Analysis of dosage effect of speciation gene Prdm9 on fertility of mouse hybrids

(cz) Fenomén hybridní sterility představuje jeden z evolučních mechanismů umožňující speciaci, tedy vznik nových druhů. Zatím bylo popsáno jen málo speciačních genů a Prdm9 (PR-domain 9) je první a zatím jediný identifikovaný u savců. Hraje totiž roli ve sterilitě hybridů vznikajících křížením poddruhů myši domácí. Ohledně vlivu Prdm9 na plodnost semifertilních hybridních samců bylo ale dosud publikováno jen omezené množství poznatků, přitom v přírodě jsou takoví samci daleko četnější než samci zcela sterilní. Pomocí panelu genetických nástrojů a baterií fenotypizačních testů dokazuje tato disertace ústřední roli Prdm9 v hybridní sterilitě na mnoha typech mezi-poddruhových kříženců s různě postiženou plodností a věkovým omezením. Přidáváním ale i ubíráním genové dávky Prdm9 se fenotyp snížené plodnosti vylepší. Také zvýšení genové dávky Prdm9 alely, která v jedné kopii zapříčiňuje sterilitu, zlepšuje parametry fertility F1 hybridů. PRDM9 protein hraje roli i v určování míst meiotické rekombinace. Práce poukazuje na princip interakce soutěže alel v určování rekombinančně preferovaných genomických lokusů - hotspotů, a tím možné způsoby propojení obou rolí Prdm9. Tato disertace shrnuje sestavu tří logicky propojených publikací, které mají ambici být významnými díly skládačky poznání mechanismu...(eng) The phenomenon of hybrid sterility represents one of the evolutionary mechanisms that enables speciation. Only a few speciation genes have been uncovered. The only one found in mammals is Prdm9 (PR-domain 9). Data in the literature on the involvement of Prdm9 in decreased fertility of various semifertile hybrid males of house mouse subspecies were scarce before the results of this thesis were completed, despite that such males are much more frequent in nature than the fully sterile ones. Utilizing a panel of genetic tools and a battery of phenotyping tests, this thesis shows a central role of Prdm9 in fecundity of hybrids, including many fertility disorders and age dependency. Both increasing and reducing the Prdm9 gene dosage significantly elevated fertility parameters. Surprisingly, even the allele that in one copy causes full hybrid sterility increased F1 hybrid fertility when present in multiple copies. The PRDM9 protein also plays a role in identifying the sites of meiotic recombination. This study also points out the principles of allelic competition in determination of the sites of preferred recombination (hotspots), which suggests a possible link between both previously described Prdm9 roles. This thesis summarizes a set of three logically interconnected publications with the ambition...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Analysis of dosage effect of speciation gene Prdm9 on fertility of mouse hybrids

(eng) The phenomenon of hybrid sterility represents one of the evolutionary mechanisms that enables speciation. Only a few speciation genes have been uncovered. The only one found in mammals is Prdm9 (PR-domain 9). Data in the literature on the involvement of Prdm9 in decreased fertility of various semifertile hybrid males of house mouse subspecies were scarce before the results of this thesis were completed, despite that such males are much more frequent in nature than the fully sterile ones. Utilizing a panel of genetic tools and a battery of phenotyping tests, this thesis shows a central role of Prdm9 in fecundity of hybrids, including many fertility disorders and age dependency. Both increasing and reducing the Prdm9 gene dosage significantly elevated fertility parameters. Surprisingly, even the allele that in one copy causes full hybrid sterility increased F1 hybrid fertility when present in multiple copies. The PRDM9 protein also plays a role in identifying the sites of meiotic recombination. This study also points out the principles of allelic competition in determination of the sites of preferred recombination (hotspots), which suggests a possible link between both previously described Prdm9 roles. This thesis summarizes a set of three logically interconnected publications with the ambition..

Mouse congenic strains.

Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Establishment and phenotyping of three mouse congenic strains.

Mouse congenic strains are widely used as an important genetic tool for various purposes of research. Congenic strains are special inbred strains in which part of the genome of one mouse is transferred to another by backcrossing the donor mouse to the recipient strain with appropriate selection. The classical ten-generation breeding protocol is commonly utilized and adapted for different experimental needs. Our department is focused on the study of hybrid sterility between two related mouse strains M. m. musculus and M. m. domesticus. The first aim of this work was the utilization of congenics to stabilize three transgenic lines, which previously helped to identify first mammalian hybrid sterility gene Hst1 as Prdm9 (Mihola et al, 2009). Bacterial artificial chromosomes (BACs) present in transgenes encompassed several genes including Prdm9. The second goal was to look for some new phenotypes of our congenic strains, which may be causally associated with genes encoded by the BACs, as well as an additional research on the hybrid sterility phenomena. We established three new congenic strains on C57BL/6J (B6) background and found significant differences between transgenics versus their littermate controls in weights of testis, gonadal fat, body, liver, and heart. Powered by TCPDF (www.tcpdf.org

Focal Adhesion Protein Vinculin Is Required for Proper Meiotic Progression during Mouse Spermatogenesis

The focal adhesion protein Vinculin (VCL) is ascribed to various cytoplasmic functions; however, its nuclear role has so far been ambiguous. We observed that VCL localizes to the nuclei of mouse primary spermatocytes undergoing first meiotic division. Specifically, VCL localizes along the meiosis-specific structure synaptonemal complex (SC) during prophase I and the centromeric regions, where it remains until metaphase I. To study the role of VCL in meiotic division, we prepared a conditional knock-out mouse (VCLcKO). We found that the VCLcKO male mice were semi-fertile, with a decreased number of offspring compared to wild-type animals. This study of events in late prophase I indicated premature splitting of homologous chromosomes, accompanied by an untimely loss of SCP1. This caused erroneous kinetochore formation, followed by failure of the meiotic spindle assembly and metaphase I arrest. To assess the mechanism of VCL involvement in meiosis, we searched for its possible interacting partners. A mass spectrometry approach identified several putative interactors which belong to the ubiquitin–proteasome pathway (UPS). The depletion of VLC leads to the dysregulation of a key subunit of the proteasome complex in the meiotic nuclei and an altered nuclear SUMOylation level. Taken together, we show for the first time the presence of VCL in the nucleus of spermatocytes and its involvement in proper meiotic progress. It also suggests the direction for future studies regarding the role of VCL in spermatogenesis through regulation of UPS

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PLoS Genet 2015 Sep 14; 11(9):e1005512

<i>Prdm9</i> Incompatibility Controls Oligospermia and Delayed Fertility but No Selfish Transmission in Mouse Intersubspecific Hybrids

<div><p>PR-domain 9 (<i>Prdm9</i>) is the first hybrid sterility gene identified in mammals. The incompatibility between <i>Prdm9</i> from <i>Mus musculus domesticus</i> (Mmd; the B6 strain) and the <i>Hstx2</i> region of chromosome (Chr) X from <i>M. m. musculus</i> (Mmm; the PWD strain) participates in the complete meiotic arrest of mouse intersubspecific (PWD×B6)F1 hybrid males. Other studies suggest that also semisterile intersubspecific hybrids are relevant for mouse speciation, but the genes responsible remain unknown. To investigate the causes of this semisterility, we analyzed the role of <i>Prdm9</i> and Chr X in hybrids resulting from the crosses of PWK, another Mmm-derived inbred strain. We demonstrate that <i>Prdm9</i> and Chr X control the partial meiotic arrest and reduced sperm count in (PWK×B6)F1 males. Asynapsis of heterosubspecific chromosomes and semisterility were partially suppressed by removal of the B6 allele of <i>Prdm9</i>. Polymorphisms between PWK and PWD on Chr X but not in the <i>Prdm9</i> region were responsible for the modification of the outcome of <i>Prdm9</i> - Chr X F1 hybrid incompatibility. Furthermore, (PWK×B6)F1 hybrid males displayed delayed fertility dependent on the <i>Prdm9</i> incompatibility. While the <i>Drosophila</i> hybrid sterility gene <i>Overdrive</i> causes both delayed fertility and increased transmission of its own chromosome to the offspring, the segregation of Chr X and the <i>Prdm9</i> region from the mouse (PWK×B6)F1 males was normal. Our results indicate extended functional consequences of <i>Prdm9</i> - Chr X intersubspecific incompatibility on the fertility of hybrids and should influence the design of fertility analyses in hybrid zones and of laboratory crosses between Mmm and Mmd strains.</p></div

Pachytene phenotypes of PWK F1 males.

<p>Age, male age (weeks); N, number of counted pachynemas from a total of two or three males; SB, AB, 0B, % pachytene spermatocytes carrying a normal sex body and all chromosomes synapsed (SB), an abnormal sex body (AB, a sex body containing unsynapsed autosomes), and neither abnormal nor sex body (0B; always carried also unsynapsed chromosomes). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095806#pone-0095806-g002" target="_blank">Figure 2A</a> for representative phenotypes and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095806#pone-0095806-t001" target="_blank">Table 1</a> for other abbreviations. Antibodies against SYCP3, SYCP1, and γH2AX were used to stage the cells on chromosomal spreads. See the text for statistical evaluations.</p