Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence.

Breslin MB, Zhu M, Notkins AL, Lan MS
Nucleic Acids Res. 2002 30 (4): 1038-45

PMID: 11842116 · PMCID: PMC100352 · DOI:10.1093/nar/30.4.1038

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/beta2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/beta2 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/beta2 gene.

MeSH Terms (23)

Base Sequence Basic Helix-Loop-Helix Transcription Factors Binding Sites Brain Cell Differentiation Consensus Sequence DNA-Binding Proteins Electrophoretic Mobility Shift Assay Eye Proteins HeLa Cells Homeodomain Proteins Humans Paired Box Transcription Factors Pancreas PAX6 Transcription Factor Promoter Regions, Genetic Protein Structure, Tertiary Repressor Proteins Sequence Alignment Trans-Activators Transcription, Genetic Tumor Cells, Cultured Zinc Fingers

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