Watanabe T et al. (2005), Functional role of a novel ternary complex comp...

XB-ART-2538

Dev Biol 2005 Jan 15;2772:508-21. doi: 10.1016/j.ydbio.2004.08.051.

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Functional role of a novel ternary complex comprising SRF and CREB in expression of Krox-20 in early embryos of Xenopus laevis.

Watanabe T , Hongo I , Kidokoro Y , Okamoto H .

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Krox-20, originally identified as a member of "immediate-early" genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. Here we cloned a genomic sequence of Xenopus Krox-20 (XKrox-20) and studied functions of a promoter element in the flanking sequence and associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, we showed that the 5' flanking sequence was sufficient to induce luciferase activities when the reporter construct was injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5' flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (-72 to -51 from the transcription initiation site), both of which were essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bound to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts were serum response factor (SRF) and a member of CRE binding protein (CREB) family proteins that bound the CArG-box and CRE, respectively. They appeared to assemble on the minimal promoter element to produce a novel ternary complex. When we injected mRNA of a dominant-negative version of Xenopus SRF (XSRFDeltaC) into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity was strongly suppressed. Suppression by XSRFDeltaC was counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element.

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Species referenced: Xenopus laevis
Genes referenced: camp chrd creb1 eef1a2 egr1 egr2 fgf2 foxg1 mafb prl.2 srf tbx2

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	Fig. 1. Structural analysis of the Xenopus Krox-20 gene and comparison with human and mouse Krox-20 genes. (A) Schematic illustration of the XKrox-20 genomic clone isolated (an uppermost trace). A part of the clone that includes two exons and an intron is zoomed and compared with a corresponding part of human Krox-20, egr-2 (lower traces). The two exons are represented by boxes, in which the consensus C2H2 type Zinc finger regions are dotted. Numbers represent those from the transcription initiation site. The translation initiation (0) and termination (A) sites are also indicated. The 3Vend of exon 2 in XKrox-20 is not determined. (B) Alignment of the nucleotide sequences around the transcription initiation site of Krox-20 genes from three different species. The respective initiation sites are indicated (!). Gaps in the sequence are positioned with dashes and numbers are indicated for the Xenopus sequence. The positions of sequence identity are also indicated (*). The previously known sequence elements are boxed. These include the Ets-binding site (EBS), the CArG-box, the cyclic AMP response element (CRE), and the TATA-box.
	Fig. 2. Analysis of promoter activity within the genomic XKrox-20 sequence. (A) Schematic illustration of subcloning strategy. In the upper trace, the coding sequence of XKrox-20 is boxed. The 5Vflanking sequence is subcloned into a site immediately upstream of the firefly-luciferase-coding sequence (Luc) in the reporter plasmid pGL3, while the intron and 3Vflanking sequences are subcloned into a site immediately downstream of the luciferase-coding sequence (the lower trace). (B) Schematic illustration of injection strategy. The luciferase reporter constructs were injected into eight-cell stage embryos together with an internal standard plasmid pRL-CMV that contained Renilla luciferase-coding sequence. Arrows indicate injection sites. AD; animal-dorsal. VV; vegetalventral. (C) The promoter activity of the genomic XKrox-20 fragments. Injected reporter constructs are shown schematically on the left of histogram. Luciferase activities were measured at stage 20 and the firefly luciferase reporter activity normalized to the pRL-CMV internal standard activity was represented in the histogram as percentages of the maximum value.
	Fig. 3. Identification of a minimal promoter element within the 5Vflanking sequence of XKrox-20. A series of deletion constructs was injected into two AD blastomeres at the eight-cell stage and relative luciferase activities are presented as described for Fig. 2C. (A and B) A series of 5Vdeletion constructs from 3987 to 56 that were indicated on the left of each histogram was assayed. (C) A series of 3Vdeletion constructs from 1 to 55, which had a fixed 5V boundary at 72, was assayed. These constructs had a heterologous TATA-box-containing sequence in the immediate upstream of the luciferase-coding sequence. Injected reporter constructs are shown schematically on the left of histogram.
	Fig. 4. Analysis of the effects of mutation of CArG-box and CRE on the minimal promoter activity. The original construct had the minimal promoter element ( 72 to 51) and the heterologous TATA-box-containing sequence as described for Fig. 3C. The CArG-box and CRE were mutated as indicated on the left of histogram ( ). Mutated promoter fragments were double-stranded synthetic oligonucleotides. The original and mutated constructs were injected into two AD blastomeres at the eight-cell stage and assayed. Relative luciferase activities are presented as percentages of the maximum value.
	Fig. 5. A ternary complex formation on the minimal promoter element by SRF and CREB proteins. Binding reactions were carried out with end-labeled probe, either wild type or mutated, in the absence or presence of whole cell extracts from stage 20 embryos. Further additives are indicated below. Reaction mixtures were electrophoresed on 3.5% native polyacrylamide gel. In A and B, end-labeled wild-type probes ( 73 to 43) were used. (A) Lane 1, probe alone; 2, probe plus extracts and no competition; 3, competition with unlabeled probe at 125-fold excess; 4, components in lane 2 plus anti-SRF; 5, components in lane 2 plus anti-pCREB; 6, components in lane 2 plus anti-SRF and anti-pCREB. (B) Lane 1: probe plus extracts and no competition; 2, competition with unlabeled CArGbox- mutated fragment at 125-fold excess; 3, competition with unlabeled CRE-mutated fragment at 125-fold excess. (C) Lane 1: end-labeled wild-type probe plus extracts; 2, end-labeled CRE-mutated probe plus extracts; 3, end-labeled CArG-box-mutated probe plus extracts; 4, components in lane 2 plus anti-SRF; 5, components in lane 3 plus anti-pCREB; 6, components in lane 1 plus anti-pCREB and anti-SRF.
	Fig. 6. Suppression of the minimal promoter activity in the reporter construct by inhibition of SRF. (A) Structural features of wild-type XSRF and its dominant-negative version employed (XSRFDC). Black and gray boxes represent the C-terminal transactivation and the N-terminal DNA binding domains, respectively. (B) Suppression of the minimal promoter activity by XSRFDC and its rescue by wild-type XSRF. Synthetic mRNAs encoding XSRFDC or XSRF were coinjected with the minimal reporter construct described in Fig. 4 and pRL-CMV into two animal blastomeres of eight-cell stage embryos. The injected amounts of XSRFDC and XSRF mRNAs were 1.5 and 4.5 pg/blastomere, respectively. The total amount of injected mRNA was adjusted to 6.0 pg/blastomere by adding control GFP mRNA. Reporter activities were analyzed and presented as in Fig. 2B.
	Fig. 7. Involvement of SRF in the expression of endogenous XKrox-20. In each panel, mRNA was injected into two animal blastomeres on one side (*) at the eight-cell stage embryo and in situ hybridization was performed at the stage 20 (C to G and I) or stage 11.5 (H). (A) XSRFDC mRNA and lineage-tracing GFP mRNA were coinjected at 60 and 6 pg/blastomere, respectively. Florescent view of a stage 20 embryo. (B) Bright-field view of A. The arrow points to the cement gland. (C) Control GFP mRNA was injected at 60 pg/blastomere. (D) XSRFDC and lineage-tracing GFP mRNAs were coinjected at 60 and 6 pg/blastomere, respectively. (E) XSRFDC and XSRF mRNAs were coinjected at 60 and 80 pg/blastomere, respectively. In C, D, and E, arrows point to the expression of XKrox-20, whereas arrowheads point to that of BF-1. (F) XSRFDC and lineage-tracing GFP mRNAs were coinjected as in D and double in situ hybridization was performed to visualize GFP mRNA (red signal), BF-1 transcript (green signal, indicated by arrowhead), and XKrox-20 transcript (green signal, indicated by arrow). (G) The same injection scheme as in F. Double in situ hybridization was performed to visualize GFP mRNA (red signal), BF-1 transcript (green signal, indicated by arrowhead), and XmafB transcript (green signal, indicated by arrow). (H) The same injection scheme as in F. Double in situ hybridization was performed to visualize GFP mRNA (red signal) and chordin transcript (green signal, indicated by arrow). (I) The same injection scheme as in F. Double in situ hybridization was performed to visualize GFP mRNA (red signal) and actin transcript (green signal, indicated by arrow).
	Fig. 8. Involvement of SRF in FGF-induced expression of XKrox-20 in ectoderm cells. (A) Experimental design for ectodermal cell culture assay used in B. Control GFP or XSRFDC mRNA was injected at 60 pg/blastomere into four animal blastomeres of eight-cell stage embryos. When they reached stage 10, ectodermal tissues were isolated. The dissociated ectodermal cells were then inoculated into microculture wells at 200 cells/well. After completion of reaggregation by brief centrifugation, cells were cultured in the presence of increasing concentrations of bFGF until control embryos reached stage 23. The transcriptional levels of XKrox-20 and BF-1 were analyzed by RT-PCR (Kengaku and Okamoto, 1995; Hongo et al., 1999). (B) Suppression of FGF-induced XKrox-20 expression in ectoderm cells by XSRFDC. Autoradiographs are shown of RT-PCR products of the transcripts from XKrox-20 and BF-1, both of which were coamplified with EF1a transcript, an internal standard (upper panels). Each RT-PCR product was quantified by a laser image analyzer and values for XKrox-20 and BF-1 transcripts with (.) or without (o) overexpression of XSRFDC, which are normalized to EF1a transcript, are presented as percentages of the respective maximum value and plotted against bFGF dose (lower graphs).