The HAT domain of p300 is critical for the basal Ig germ-line α promoter activity while Stat-1 and E1A act as strong repressors

초록

p300 acts as a coactivator for TGF-beta 1-induced Ig germ-line (GL) alpha transcription. To understand the exact role of p300, we analyzed known domains of p300 using several deletion mutants. p300 mutants lacking both the histone acetyltransferase (HAT) and Smad-binding domains did not enhance GL alpha promoter activity. In addition, Smad-binding domain within p300 was not sufficient for the promoter activity. Surprisingly, overexpression of a p300 mutant retaining the HAT domain but lacking the rest of downstream segment, p300[N1736], greatly augmented the GL alpha promoter activity, regardless of TGF-beta 1 treatment, to levels much higher than that of p300[WT]. Together, these results suggest that HAT activity of p300 is required for GL alpha promoter activity. We found that p300[N1736] binds to the GL alpha promoter using ChIP assays. Further analysis of p300[N1736] indicated that both Stat1 and HDAC1 can inhibit the enhancing effect of p300[N1736], suggesting the importance of the Stat1-binding domain and the HAT domain. E1A, a repressor of p300[WT], had no effect on the activity of p300[N1736], which also lacks the E1A-binding domain. Finally, when the amount of transfected p300[N1736] was reduced 40-fold, p300[N1736] functioned similarly to overexpressed p300[WT]. Thus, p300 [N1736], though not having a Smad3-binding domain, augmented TGF-beta 1-induced GL alpha promoter activity, which was inhibited by IFN-gamma/Stat-1. Taken together, these results indicate that p300 acts as a basal element in GL alpha transcription without binding to Smad3. Additionally, E1A, Stat1, and HDAC1 act as repressors of p300 in such transcription.

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