Paterno GD et al. (1997), cDNA cloning of a novel, developmentally regula...

XB-ART-15880

J Biol Chem 1997 Oct 10;27241:25591-5. doi: 10.1074/jbc.272.41.25591.

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cDNA cloning of a novel, developmentally regulated immediate early gene activated by fibroblast growth factor and encoding a nuclear protein.

Paterno GD , Li Y , Luchman HA , Ryan PJ , Gillespie LL .

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We have utilized the polymerase chain reaction (PCR)-based differential display methodology (Liang, P., and Pardee, A. B. (1992) Science 257, 967-969) to identify a novel transcript whose expression levels increased in Xenopus embryo explants during mesoderm induction by fibroblast growth factor. The PCR product was used to clone a 2.3-kilobase pair cDNA representing this transcript, which we have named er1 (early response 1). The er1 cDNA contained a single open reading frame predicted to encode a protein of 493 amino acid residues. A data base homology search revealed that the predicted ER1 amino acid sequence contains three regions of similarity to the rat and human proteins encoded by the metastasis-associated gene, mta1, and two regions of similarity to the Caenorhabditis elegans sequence that is similar to mta1. The fibroblast growth factor-induced increase in er1 steady-state levels was not dependent on de novo protein synthesis, demonstrating that er1 is an immediate-early gene. Northern blot analysis revealed a single 2.8-kilobase pair mRNA that was observed predominantly during the initial cleavage and blastula stages of Xenopus development, with little or no detectable mRNA during subsequent development. Quantitative PCR analysis of early developmental stages showed that er1 peaked during late blastula. Computer-assisted analysis of the predicted ER1 amino acid sequence revealed two putative nuclear localization signals, four highly acidic regions clustered at the N terminus and a proline-rich region located near the C terminus. Subcellular localization by immunocytochemistry revealed that the ER1 protein was targeted exclusively to the nucleus. Transactivation assays using various regions of ER1 fused to the DNA binding domain of GAL4 demonstrated that the N-terminal acidic region is a potent transactivator. These data suggest that ER1 may function as a transcription factor.

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Species referenced: Xenopus laevis
Genes referenced: lgals4.2 mier1 mta1 tbx2
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	Nuclear localization of ER1. A, immunoprecipitation of in vitro translation products with anti-ER1. Rabbit reticulocyte lysates programmed with er1cDNA in pcDNA3 were immunoprecipitated with either preimmune (lane 2) or anti-ER1 (lane 3) serum prepared in our laboratory. Total translation products representing one-half of the input into each immunopreciptation are shown in lane 1.B, ER1 is localized within the nucleus in transfected NIH 3T3 cells. NIH 3T3 cells were transfected with either the pcDNA3 vector alone (top) or er1-pcDNA3 (bottom). After 48 h, cells were fixed and stained with anti-ER1 as described under experimental Procedures.
	Figure 4 Expression of er1 is restricted to early developmental stages in Xenopus. A, Northern blot analysis of er1 expression. Total RNA was isolated from the following developmental stages: stage 2 (2-cell;lane 1), stage 6 (64-cell; lane 2), stage 7 (early blastula; lane 3), stage 8 (mid-blastula; lane 4), stage 12 (mid-gastrula; lane 5), stage 17 (neurula;lane 6), stage 22 (tailbud; lane 7), stage 30 (lane 8), and stage 41 (tadpole; lane 9). Northern analysis was performed as in Sambrook et al. (20) using 32P-labeled 2.3-kb er1 cDNA as a probe. The blot was stripped and reprobed with 32P-labeled histone H4 cDNA. B, quantitative PCR analysis ofer1 levels during blastula and gastrula stages of development. Total RNA was isolated at 1-h intervals during blastula stages, beginning at stage 7 (lane 1) and ending with stage 9 (lane 4). For gastrula stages in lanes 5, RNA was isolated at stages 10, 10.5, and 12, respectively, according to morphological criteria (29). RT-PCR and analysis were performed as described in the legend to Fig. 3.
	Figure 3 er1 is an FGF immediate-early response gene. A, FGF-stimulated increase in steady-state levels ofer1. Explants (5 per sample) from stage 8 Xenopusblastulae were treated for 30 min in the presence (lane 2) or the absence (lane 1) of 100 ng/ml Xenopusbasic FGF. Total RNA was extracted, and RT-PCR analysis was performed as described under xperimental Procedures.B, FGF-stimulated increase of er1 in the absence of protein synthesis. Explants were preincubated for 30 min with (lanes 3 and 4) or without (lanes 1 and2) 5 μg/ml cycloheximide; 100 ng/ml Xenopusbasic FGF was added to the samples in lanes 2 and4, and all samples were incubated for an additional 30 min. Extraction and analysis were performed as described for A.CHX, cycloheximide.