Abstract
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.
Highlights
To maintain the consistency of the nomenclature and to establish a connection between the different experiments, we have introduced a schematic figure as supplemental Fig. 1, where specific nomenclature regarding promoter regions used in luciferase studies, chromatin immunoprecipitation, and the oligonucleotides used in Electromobility Shift Assays (EMSA)
Failure of any of these processes results in a neural tube defect, characterized by exencephaly and anencephaly in the rostral part and as spina bifida in the caudal part of the neural tube [24]
Neural crest cells emerge from the dorsal neural tube and migrate throughout the developing embryo, where they give rise to a range of cell types, including neurons and glial cells of the peripheral nervous system and melanocytes
Summary
E, embryonic day; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; DMEM, Dulbecco’s modified Eagle’s medium; HDAC1, histone deacetylases 1; SIRT1, silent information regulator 1 (Sir2␣); TRP-1, tyrosinase-related protein-1; PDBS, paired domain binding site; HDBS, homeodomain-binding site; PBS, phosphatebuffered saline; TGF, transforming growth factor-. The expression of one of these genes, human TGF2, was increased in two of the three Pax transfectants studied, and this gene showed the highest putative Pax3-binding motif score, making it a likely gene regulated by Pax. Our microarray data [6], along with the present findings in this paper in combination with the results of Sanford et al [10], indicate that TGF2 is regulated by Pax and could be involved in early embryonic development
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