DNA-Binding Specificity of the Cut Repeats from the Human Cut-Like Protein

Abstract

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells

“…The alignment of these sequences revealed the consensus, 5 0 -AATCG(A)ATA(C)-3 0 (Figure 4b), which overlapped significantly with those of the cut domain of human CDP (5 0 -ATCGAT-3 0 ) and OC1 (5 0 -ATCAAT-3 0 ). 20,22 Based on the consensus-binding sequence of OC2, we searched for candidate target genes encoding proteins related to Th1 function by checking the genome sequences of the promoter region. We found tandem repeats of the consensus sequence 5 0 upstream of the human T-bet gene, 5 0 -ATCAATAAAGATCGAT-3 0 , in the region from À490 to À475 (the consensus sequences are underlined and designated as 'site 1' and 'site 2', Figure S1) were mutated in the reporter containing OC2 promoter (À451 to þ 124) and the reporter activities of each construct, as well as the wild-type (top) and OC2 promoter (À347 to þ 124, bottom), were examined in the presence or absence of T-bet in COS7 cells.…”

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

“…1). CDP CR1, a negative regulator of many genes, is involved in cell specification in several tissues, including the external sense organs, the malpighian tubules, muscles, and the tracheal system [34]. Pax-6, an activator, binds to islet hormone promoters (glucagon, insulin and somastatin) and operates as an activator [35].…”

CDP Is a Repressor of Mouse Mammary Tumor Virus Expression in the Mammary Gland

“…CDP has four different DNA-binding domains, and each of these domains has a different specificity for DNA, although the CR2 and CR3 domains appear to have the most similar DNAbinding properties (3,27). Thus, the presence of CR1 in the full-length protein may decrease its affinity for the 3Ј site or increase its affinity for the 5Ј site in the pNRE.…”