Two isoforms of the RAC-specific guanine nucleotide exchange factor TIAM2 act oppositely on transmission ratio distortion by the mouse t-haplotype

Abstract

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.

Fig 1. Two transcript isoforms of Tiam2 are expressed in spermatogenic cells.

(A) Schematic drawing of the wild type t-complex (+) and the t-haplotype (t). Genes with proven function in TRD are boxed; inversions are indicated by arrows. (B) Northern blot analysis of Tiam2 expression in testes of wild-type and t-haplotype mice. (C) RT-qPCR analysis of Tiam2 transcripts (L is Tiam2l and S is Tiam2s) in testes of C57BL/6 wild-type mice at day 7 to day 24 after birth reflecting the first round of spermatogenesis, and in adult mouse testis. (D) in situ hybridization with a Tiam2 probe. (E) RT-qPCR analysis of Tiam2 transcripts in testis RNA derived from C57BL/6 or from mice carrying t haplotypes on the C57BL/6 background; all values were calculated relative to Tiam2l expression in one C57BL/6 sample set to 1. Abbr.: Mb, Megabases; T, Brachyury; tf, tufted; H-2, mouse MHC complex; Inv(17)1 to Inv(17)4, Inversions 1 to 4 spanning the t-complex; se, Sertoli cell; sg, spermatogonium; sc, spermatocyte; sz, spermatozoa; rs, round spermatid.

Fig 2. Transgenic overexpression of Tiam2l and Tiam2s.

(A) Transgenic constructs for overexpression of Tiam2l and Tiam2s during spermatogenesis. The TIAM2 domain structure and abbreviations are according to [27] (B, C) RT-qPCR analysis verifying RNA expression of the transgenic constructs in the testis. (D, E) t-haplotype transmission ratio of TgL transgenic mice and non-transgenic control animals. Horizontal bars denote average t-transmission ratio (%); the number of t carrying out of the total offspring is given in brackets below. Dots indicate the number of offspring sired by individual males. See also S3 Table for total results. Abbr.:Ins, 2x chicken beta-globin insulator; kb, kilobases; pA, polyadenylation signals of rabbit beta-globin and SV40;Prom, promoter of the angiotensin converting enzyme.

Fig 3. Targeted inactivation of the Tiam2 gene.

(A) Gene targeting strategy. Tiam2 was inactivated by introducing a neomycin selection cassette deleting exons 16 and 17. Introns are depicted as line, exons as boxes and targeted exons as shaded boxes. (B) Confirmation of correct homologous recombination. Southern blot analysis of NheI digested genomic DNA of ES-cells and mice with the right (3’) and left (5’) external probes. (C) Loss of Tiam2 transcription upon targeting of the Tiam2 locus as determined by RT-PCR expression analysis of both Tiam2l and Tiam2s in wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mutant testes using a primer pair detecting both transcripts (see S6 Table). (D, E) t-haplotype transmission ratio of Tiam2^LS/t males and Tiam2^+/t control animals, without (D) or carrying in addition the transgene construct Tg1S (E). Horizontal bars denote average t-transmission ratio (%); the number of t carrying out of the total offspring is given in brackets below. Dots indicate the number of offspring sired by individual males. See also S4 and S5 Tables for total results. Abbr.: wt, wild-type; ko, knockout; +, wild-type allele;—, knock-out allele; 3xpA, polyadenylation signal.

Fig 4. Molecular model of transmission ratio distortion comprising the opposing roles of TIAM2L and TIAM2S.

Schematic drawing representing haploid sperm cells of a syncytium of a t/+ heterozygous male undergoing spermatogenesis, the blue cell containing the t-haplotype expressing Smok, Smok^Tcr and the t-distorter alleles, the green cell carrying the wild-type t-complex expressing Smok and wild type distorter alleles. While the distorter products are exchanged between the cells (i.e. they act in trans), and together impair sperm motility, the SMOK proteins and SMOK^TCR are retained within the cell carrying the gene leading to a phenotypic difference of the spermatozoa, such that t-sperm (blue) are functionally rescued and show normal motility (indicated by a wavy tail) while +-sperm (green) remain impaired. Thus offspring of the t/+ male preferentially inherit the t-haplotype. For details of the molecular model see the text. Red upward pointing arrows at distorter proteins indicate up-regulation, blue down-pointing arrows down-regulation; the green down-pointing arrow to the axoneme symbolizes rescued, the dark-red down-pointing arrow impaired flagellar motility.