ARS | Publication request: Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding dEF1

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 22, 2008
Publication Date: N/A

Interpretive Summary: The chicken infectious anemia virus (CAV) gene promoter fused to the gene for enhanced green fluorescent protein (EGFP) was used to measure virus activity in response to estrogen. This construct had increased expression in an estrogen receptor-enhanced cell line when treated with estrogen. The virus promoter-enhancer DNA sequence was also found to bind some of the proteins that recongnize a consensus estrogen response element (ERE) (Miller et al., J Virol, 79 2005), a DNA sequence that is responsible for the regulation of estrogen responsive genes in animals. Cotransfection assays were done, using expression vectors for thyroid receptor (TR) or chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) to increase the levels of these hormone receptors in the cells containing the CAV promoter driving expression of EGFP. This tested for transcription regulation by these members of the nuclear receptor family that also recognize a consensus ERE. Coexpression with COUP-TF1 but not TR or a control RSV promoter vector was found to decrease expression of EGFP by 50-60% in fibrocytic and liver cell lines. A long virus promoter construct that included sequences downstream from the transcription start point (TSP) expressed lower levels of EGFP than a shorter sequence that stopped at the TSP. Mutation of an E box-like sequence at the TSP restored expression of this longer promoter to the same level as the short promoter. Deletions at the 3' end of the long promoter that left the E box intact did not restore promoter activity. Electromobility shift assays were done to find if proteins from the nucleus bind to the DNA sequence at the TSP. Assays showed that multiple proteins can bind to this sequence and that one of these is the transcription regulator delta-EF1 (dEF1). These findings indicate that the CAV promoter activity can be repressed directly or indirectly by COUP-TF1 and dEF1.

Technical Abstract: Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an estrogen receptor-enhanced cell line when treated with estrogen. This promoter-enhancer also binds unidentified proteins that recognize a consensus estrogen response element (ERE) (Miller et al., J Virol, 79 2005). Cotransfection assays, using expression vectors for thyroid receptor (TR) or chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) and the CAV promoter driving expression of EGFP, were used to test for transcription regulation by these members of the nuclear receptor family that also recognize a consensus ERE. Coexpression with COUP-TF1 but not TR or a control RSV promoter vector was found to decrease expression of EGFP by 50-60% in DF-1 and LMH cells. A long promoter construct that included sequences downstream from the transcription start point (TSP) expressed lower levels of EGFP than a construct that stopped at the TSP. Mutation of a putative E box at the TSP restored expression of the long promoter to the same level as the short promoter. Deletions at the 3¿ end of the long promoter that left the E box intact did not restore promoter activity. Electromobility shift assays showed that the transcription regulator delta-EF1 (dEF1) is one of the proteins that recognize the E box region. These findings indicate that the CAV promoter activity can be repressed directly or indirectly by COUP-TF1 and dEF1.