The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system - PubMed (original) (raw)

The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system

T Schlake et al. Proc Natl Acad Sci U S A. 1997.

Abstract

In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to a 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulated reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution.

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Figures

Figure 2

Figure 2

Protein alignment of Whn DNA binding domains. (A) Genomic structure of whn genes. Introns are indicated by thin lines, exons by rectangles. Shading indicates regions encoding the DNA binding domain. Note the highly variable sizes of introns. (B) Alignment of all known Whn DNA binding domains (uppercase letters) and flanking sequences; amino acids are abbreviated in single-letter code. The phase of introns is indicated in brackets. Asterisks indicate that splice junctions were determined by comparison with other whn genes, rather than by comparison of genomic and cDNA sequences. The sequences for mouse (4), rat (4, 8, 15) and fugu (8) have been described earlier. Residues identical in all eight genes are given in the consensus line; some conservative changes are denoted by number: 1, negatively charged amino acid (E, D); 2, positively charged amino acid (K, R). The bottom line indicates the presumed secondary structure characteristics of the Whn winged-helix domain based on the structure of the DNA binding domain of HNF3γ (16). h, helix; s, β-sheet; w, loop (wing). Pairwise comparisons indicate that Whn DNA binding domains from human and amphioxus are 80% identical. Characteristic amino acid signatures are highlighted in different colors. Note that the exon containing the Whn DNA binding domain in amphioxus extends further into the 5′ direction.

Figure 1

Figure 1

Binding site selection for mouse Whn DNA binding domain. Double-stranded oligonucleotides randomized at 20 positions were incubated with the bacterially expressed mouse Whn DNA binding domain. After six rounds of affinity purification, bound oligonucleotides were cloned and sequenced. All sequences contained an identical tetranucleotide, 5′-ACGC. The frequency of nucleotides occurring in the flanking regions is indicated by a percentage. No sequence specificity was detected upstream or downstream of the shown 11-bp region.

Figure 3

Figure 3

Absence of _whn_-related genes in mouse, zebrafish, and amphioxus. Hybridization analysis was performed under low stringency conditions (6× SSC at 65°C) with cDNA probes spanning the DNA binding domain from the indicated species. DNAs were digested with _Hin_dIII (mouse), _Spe_I (zebrafish), and _Dra_I (amphioxus) to reveal single fragments for the known whn genes. Note the absence of additional hybridizing bands.

Figure 4

Figure 4

Recognition of Whn binding sites in vitro and in vivo. (A) Electrophoretic mobility shift assays using mouse, zebrafish, and amphioxus DNA binding domain peptides. A double-stranded oligonucleotide obtained via in vitro selection was used in wild-type configuration (core sequence ACGC), in modified forms (changed nucleotides in core sequence are indicated by lowercase letters), or in in vitro methylated form (m denotes 5-methylcytosine). (B) Transactivation of a luciferase reporter gene after transient transfection into BHK cells. A luciferase gene with a minimal promotor (11) was cotransfected with a mouse whn (DBDMm) expression plasmid or with constructs in which the mouse DNA binding domain was changed to that of zebrafish (DBDDr) or amphioxus (DBDBl) to establish a luciferase baseline activity. These values were compared with reporter constructs in which a whn response element was positioned upstream of the minimal promotor and expressed as fold transactivation. Values shown represent the average of two experiments with a variation of less than 20%. I refers to expression constructs with an N-terminal MYC tag; II refers to constructs with a C-terminal MYC tag. DBD, DNA binding domain; AD, transcriptional activation domain (8). In control experiments, the transfection of whn expression constructs in anti-sense orientation had no effect on luciferase activity.

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