Transcript profiling using a zebrafish heart cDNA library previously revealed abundant expressed sequence tags (ESTs) upregulated in zebrafish embryos treated with the aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here, we identify those ESTs as LTR-containing retroelements termed EZR1 (Expressed-Zebrafish-Retroelement group 1). EZR1 is highly redundant in the genome and includes canonical long terminal repeats (LTRs) flanking an integrase-like open reading frame and a region similar to retroviral envelope protein genes. EZR1 sequences lack reverse transcriptase, RNase H, or protease, indicating retrotransposition would be nonautonomous. No AHR binding motifs were found in the EZR1 promoter region. A putative NF-jB-binding site was found, and TCDD-treated zebrafish embryos had significantly increased levels of nuclear protein(s) binding to this sequence. Protein-EZR1 DNA complex formation was partially competed by a mammalian consensus jB sequence, consistent with NF-jB-like activation contributing to increased protein binding to this site. Mobility of the TCDD-induced protein-EZR1 complex differed from that of authentic NF-jB protein bound to the consensus jB site. The results suggest that EZR1 is regulated by interaction with NF-jB or NF-jB-like protein(s) different from the NF-jB protein binding to the consensus jB site. The nature of the NF-jB-like protein and the relationship between EZR1 induction and cardiovascular toxicity caused by TCDD warrant further investigation.

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In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95°C for 5 min and subsequent re-annealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.

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