Nucleic Acids Research, 2002, Vol. 30, No. 4 1038-1045
© 2002 Oxford University Press
Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence
Research Institute for Children, Children’s Hospital, Departments of Pediatrics and Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA and 1Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the majority of the repressor activity. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST–IA-1DBD fusion protein (257–510 a.a.), we identified the consensus IA-1 binding sequence, TG/TC/TC/TT/AGGGGG/TCG/A. Further experiments showed that zinc-fingers 2 and 3 of IA-1 are sufficient to demonstrate transcriptional activity using an IA-1 consensus site containing a reporter construct. A database search with the consensus IA-1 binding sequence revealed target sites in a number of pancreas- and brain-specific genes consistent with its restricted expression pattern. The most significant matches were for the 5'-flanking regions of IA-1 and NeuroD/ß2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/ß2 promoter/CAT construct modulated CAT activity. These findings suggest that the IA-1 protein may be auto-regulated and play a role in pancreas and neuronal development, specifically in the regulation of the NeuroD/ß2 gene.
* To whom correspondence should be addressed at: Research Institute for Children, Children’s Hospital, Research and Education Building, Room 2211, 200 Henry Clay Avenue, New Orleans, LA 70118, USA. Tel: +1 504 896 2705; Fax: +1 504 896 2722; Email: mlan{at}chnola-research.org
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