Identification of the DNA binding specificity of the human ZNF219 protein and its function as a transcriptional repressor.
The ZNF219 gene is a member of the Kruppel-like zinc finger gene family that is involved in a diverse range of biological processes. The ZNF219 gene encodes a 77-kDa nuclear protein containing nine sets of C2H2 zinc finger structures. By using a random oligonucleotide selection assay and the electromobility gel shift assay, we have revealed that the ZNF219 protein recognizes two copies of CCCCCA. The DNA binding core element is CCCCC. 3' flanking A residues enhance binding of the ZNF219 protein. Use of the various truncated ZNF219 constructs demonstrated that zinc finger 1 to 3 or zinc finger 5 and 6 domains are sufficient to allow specific DNA binding. Both domains independently recognized the same consensus sequence, CCCCCA. Proteins expressed from human cDNA clones KIAA0390 and KIAA0222, which have partial similarities to ZNF219, also showed specific binding to the same core DNA sequence. Potential ZNF219 binding sites were found in the HMGN1 promoter. To examine the function of ZNF219 in the modulation of transcription, we constructed Gal4 DNA binding domain (DBD)/ZNF219 fusion proteins and demonstrated that ZNF219 functioned as a transcriptional repressor for the HMGN1 promoter. Experiments with the truncated ZNF219 constructs suggest that the proline-rich sequence (226-272 a.a., proline content 49%) was responsible for part of the observed repression. These findings provide us with an important start point in our understanding of the functional role of ZNF219 in vivo.
Sakai
Tsuyoshi
T
Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi, Hatiouji, Tokyo 192-8977, Japan. sakai@t.soka.ac.jp
Hino
Kimihiro
K
Wada
Shinya
S
Maeda
Hidekatsu
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D030562:Databases, Protein; D024202:Electrophoretic Mobility Shift Assay; D005057:Eukaryotic Cells / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D017385:Sequence Homology; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects*; D016335:Zinc Fingers / Q000502:physiology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C413370:ZNF219 protein, human