Abstract
Thyroid hormone (T(3)) induces gene regulation programs necessary for tadpole metamorphosis. Among the earliest responses to T(3) are the up-regulation of T(3) receptor beta (TRbeta; autoinduction) and BTEB1 (basic transcription element-binding protein 1). BTEB1 is a member of the Krüppel family of transcription factors that bind to GC-rich regions in gene promoters. The proximal promoter of the Xenopus laevis TrbetaA gene has seven GC-rich sequences, which led us to hypothesize that BTEB1 binds to and regulates TrbetaA. In tadpoles and the frog fibroblast-derived cell line XTC-2, T(3) up-regulated Bteb1 mRNA with faster kinetics than TrbetaA, and Bteb1 mRNA correlated with increased BTEB1 protein expression. BTEB1 bound to GC-rich sequences in the proximal TrbetaA promoter in vitro. By using chromatin immunoprecipitation assay, we show that BTEB1 associates with the TrbetaA promoter in vivo in a T(3) and developmental stage-dependent manner. Induced expression of BTEB1 in XTC-2 cells caused accelerated and enhanced autoinduction of the TrbetaA gene. This enhancement was lost in N-terminal truncated mutants of BTEB1. However, point mutations in the zinc fingers of BTEB1 that destroyed DNA binding did not alter the activity of the protein on TrbetaA autoinduction, suggesting that BTEB1 can function in this regard through protein-protein interactions. Our findings support the hypothesis that BTEB1 associates with the TrbetaA promoter in vivo and enhances autoinduction, but this action does not depend on its DNA binding activity. Cooperation among the protein products of immediate early genes may be a common mechanism for driving developmental signaling pathways.
Highlights
Basic transcription element-binding protein 1 (Bteb1) is an immediate early gene induced by T3 in most tadpole tissues during metamorphosis
We show that the kinetics of Bteb1 mRNA up-regulation in response to T3 are faster than TrA and that BTEB1 binds to regions of the proximal TrA promoter that contain GC boxes
Tadpole Brain—Similar to results that we reported earlier [13], we observed a strong increase of BTEB1 protein expression in premetamorphic tadpole brain (NF stage 52) following treatment with T3 (10 nM for 24 h; representative brain sections shown in Fig. 1B, panels 1 and 2)
Summary
T3, 3,5,3Ј-L-triiodothyronine; TR, T3 receptor ; TRE, thyroid hormone-response element; UTR, untranslated region; RT, reverse transcription; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; DBD, DNA binding domain; RTqPCR, quantitative real time PCR; BTE, basic transcription element; ANOVA, analysis of variance; NF, Nieuwkoop and Faber. BTEB proteins have been reported to activate or repress transcription depending on the number of GC boxes present in the promoter construct tested, the target gene analyzed, and the cell type (13–15, 27, 30 –36). Whether these proteins function as transcriptional activators or repressors may depend on the architecture of the specific promoter and the chromatin environment [15]. Forced expression of BTEB1 in the X. laevis fibroblast cell line XTC-2 [37] accelerates the activation of the TrA promoter and expression of endogenous TrA mRNA in response to T3 This action depends on the first 30 amino acids of BTEB1, but not on its DNA binding capacity, because point mutations in the zinc fingers did not alter the activity. 5Ј-TTTGCTGAAAGAAATGGCTACATC-3Ј 5Ј-CAC GGC CTG GAT CAT GGA-3Ј VIC-AGG GTA TTG TGA AAG ACA-MGBNFQ
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