Human subtelomeric WASH genes encode a new subclass of the WASP family

Abstract

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences.

Figure 1. Organization of WASH Loci in the Current Genome Assembly and Representative Transcripts

Only the 9p copy (bold) has a full-length intact ORF; others are partial or disrupted by frameshifts or in-frame stop codons. One copy assigned to 1p is more likely to be a variant allele of 19p [4]. Coding exons are numbered 1 through 10; thin bars represent non-coding exons. Sequence indicated in gray is shared only by Xq/Yq and 16p and lacks the N-terminal portion of WASH. Black and gray arrowheads indicate terminal and degenerate internal telomere-repeat arrays, respectively.

Figure 2. LogoPlot of Protein Alignment of WASH Orthologs from 21 Diverse Species from Mammals to Entamoeba

Heights of letters indicate degree of conservation in units of maximum entropy expressed in bits. Colors indicate amino-acid similarity: KRH, green; DE, blue; and AVLIPWFM, red. Conserved domains discussed in the text are labeled at their approximate midpoints. Gaps indicate regions in the alignment where only a minority of the sequences has residues present. The actual alignment is provided in Figure S4.

Figure 3. WASH Proteins Are Phylogenetically and Structurally Distinct from Known WASP Family Members

(A) WASP family members and characteristic domains, adapted from reference [16] to show the new subfamily of WASH orthologs. Humans possess multiple WASH genes distributed in their subtelomeres, including short forms like one of two Entamoeba orthologs. Excluding Entamoeba, all other non-primate species examined have one WASH ortholog, except S. cerevisiae, in which there is only a WASP-related homolog. P, proline-rich domain; VCA, actin-binding and polymerizing domains; WHD1 and WHD2, subfamily-specific N-terminal domains of WASH (Figure S7). (B) Neighbor joining tree of WASP family members based on C-terminal (VCA region) alignment (Figure S6). Bootstrap values were calculated over 1,000 iterations. Vertebrate clades with high bootstrap support are in color. Species are indicated using two-letter abbreviations in front of each protein name (see Text S1).

Figure 4. Colocalization of Transiently Expressed, GFP-Tagged Full-Length Human WASH with Actin in Filopodia (Two Indicated by Arrowheads) and Lamellipodia.

(A) and (D), GFP-WASH (green) and DAPI to stain nuclei (blue); (B) and (E), Texas-red phalloidin to stain actin (red) and DAPI; (C) and (F), three-color overlay. Size bar, 10 μm.

Figure 5. WASH Promotes Actin Nucleation by the Arp2/3 Complex

Pyrene-actin polymerization assays were conducted with the concentrations indicated for WASH VCA and N-WASP VCA (A). Similar to N-WASP VCA (B), WASH VCA (C) does not promote actin nucleation in the absence of Arp2/3. In the presence of Arp2/3, WASH promotes actin nucleation in a concentration-dependent manner (C).

Figure 6. Analysis of washout Mutant Flies

(A) A schematic of the washout coding region showing the original P-element insertion allele (wash ^EY15549) and a precise (wash ^exc15) and imprecise (wash ^Δ185) excision allele. Sequence analysis of wash ^Δ185 reveals two stop codons at positions 11 and 12 due to the internal deletion of 1,029 nucleotides and insertion of six nucleotides (underlined) at the junction. (B) Genetic analysis of washout alleles. No flies homozygous for the wash ^Δ185 excision allele were recovered. CyO is a balancer chromosome that is homozygous lethal. (C) The wash ^Δ185 mutant phenotype. Bright-field micrograph of pupae heterozygous (left) or homozygous (right) for the wash ^Δ185 allele. The homozygous mutant displays an elongated phenotype and spiracle-eversion (arrows) defects, while the heterozygote is phenotypically normal.

Figure 7. Summary of Locations of WASH Detected by FISH in Unrelated Individuals

The signal-scoring criteria used to deem a location as homozygous (two bars) or heterozygous (one bar) for the presence of WASH sequence are supplied in Text S1. Here, we indicate human WASH locations excluding partial duplicates on Xqter, Yqter, and some 16pter alleles, which were detected only with FISH probes including the distal portion of WASH. For the nonhuman primates, we used a pool of probes completely spanning WASH in order to report all WASH locations in these species. Nonhuman primate chromosomes are numbered according to the corresponding chromosome in the human karyotype.