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Regulation of murine TGFbeta2 by Pax3 during early embryonic development.

Mayanil CS, Pool A, Nakazaki H, Reddy AC, Mania-Farnell B, Yun B, George D, McLone DG, Bremer EG
The Journal of biological chemistry


Previously our laboratory identified TGFbeta2 as a potential downstream target of Pax3 by utilizing microarray analysis and promoter data base mining (Mayanil, C. S. K., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B. J., McLone, D. G., and Bremer, E. G. (2001) J. Biol. Chem. 276, 49299-49309). Here we report that Pax3 directly regulates TGFbeta2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGFbeta2 promoter (GenBanktrade mark accession number AF118263). Both TGFbeta2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGFbeta2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGFbeta2 cis-regulatory elements between bp 741-940 and bp 1012-1212 bind acetylated Pax3 and are associated with p300/CBP and histone deacetylases. The cis-regulatory elements between bp 741 and 940 in addition to associating with acetylated Pax3 and HDAC1 also associated with SIRT1. Whole mount in situ hybridization and quantitative real time reverse transcription-PCR showed diminished levels of TGFbeta2 transcripts in Pax3(-/-) mouse embryos (whose phenotype is characterized by neural tube defects) as compared with Pax3(+/+) littermates (embryonic day 10.0; 30 somite stage), suggesting that Pax3 regulation of TGFbeta2 may play a pivotal role during early embryonic development.

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