the expression of markers of RPE cell differentiation and are expressed in RPE cells of PVR ERMs. These studies applied computational modeling and biochemical verification to identify biologically relevant transcription factors that regulate RPE cell phenotype and possibly pathological changes in RPE in response to diseases or trauma. These TFs may also provide potential therapeutic targets for the prevention and treatment of ocular proliferative disorders such as PVR. ^
Biology, Molecular|Biology, Cell|Biology, Bioinformatics
Craig Herbert Pratt,
"Transcriptional regulatory network analysis of retinal pigment epithelial cells during epithelial-mesenchymal transformations"
(January 1, 2007).
ETD Collection for Thomas Jefferson University.