Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence.
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.
Breslin
Mary B
MB
Research Institute for Children, Children's Hospital, Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Zhu
Min
M
Notkins
Abner L
AL
Lan
Michael S
MS
D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002454:Cell Differentiation; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D005136:Eye Proteins; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010179:Pancreas / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D015534:Trans-Activators; C093970:NEUROD1 protein, human; C000605730:PAX6 protein, human; C087342:INSM1 protein, human