In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis.

Epigenetic origin of adaptive phenotypic variants in the human blood fluke Schistosoma mansoni.

BACKGROUND: Adaptive evolution is not possible without the generation of phenotypic variants. The origin of these variations has been a central topic in evolutionary biology. Up to now, it was commonly accepted that standing genetic variation is the only cause of phenotypic variants. However, epigenetic information is emerging as a complementary source of heritable phenotypic variation that contributes to evolution. The relative importance of genetics and epigenetics in generating heritable phenotypic variation is nevertheless a matter of debate. RESULTS: We used a host-parasite system to address this question. The human blood fluke Schistosoma mansoni can adapt rapidly to new intermediate snail hosts. The interaction between parasite and mollusk is characterized by a compatibility polymorphism illustrating the evolutionary dynamics in this system. The principal molecular marker for compatibility (infection success) is the expression pattern of a group of polymorphic mucins (SmPoMuc) in the parasite. We show here that chromatin structure changes as the SmPoMuc promoters are the cause for SmPoMuc transcription polymorphism leading to phenotypic novelty and increase in infection success, i.e., fitness. CONCLUSION: We establish that epigenetic changes can be the major if not only cause of adaptive phenotypic variants in Schistosoma mansoni, suggesting that epimutations can provide material for adaptive evolution in the absence of genetic variation in other systems. In addition, our results indicate that epidrugs can be used to control parasite development but also parasite evolution.

Diversity of Prdm9 zinc finger array in wild mice unravels new facets of the evolutionary turnover of this coding minisatellite.

In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons -1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.

Private selective sweeps identified from next-generation pool-sequencing reveal convergent pathways under selection in two inbred Schistosoma mansoni strains.

BACKGROUND: The trematode flatworms of the genus Schistosoma, the causative agents of schistosomiasis, are among the most prevalent parasites in humans, affecting more than 200 million people worldwide. In this study, we focused on two well-characterized strains of S. mansoni, to explore signatures of selection. Both strains are highly inbred and exhibit differences in life history traits, in particular in their compatibility with the intermediate host Biomphalaria glabrata. METHODOLOGY/PRINCIPAL FINDINGS: We performed high throughput sequencing of DNA from pools of individuals of each strain using Illumina technology and identified single nucleotide polymorphisms (SNP) and copy number variations (CNV). In total, 708,898 SNPs were identified and roughly 2,000 CNVs. The SNPs revealed low nucleotide diversity (π = 2 × 10(-4)) within each strain and a high differentiation level (Fst = 0.73) between them. Based on a recently developed in-silico approach, we further detected 12 and 19 private (i.e. specific non-overlapping) selective sweeps among the 121 and 151 sweeps found in total for each strain. CONCLUSIONS/SIGNIFICANCE: Functional annotation of transcripts lying in the private selective sweeps revealed specific selection for functions related to parasitic interaction (e.g. cell-cell adhesion or redox reactions). Despite high differentiation between strains, we identified evolutionary convergence of genes related to proteolysis, known as a key virulence factor and a potential target of drug and vaccine development. Our data show that pool-sequencing can be used for the detection of selective sweeps in parasite populations and enables one to identify biological functions under selection.

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse.

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11(-/-) mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

Playing hide and seek with mammalian meiotic crossover hotspots.

Crossovers (COs) are essential for meiosis and contribute to genome diversity by promoting the reassociation of alleles, and thus improve the efficiency of selection. COs are not randomly distributed but are found at specific regions, or CO hotspots. Recent results have revealed the historical recombination rates and the distribution of hotspots across the human genome. Surprisingly, CO hotspots are highly dynamic, as shown by differences in activity between individuals, populations and closely related species. We propose a role for DNA methylation in preventing the formation of COs, a regulation that might explain, in part, the correlation between recombination rates and GC content in mammals.

A fast and specific alignment method for minisatellite maps.

BACKGROUND: Variable minisatellites count among the most polymorphic markers of eukaryotic and prokaryotic genomes. This variability can affect gene coding regions, like in the prion protein gene, or gene regulation regions, like for the cystatin B gene, and be associated or implicated in diseases: the Creutzfeld-Jakob disease and the myoclonus epilepsy type 1, for our examples. When it affects neutrally evolving regions, the polymorphism in length (i.e., in number of copies) of minisatellites proved useful in population genetics. MOTIVATION: In these tandem repeat sequences, different mutational mechanisms let the number of copies, as well as the copies themselves, vary. Especially, the interspersion of events of tandem duplication/contraction and of punctual mutation makes the succession of variant repeats much more informative than the sole allele length. To exploit this information requires the ability to align minisatellite alleles by accounting for both punctual mutations and tandem duplications. RESULTS: We propose a minisatellite maps alignment program that improves on previous solutions. Our new program is faster, simpler, considers an extended evolutionary model, and is available to the community. We test it on the data set of 609 alleles of the MSY1 (DYF155S1) human minisatellite and confirm its ability to recover known evolutionary signals. Our experiments highlight that the informativeness of minisatellites resides in their length and composition polymorphisms. Exploiting both simultaneously is critical to unravel the implications of variable minisatellites in the control of gene expression and diseases.

Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21.

Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.

Manipulating multiple sequence alignments via MaM and WebMaM.

MaM is a software tool that processes and manipulates multiple alignments of genomic sequence. MaM computes the exact location of common repeat elements, exons and unique regions within aligned genomics sequences using a variety of user identified programs, databases and/or tables. The program can extract subalignments, corresponding to these various regions of DNA to be analyzed independently or in conjunction with other elements of genomic DNA. Graphical displays further allow an assessment of sequence variation throughout these different regions of the aligned sequence, providing separate displays for their repeat, non-repeat and coding portions of genomic DNA. The program should facilitate the phylogenetic analysis and processing of different portions of genomic sequence as part of large-scale sequencing efforts. MaM source code is freely available for non-commercial use at http://compbio.cs.sfu.ca/MAM.htm; and the web interface WebMaM is hosted at http://atgc.lirmm.fr/mam.

Male cell microchimerism in normal and diseased female livers from fetal life to adulthood.

Male microchimerism is frequent in the adult female liver and is attributed to fetal cells originating from previous male offspring. It has never been studied in pregnant women, female children, or fetuses. We examined its frequency and cellular nature in normal and diseased female livers from fetal life to adulthood. Forty-six liver samples from 29 women, 6 female children, and 11 female fetuses were screened for the Y chromosome via polymerase chain reaction (PCR) assay and fluorescent in situ hybridization (FISH). The X chromosome was used as an internal control. A third PCR assay was used for Y genotyping. The Y chromosome was detected in 5 of 6 children, 7 of 11 fetuses, 3 of 9 women with normal liver, 7 of 10 women with chronic hepatitis C, 5 of 6 women with acute liver disease during pregnancy with male offspring, and 2 of 4 nonpregnant women with fulminant hepatitis. In positive samples, the mean XY/XX ratio was 0.012 (+/-0.004). In women, male microchimerism was correlated with previous male offspring. Male hepatocytes, detected via FISH combined with anti-hepatocyte immunohistochemistry, were observed only in fetuses (4/9) and in postpartem women (4/6). Y genotypes were different from each other in 4 of 5 female livers. In conclusion, male liver microchimerism is frequent in normal and diseased female livers. The presence of male cells in the liver of female children and fetuses is probably due to the transplacental transmission of fetal cells preexisting in the mother and acquired either from previous pregnancy with male offspring or during the mother's own fetal life.

Recombination across the centromere of disjoined and non-disjoined chromosome 21.

Meiotic recombination is generally suppressed across the centromere of eukaryotic chromosomes. In human, megabase-long satellite sequences and contiguous segmental duplications hamper both physical and fine scale genetic mapping in regions flanking centromeric DNA. We have developed polymorphic microsatellite markers embedded within the duplicated most proximal sequences of the long arm and of the short arm of chromosome 21 by using paralogous specific bases as anchor points for their specific detection. Segregation analysis in CEPH reference pedigrees shows that recombination is repressed significantly across the centromere of chromosome 21 both in male and in female but not in the most proximal 21q region in female. Extreme size variations of the alpha-satellite I blocks transmitted in these families and deduced from quantitative FISH analysis are not correlated with the inter-individual variations of recombination activity observed in the peri-centromeric region. Finally, none of 28 families with a trisomy 21 child previously associated with a nullitransitional meiosis I non-disjunction event presents a recombination exchange across the centromere. This confirms that, for this group of errors, the lack of recombination is the primary susceptibility factor, not abnormal recombination in the centromeric region.

Instability of the human minisatellite CEB1 in rad27Delta and dna2-1 replication-deficient yeast cells.

Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them. Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations. We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Delta cells. Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Delta cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit. Altogether, these results suggest that the improperly processed 5' flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events. This replication-dependent phenomenon differs from the recombination-induced instability in yeast meiotic cells.

Evolutionary fate of an unstable human minisatellite deduced from sperm-mutation spectra of individual alleles.

Although mutation processes at some human minisatellites have been extensively characterized, the evolutionary fate of these unstable loci is unknown. Minisatellite instability is largely germline specific, with mutation rates up to several percent and with expansion events predominating over contractions. Using allele-specific small-pool polymerase chain reaction, we have determined sperm-mutation spectra of individual alleles of the highly unstable human minisatellite CEB1 (i.e., D2S90). We show that, as allele size increases, the proportion of contractions rises from <5% to 50%, with the average size of deletion increasing and eventually exceeding the average size of expansion. The expected net effect of these trends after many generations is an equilibrium distribution of allele sizes, and allele-frequency data suggest that this equilibrium state has been reached in some contemporary human populations.