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RANBP17, A Novel Non-bHLH Binding Partner of bHLH Transcription Factor E12

by Lee, Jun-Ho

Abstract (Summary)
E12 is a ubiquitously expressed basic helix-loop-helix (bHLH) transcription factor regulating various developmental processes via homo- or heterodimerization with other bHLH family members and it also has a number of non-bHLH binding partners in diverse biological settings. To search for novel binding partners of E12, the bHLH domain of E12 was utilized as bait in yeast two-hybrid screening of a DU145 prostate cancer cell library. As a consequence, RANBP17, a member of the importin-? superfamily was identified as a binding partner of E12. The interaction of E12 and RANBP17 was also confirmed in mammalian two hybrid analysis evidenced by significantly enhanced relative luciferase activity in HeLa cells. Semiquantitative RTPCR, quantitative real-time PCR and Northern blot analyses revealed that RANBP17 is present as a major ~4.5 kb transcript showing somewhat ubiquitous expression across a panel of human cancer cell lines. Immunostaining and subcellular fractionation of nuclear and cytoplasmic cell extracts demonstrated that RANBP17 is localized to both cytoplasm and nucleus. Upon co-expression with E12, the intracellular localization of RANBP17 showed a shift to a predominant nuclear localization implicating the dominant role of E12 in nuclear localization of RANBP17 and also supporting the interaction of E12 with RANBP17. The interaction of E12 and RANBP17 is further verified by coimmunoprecipitation and E-box dependent functional assay wherein co-expression of RANBP17 with E12 and MyoD revealed that RANBP17 can induce E12/MyoDmediated transcriptional activation. Several proteins related to E12 and RANBP17 such as E47, short form of RANBP17 and RANBP16 also showed similar E12/MyoD mediated transactivation in this assay and taken together, these observations substantiate the specific interaction of E12 and RANBP17 and support the conclusion that the bHLH domain of E12 and the Crm1 domain of RANBP17 are responsible for this interaction. Based on our retroviral infection studies in C2C12 cells, it is considered that RANBP17 exerts its effect on transcriptional regulation of a subset of E12-regulated genes in a promoter-specific manner under physiological conditions.
Bibliographical Information:

Advisor:

School:University of Toledo Health Science Campus

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:ranbp17 binding partner bhlh transcription factor e12 e2a interaction

ISBN:

Date of Publication:01/01/2008

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