atRA Regulation of NEDD9, a gene involved in neurite outgrowth and cell adhesion.
We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from -1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from -475 to -445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.
Knutson
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Interdepartmental Graduate Program in Nutritional Sciences, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA.
Clagett-Dame
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D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons; D005786:Gene Expression Regulation; D006801:Humans; D016501:Neurites / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D020218:Response Elements; D047488:Retinoid X Receptors / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology / Q000502:physiology*; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; C099942:NEDD9 protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014212:Tretinoin