Conserved alternative and antisense transcripts at the programmed cell death 2 locus

<p>Abstract</p> <p>Background</p> <p>The programmed cell death 2 (<it>Pdcd2</it>) gene on mouse chromosome 17 was evaluated as a member of a highly conserved synteny, a candidate for an imprinted locus, and a candidate for the Hybrid sterility 1 (<it>Hst1</it>) gene.</p> <p>Results</p> <p>New mouse transcripts were identified at this locus: an alternative <it>Pdcd2 </it>mRNA skipping the last two coding exons and two classes of antisense RNAs. One class of the antisense RNA overlaps the alternative exon and the other the entire <it>Pdcd2 </it>gene. The antisense RNAs are alternative transcripts of the neighboring TATA-binding protein gene (<it>Tbp</it>) that are located mainly in the cell nucleus. Analogous alternative PDCD2 forms truncating the C-terminal domain were also detected in human and chicken. Alternative transcripts of the chicken <it>PDCD2 </it>and <it>TBP </it>genes also overlap. No correlation in the transcription of the alternative and overlapping mRNAs was detected. Allelic sequencing and transcription studies did not reveal any support for the candidacy of <it>Pdcd2 </it>for <it>Hst1</it>. No correlated expression of <it>Pdcd2 </it>with the other two genes of the highly conserved synteny was observed. <it>Pdcd2</it>, <it>Chd1</it>, and four other genes from this region were not imprinted in the embryo.</p> <p>Conclusion</p> <p>The conservation of alternative transcription of the <it>Pdcd2 </it>gene in mouse, human and chicken suggests the biological importance of such truncated protein. The biological function of the alternative <it>PDCD2 </it>is likely to be opposite to that of the constitutive form. The ratio of the constitutive and alternative <it>Pdcd2 </it>mRNAs differs in the tissues, suggesting a developmental role.</p> <p>The identified <it>Tbp-</it>alternative <it>Pdcd2</it>-antisense transcripts may interfere with the transcription of the <it>Pdcd2 </it>gene, as they are transcribed at a comparable level. The conservation of the <it>Pdcd2</it>/<it>Tbp </it>sense-antisense overlap in the mouse and chicken points out its biological relevance. Our results also suggest that some cDNAs in databases labeled as noncoding are incomplete alternative cDNAs of neighboring protein-coding genes.</p

Fine Haplotype Structure of a Chromosome 17 Region in the Laboratory and Wild Mouse

Extensive linkage disequilibrium among classical laboratory strains represents an obstacle in the high-resolution haplotype mapping of mouse quantitative trait loci (QTL). To determine the potential of wild-derived mouse strains for fine QTL mapping, we constructed a haplotype map of a 250-kb region of the t-complex on chromosome 17 containing the Hybrid sterility 1 (Hst1) gene. We resequenced 33 loci from up to 80 chromosomes of five mouse (sub)species. Trans-species single-nucleotide polymorphisms (SNPs) were rare between Mus m. musculus (Mmmu) and Mus m. domesticus (Mmd). The haplotypes in Mmmu and Mmd differed and therefore strains from these subspecies should not be combined for haplotype-associated mapping. The haplotypes of t-chromosomes differed from all non-t Mmmu and Mmd haplotypes. Half of the SNPs and SN indels but only one of seven longer rearrangements found in classical laboratory strains were useful for haplotype mapping in the wild-derived M. m. domesticus. The largest Mmd haplotype block contained three genes of a highly conserved synteny. The lengths of the haplotype blocks deduced from 36 domesticus chromosomes were in tens of kilobases, suggesting that the wild-derived Mmd strains are suitable for fine interval-specific mapping

Data from: Hybrid sterility locus on Chromosome X controls meiotic recombination rate in mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies

PRDM9 can form heteromeric complexes at hotspots.

<p>(A) PRDM9 protein variants can form homo- and heteromeric complexes. Western blot of various tagged versions of human PRDM9 detected either with anti-FLAG or anti-V5 antibodies (asterisk—non-specific band). V5-PRDM9^C (V5-C) is found in association with FLAG-PRDM9^A (FLAG-A) or FLAG-PRDM9^C (FLAG-C) after FLAG or V5 IP. (B) PRDM9^A is bound to the PRDM9^C-activated hotspot 5A when co-expressed with PRDM9^C. Tagged <i>PRDM9</i> alleles, indicated on the x-axis, were transfected into HEK293 cells and subsequently subjected to ChIP against FLAG antibody; qPCR was performed on ChIP DNA samples at the center of the 5A hotspot (circles—individual biological replicates, lines—mean). (C) and (D) Similar to B showing ChIP results for PRDM9^C-activated hotspots at Chr 3 114 Mb and Chr 3 4 Mb respectively. (E) PRDM9^A can bind A-hotspots. Similar to B-D showing ChIP results for PRDM9^A-activated hotspot at Chr 3 54 Mb. (F) PRDM9^A can trimethylate C hotspots when in complex with PRDM9^C. Heat map of H3K4me3 ChIP-seq read counts from HEK293 cells for a 2 kb window surrounding PRDM9^C hotspots from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005512#pgen.1005512.g002" target="_blank">Fig 2D</a>.</p

<i>Prdm9</i>^<i>Dom2</i> is suppressed by competition between alleles.

<p>(A) PRDM9^Cst can suppress PRDM9^Dom2-directed recombination at <i>Ush2a</i>. To detect either parental (top) or recombinant (bottom) molecules, nested PCR was performed on DNA from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for <i>Prdm9</i>^<i>Dom2</i>. (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9^Cst or PRDM9^Dom2. (C) PRDM9^Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in <i>Prdm9</i>^{<i>Dom2/-</i>} heterozygous mice.</p

Histone methyltransferase PRDM9 is not essential for meiosis in male mice.

A hallmark of meiosis is the rearrangement of parental alleles to ensure genetic diversity in the gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSBs occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance

Expression of <i>PRDM9</i> in HEK293 cells recapitulates allele-specific hotspot activation in vivo.

<p>(A) Western blot showing that expression of PRDM9 in HEK293 cells increases global H3K4me3 levels dependent on the PR/SET domain. (B) and (C) qPCR was performed on enriched DNA from ChIP against H3K4me3 at various intervals across the hotspots indicated by the position of the symbols after expression of PRDM9 protein variants. (B) PRDM9^A-defined recombination hotspots S and F have increased H3K4me3 after expression of <i>PRDM9</i>^<i>A</i>. (C) PRDM9^C-defined recombination hotpots 5A and 22A are enriched for H3K4me3 after expression of <i>PRDM9</i>^<i>C</i>. (D) Expression of PRDM9 in HEK293 cells results in allele-specific H3K4me3 at in vivo DSB hotspots. Heat map of H3K4me3 ChIP-seq (HEK293 cells) and DMC1 SSDS (men) signals for a 2 kb window centered on PRDM9 motifs. DMC1 SSDS data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005512#pgen.1005512.ref028" target="_blank">28</a>]. (E) Aggregate plot of H3K4me3 (grey) and DMC1 (purple) signals from A-hotspots after expression of <i>PRDM9</i>^<i>A</i>. PRDM9^A motif derived from the hotspot nucleosome-depleted regions is shown above. (F) Similar to <i>E</i> except showing the result for PRDM9^C.</p