Homeodomain Determinants of Major Groove Recognition

Abstract

The homeodomain is a highly conserved structural module that binds DNA and participates in protein-protein interactions. Most homeodomains contain residues at positions 47 and 51 which mediate recognition of a TAAT core binding sequence in the major groove. The constraints imposed on the identity of these residues by homeodomain structure and DNA docking have been examined in the context of the POU domain of the Oct-1 transcription factor. A bacterial library, in which POU homeodomain residues 47 and 51 have been randomized, was probed on nitrocellulose filters for the binding of DNA fragments containing the consensus octamer sequence. The residues which provide for the highest affinity interaction with the octamer consensus sequence, and the greatest specificity, are the highly conserved wild-type residues valine 47 and asparagine 51. Interestingly, a class of variants containing arginine at position 51 was also detected in the screen and found to have moderate affinity for the consensus sequence but reduced specificity compared to the wild-type protein. A single variant containing arginine at both positions 47 and 51 was detected when the library was probed with fragments containing nucleotide substitutions at positions expected to be contacted by residues 47 and 51. This variant was used to alter the DNA-binding specificity of a transcriptional regulatory complex which depends upon Oct-1 for DNA recognition. These findings suggest that homeodomain structure and DNA docking constrain in the versatility of the domain in that only a limited set of amino acid determinants can endow the domain with specific, high-affinity DNA binding.

Molecular Cloning of LIM Homeodomain Transcription Factor <i>Lhx2</i> as a Transcription Factor of Porcine Follicle-Stimulating Hormone Beta Subunit (FSHβ) Gene

“…Taken together, Fshβ as well as Cga may be controlled by a large regulatory complex formed by a protein-protein interaction linkage of LHX2 and the LIM domain family. A core binding motif, TAAT/ATTA, of the homeodomain transcription factors is well characterized [31][32][33][34]. As expected, 14 out of the 15 binding sites for LHX2 identified in the porcine Fshβ promoter contain a TAAT/ATTA motif ( Table 1).…”

“…We previously cloned CLIM2 as the LHX2-binding protein and demonstrated that the interaction of CLIM2 represses the LHX2-dependent stimulation of porcine Cga expression [40]. CLIM2 is known to participate in transcriptional A core binding motif, TAAT/ATTA, of the homeodomain transcription factors is well characterized [31][32][33][34]. As expected, 14 out of the 15 binding sites for LHX2 identified in the porcine Fshβ promoter contain a TAAT/ATTA motif (Table 1).…”

“…In the distal region over Table 1, which shows that they consisted mostly of A/T-rich sequences. Notably, with the exception of region −1915/−1899 b, they contained TAAT/ATTA, known as a core binding motif for homeodomain transcription factors [31][32][33][34].…”

Regulation of Porcine Pituitary Glycoprotein Hormone Alpha Subunit Gene with LIM-Homeobox Transcription Factor Lhx3

“…The region contains many TAAT/ATTA core motifs, a binding element for multiple members of the homeodomain family [29][30][31][32]. In the present study, we demonstrated that Lhx3 definitely activates the expression of porcine Cga by binding to the sites in both the distal and proximal regions, which mostly contain TAAT/ATTA core motif.…”

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

“…Generally, most homeodomain factors recognize nucleotide sequences containing a TAAT core motif (Catron et al 1993, Damante et al 1994, Jagla et al 1994, Pomerantz & Sharp 1994. Prop-1 is a homeodomain transcription factor and known to bind to the consensus sequence, PRDQ9 (5 0 -TAATtgaATTA-3 0 , a palindromic sequence of a TAAT core motif; Sornson et al 1996).…”