XB-ART-36858J Med Genet. January 25, 2008; 283 (4): 2275-85.
A role for basic transcription element-binding protein 1 (BTEB1) in the autoinduction of thyroid hormone receptor beta.
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.
PubMed ID: 18045867
Article link: J Med Genet.
Grant support: 1 R01 NS046690 NINDS NIH HHS , T32 ES007062 NIEHS NIH HHS , 1 R01 NS046690 NINDS NIH HHS
Genes referenced: fabp2 klf9 rpl8 thrb
Article Images: [+] show captions
|FIGURE 1. Thyroid hormone up-regulates Bteb1 mRNA in tadpole brain with faster kinetics than TrβA mRNA. Increased Bteb1 mRNA correlates with elevated BTEB1 protein. A, up-regulation of Bteb1 (top) and TrβA (bottom) mRNAs in premetamorphic X. laevis tadpole brain (NF stage 52) following exposure to T3 (10 nM) added to the aquarium water (n = 4/time point). Gene expression was analyzed by RTqPCR. Asterisks designate significant differences from the zero time point (p < 0.0001; Scheffe's test). B, treatment with T3 increases BTEB1 protein expression in X. laevis tadpole brain. Panel 1, BTEB1 protein is expressed at a very low level in NF stage 52 tadpole brain (optic tectum shown) but is increased dramatically by T3 treatment (panel 2;10 nM in aquarium water for 24 h). Panel 3, representative sagittal brain section (hypothalamic region) from an NF stage 52 tadpole treated with T3 in the aquarium water (10 nM; 24 h). Strong BTEB1 staining was restricted to cell nuclei. Panel 4, immunostaining for BTEB1 was eliminated by preabsorption with GST-xBTEB. BTEB1 immunoreactivity was detected by Cy3 immunofluorescence (panels 1 and 2) or by horseradish peroxidase staining (panels 3 and 4). C, Western blot analysis of xBTEB1 in protein extracts from pCS2 or pCS2-xBTEB1-transfected XTC-2 cells (upper panel); endogenous BTEB1 in protein extracts of XTC-2 cells were treated with or without T3 (30 nM) for 24 h (lower panel). This dose of T3 causes a maximal response in TrβA and Bteb1 mRNA (data not shown). Immunoblotting was conducted using affinity-purified IgG that recognizes the N-terminal region of xBTEB1 (see “Experimental Procedures”). Arrows point to the two BTEB1 bands.|
|FIGURE 2. Schematic representation of the X. laevis TrβA gene with locations of GC boxes and regions analyzed by EMSA and ChIP assay. The bars with letters below indicate the general regions of the TrβA gene targeted for analysis by EMSA and in the ChIP assay and correspond to the specific sequences given in supplemental Table 1. The numbering of the seven GC boxes corresponds to that given in Table 4. The dark gray filled box represents the upstream region, the S indicates the transcription start site, the light gray filled box represents the 5′-UTR, and the asterisk indicates a TRE that has been characterized and proposed to mediate T3-dependent transactivation (40, 58).|
|FIGURE 3. A, binding of GST-xBTEB1[DBD] to regions of the proximal X. laevis TrβA promoter in vitro. We used EMSA to test the ability of radioinert DNA fragments (1.89 μM/reaction) corresponding to different regions of the proximal TrβA promoter (generated by PCR; see Fig. 2 and supplemental Table 1) to displace GST-xBTEB1[DBD] binding to the 32P-BTE probe. mBTE, mutated BTE. B, binding of GST-xBTEB1[DBD] to GC-rich regions of the proximal TrβA promoter. We used EMSA to test whether GST-xBTEB1[DBD] could bind to short 32P-labeled oligonucleotides encompassing one or two GC boxes in the TrβA promoter. The numbering of the GC boxes included in each oligonucleotide probe is based on that given in Fig. 2 and Table 4. In each case homologous, radioinert competitors (1.89 μM) were used to displace binding.|
|FIGURE 4. BTEB1 associates with the proximal TrβA promoter in vivo in a T3 and developmental stage-dependent manner. ChIP assay was conducted using an affinity-purified IgG directed against the N-terminal region of X. laevis BTEB1. A, T3-dependent association of BTEB1 with the proximal TrβA promoter in tadpole brain and tail. Premetamorphic (NF stage 52) X. laevis tadpoles were treated with 10 nM T3 added to the aquarium water for 48 h prior to tissue collection for ChIP assay (see “Experimental Procedures”). The lettered TrβA promoter regions analyzed correspond to those given in Fig. 2 and supplemental Table 1. The TrβA exon 3/exon 4, and the Efα1 and Ifabp promoters were used as negative controls. B, developmental stage-dependent association of BTEB1 with the proximal TrβA promoter in early prometamorphic (NF stage 54) and climax stage (NF stage 62) X. laevis tadpole brain. Only region A of TrβA promoter, which showed robust T3-dependent association of BTEB1 was targeted for ChIP analysis in this experiment. Each of the ChIP experiments was repeated three times with similar results.|