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J. Biol. Chem., Vol. 281, Issue 34, 24544-24552, August 25, 2006

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Regulation of Murine TGF2 by Pax3 during Early Embryonic Development^*

Chandra S. K. Mayanil¹, Angela Pool², Hiromichi Nakazaki, Anvesh C. Reddy, Barbara Mania-Farnell, Beth Yun^¶3, David George, David G. McLone, and Eric G. Bremer^¶

From the Laboratory of Neural Tube Research, Department of Pediatric Neurosurgery, Children's Memorial Research Center and Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60614, the Department of Biology, Purdue University at Calumet, Hammond, Indiana 46323, and the ^¶Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Previously our laboratory identified TGF2 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 TGF2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGF2 promoter (GenBank^TM accession number AF118263 [GenBank] ). Both TGF2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGF2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGF2 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 TGF2 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 TGF2 may play a pivotal role during early embryonic development.

Received for publication, November 21, 2005 , and in revised form, April 27, 2006.

^* This work was supported in part by a State of Illinois Excellence in Academic Medicine award (to C. S. K. M.) and a grant from Spastic Paralysis Research Foundation of Illinois-Eastern Iowa District of Kiwanis (to C. S. K. M. and D. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

² Present address: Graduate Program in Neurobiology, UCLA, Los Angeles, CA 90095.

³ Present address: Dept. of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611.

¹ To whom correspondence should be addressed: Neurobiology Program, Children's Memorial Research Center, M/c 226, 2430 N. Halsted, Chicago, IL 60614. Tel.: 773-755-6530; Fax: 773-755-6363; E-mail: smayanil{at}northwestern.edu.

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