Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila

Abstract

The ventral nervous system defective (vnd)͞NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd͞NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd͞NK-2 homeodomain binding site was shown to be T(T͞C)AAGTG(G͞C). The apparent equilibrium dissociation constant (KD) of the vnd͞NK-2 homeodomain for the consensus sequence is 1.9 ؋ 10 ؊10 M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd͞NK-2 homeodomain and determination of the apparent KD values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.homeobox ͉ DNA binding site ͉ neurobiology H omeodomain (HD) proteins that are involved in sequencespecific recognition of DNA are transcription factors regulating multiple genes during development (1, 2). They use a helix-turn-helix motif to contact DNA in the major groove and an N-terminal arm to contact DNA in the minor groove (3, 4). HDs of some homeotic proteins in Drosophila (Hom) and mammals (Hox A-D) recognize a TAAT core (1). Hox proteins regulate developmental pathways as monomers (5, 6), but a few bind to cofactors, thereby increasing the DNA-binding specificity (7). The Drosophila ventral nervous system defective (vnd)͞NK-2 is an HD-containing protein (8, 9) that initiates neural development in the ventral column of neuroectoderm that gives rise to part of the ventral nerve cord in embryos (10, 11). Dichaete acts in concert with vnd͞NK-2 and intermediate neuroblasts defective to regulate cell fate and neuroblast formation (12). Regulation of vnd͞NK-2 expression is required for both neural and glial specification (13). The three-dimensional structure of the vnd͞NK-2 HD and its interaction with a target oligodeoxynucleotide (oligonucleotide) have been reported (14-17). Site mutations (single, double, or triple sites) involving Tyr-54 (Y54M) all showed a decrease of one order of magnitude in their binding affinities to the vnd͞NK-2 consensus DNA binding site (18). The NK-2 gene family now has been found in diverse phyla and has been shown to play important roles in organogenesis during development. These include vnd͞NK-2 (9, 10, 19, 20), Nkx2-1͞TTF1 (21, 22), Nkx2-2 (21), Nkx2-9 (23), or Nkx6-1 (24) for the nervous system; Tinman͞NK-4 (25), 27), for the heart; Nkx2-5͞Csx or Nkx2-6 for the pharynx (29); Nkx2-1͞TTF-1 for the thyroid and the lung (30); Nkx2-2 for pancreatic ␤ cells (31); Nkx2-8 for the liver (32); and Nkx5-1 for inner ear (33). An orphan homeobox mouse gene Hox11, which was proposed to belong to the NK family (33), controls the genesis of the spleen (34). Nkx3-1 is expressed in many tissues in the embryo; the expression is restricted primarily to the ...

Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: Transgenic alterations and tertiary structure

“…The apparent K d values were determined from EMSAs. Although the apparent K d values differ somewhat from those previously obtained (12,18,33), probably because of slightly different experimental conditions, the results (Table 2) show that the Y54M replacement reduces the affinity for the NK-2 binding site (18NK-2 no. 1) by Ϸ10-fold.…”

“…One might suspect that the apparent inability of the Y54M mutant vnd͞NK-2 protein to repress the ind and msh genes may be because of the decreased level of UAS-vnd͞NK-2 Y54M mRNA compared with the WT or other mutant vnd͞NK-2 mRNA levels (18,25) seen at stage 10 ( Fig. 1).…”

“…Within the NK family of homeobox genes, the NK-2 class, whose founding member is vnd͞NK-2 from Drosophila melanogaster (8)(9)(10), is defined by the presence of tyrosine in position 54 of the homeodomain (11,12). This tyrosine is the major determinant of the nucleotide-binding specificity (13,14) to a sequence of DNA that contains 5Ј-CAAGTG-3Ј as its core (15)(16)(17)(18) through direct interaction between the side chain of Y54 and the cytosine that complements the guanine (in bold) of the consensus DNA (14). This core DNA sequence is recognized by all members of the Nirenberg and Kim (NK)-2 class of homeodomains (15)(16)(17)(18).…”

“…This tyrosine is the major determinant of the nucleotide-binding specificity (13,14) to a sequence of DNA that contains 5Ј-CAAGTG-3Ј as its core (15)(16)(17)(18) through direct interaction between the side chain of Y54 and the cytosine that complements the guanine (in bold) of the consensus DNA (14). This core DNA sequence is recognized by all members of the Nirenberg and Kim (NK)-2 class of homeodomains (15)(16)(17)(18). The vnd͞NK-2 promoter has been identified and contains several copies of this consensus sequence (19).…”

Mapping activation and repression domains of the vnd/NK-2 homeodomain protein

“…Whereas much is known about the interactions between vnd͞NK-2 HD and DNA (29,30,(35)(36)(37), little is known about activation or repression domains of this protein. In this report, we define the locations of repressor and activation domains of vnd͞NK-2 HD protein.…”