Identification and analysis of vnd/NK-2 homeodomain binding sites in genomic DNA

Abstract

Vnd͞NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by the vnd͞NK-2 homeodomain. Sequencing the selected genomic DNA fragments led to the identification of 77 Drosophila DNA fragments that were grouped into 42 vnd͞NK-2 homeodomain-binding loci. Most loci were within upstream or intronic regions, especially first introns. Nineteen of the Drosophila DNA fragments cloned correspond to one locus, termed Clone A, which is 312 bp in length and contains five vnd͞NK-2 homeodomain core consensus binding sites, 5 -AAGTG, and is part of the first intron of the Beadex gene. We further analyzed the interactions between Clone A and vnd͞NK-2 homeodomain protein by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. The DNase I footprinting analysis of Clone A with vnd͞NK-2 homeodomain protein revealed three strong binding sites and one weak binding site between 15 and 130 bp of Clone A. We also analyzed binding of the vnd͞NK-2 homeodomain to the 5 -flanking sequence of vnd͞NK-2 genomic DNA. The DNase I footprinting result showed that there are two strong binding sites and five weak binding sites in the fragment between ؊385 and ؊675 bp from the transcription start site of the vnd͞NK-2 gene.homeobox ͉ neurobiology ͉ Drosophila T he Drosophila ventral nervous system defective (vnd)͞NK-2 homeodomain (HD) protein contains a HD (1, 2) that recognizes the consensus nucleotide sequence 5Ј-T(T͞ C)AAGTG(G͞C) with a core sequence of 5Ј-AAGTG (3). The vnd͞NK-2 protein also contains repression and activation domains (4). The vnd͞NK-2 gene initiates neural development in the ventral column of neuroectoderm that gives rise to part of the ventral nerve cord of embryos (5-8). During embryonic development, the vnd͞NK-2 protein, directly or indirectly, activates achaete (9) and NK-6 (10), represses ind and msh (11), and autoregulates its own gene expression by positive feedback (6,8,12). The three-dimensional structure of the vnd͞NK-2 HD and its interaction with a target oligodeoxynucleotide, which contains the sequence 5Ј-TCAAGTGG, has been reported (13-16). The interaction of tyrosine in position 54 (Y54) of the HD with DNA is the major specificity determinant of the vnd͞NK-2 HD-DNA complex (15, 17). The Y54M mutant HD has a reduced affinity for the vnd͞NK-2 target DNA sequence and does not repress ind or msh (18). The A35T mutant is embryonically lethal, and the mutant protein has reduced binding affinity for DNA (19,20). The NK-2 HD has been highly conserved during evolution, and all members of the NK-2 class of HDs have Y54 in their HDs (5, 21). The DNA binding sites of many NK-2 family proteins that have been studied contain the core nucleotide sequence 5Ј-AAGTG (3). The NK-2 family of proteins plays important roles in the morphogenesis of different organs (3, 21).There are gene mutations in the NK-2 family that cause human diseases. Mutations in human NKX2-1 result in a complex disease including neurological, thyroid, and respiratory problems...

Identification of an upstream regulatory element reveals a novel requirement for Ind activity in maintaining ind expression

“…However, elements controlling expression in neuroblasts are located upstream of the coding sequence (Shao et al, 2002). Moreover, Vnd can bind to the upstream element and regulate reporter gene expression from it (Saunders et al, 1998;Wang et al, 2005;Yu et al, 2005). It should be noted, that these results are based on tissue culture reporter assays and not in vivo results.…”

“…In addition, it has been suggested that Vnd is capable of activating its own expression (Yu et al, 2005). Although there is no in vivo evidence of Vnd auto-regulation, in cultured cells Vnd is capable of binding to and activating transcription from an enhancer located upstream of its coding sequences (Wang et al, 2005;Yu et al, 2005). This suggests that regulation of Vnd expression may involve modular activity of multiple enhancer elements.…”

Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target

“…The LMO3 gene has also recently been reported to be translocated in T-ALL (Simonis et al 2009) and implicated in promoting tumorigenesis of neuroblastoma (Aoyama et al 2005). Moreover, a Drosophila homolog of NKX2-1, vnd, most preferentially binds to the Beadex or dLMO gene, the homolog of the LMO family in Drosophila (Wang et al 2005). Indeed, expression of LMO3 was the most significantly correlated of any gene with expression of NKX2-1 in the analyzed lung adenocarcinoma tumor data set ( Fig.…”

The alternative protein isoform NK2B, encoded by the vnd/NK-2 proneural gene, directly activates transcription and is expressed following the start of cells differentiation

“…The canonical NK-2 binding site, having the highest affinity for NK-2 HD, was determined ( 13 , 14 ). The sites of NK-2 protein binding, slightly varying in nucleotide sequences, were found in the regulatory regions of different Drosophila genes, including NK-2 itself ( 15 ). The TN domain located at the N-terminal part of NK-2 proteins is involved in protein–protein interactions.…”