Combination of a zinc finger and homeodomain required for protein-interaction.
The Zinc Finger Homeodomain Enhancer-binding Protein (Zfhep) is involved in skeletal patterning, immune cell, muscle, and brain development, and is necessary for life. Zfhep contains a single central homeodomain (HD) adjacent to an isolated zinc finger, the function of which is unknown. The placement of a zinc finger so close to a homeodomain is novel in nature. The aim of this work was to characterize the Zfhep homeodomain (HD) or the zinc finger homeodomain (ZHD), with respect to DNA-binding and protein-protein interactions. Glutathione-S-transferase (GST) fusion proteins containing either just the HD or both the zinc finger and HD (ZHD) were expressed in E. coli. The GST fusion protein affinity-binding assay demonstrated that Zfhep ZHD interacts specifically with the POU domain of the Oct-1 transcription factor. The adjacent zinc finger is required since Zfhep HD alone does not interact with Oct-1 POU domain. Furthermore, ZHD does not bind to the POU homeodomain lacking the POU specific region. These results demonstrate that the Zfhep zinc finger homeodomain motif functions as a protein-binding domain in vitro, and suggests that Zfhep may modulate the activity of POU domain transcription factors. However, neither the Zfhep ZHD nor the HD bound DNA in EMSA or selected a DNA-binding site from a pool of random oligonucleotides. This is the first demonstration of a function for the HD region of Zfhep, which is the first case of a bi-partite domain requiring both a zinc finger and a HD for binding to protein.
Smith
Gregory E
GE
Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville Health Sciences Center, Louisville, KY 40292, USA.
Darling
Douglas S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051863:Host Cell Factor C1; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071799:Zinc Finger E-box-Binding Homeobox 1; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494168:Pou2f1 protein, rat; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C000630720:ZEB1 protein, rat; D000071799:Zinc Finger E-box-Binding Homeobox 1