Selection of DNA binding sites for human transcriptional regulator GATA-6.
The DNA-binding sites for transcriptional regulator GATA-6 were selected and amplified by means of a PCR-mediated random-site selection method involving filter binding and gel-mobility shift analysis using the zinc finger region of human GATA-6 fused with GST. Sequencing and comparison of the selected clones suggested that (A/T/C)GAT(A/T)(A) is the consensus binding sequence, which is similar to the GATA motif (A/T)GATA(A/G) and the gastric motif (G/C)PuPu(G/C)NGAT(A/T)PuPy. GATA-6 also binds to a similar sequence containing the GATC but not the GATG sequence. These results indicated that GATA-6 shows broader sequence specificity as to DNA binding. However, adenine is favored on both sides of the core for strong binding [AGAT(A/T)A], and the order of binding is GATA > GATT > GATC. Full-length human GATA-6 expressed in COS-1 cells showed essentially the same binding specificity, suggesting that the zinc finger region of GATA-6 mainly contributes to the selection of the binding sequence. Furthermore, the non-RI method presented in this paper is convenient for determination of the binding sites of other DNA-binding proteins.
Sakai
Y
Y
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Osaka, Suita, 565-0871, Japan.
Nakagawa
R
R
Sato
R
R
Maeda
M
M
D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D050993:GATA6 Transcription Factor; D006801:Humans; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050993:GATA6 Transcription Factor; C494759:GATA6 protein, human; D014157:Transcription Factors; D004247:DNA