Inhibition of activating transcription factor 1- and cAMP-responsive element-binding protein-activated transcription by an intracellular single chain Fv fragment.
Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B. , and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254-32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VL kappa-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB.
Bosilevac
J M
JM
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495, USA.
Gilchrist
C A
CA
Jankowski
P E
PE
Paul
S
S
Rees
A R
AR
Hinrichs
S H
SH
D051697:Activating Transcription Factor 1; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000276:immunology; D014158:Transcription, Genetic
D051697:Activating Transcription Factor 1; D000911:Antibodies, Monoclonal; D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors