Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage
ABSTRACT: We report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9Dom2 variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina. These results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination. The terminal zinc finger domain of PRDM9Dom2 (PRDM9ΔZnF1Dom2, 412–847 aa), the allele present in C57BL/6J (B6) mice was cloned and tagged with 6His-HALO and then expressed in E. coli. DNA sheared to 180–200 bp is provided in considerable excess to provide competition between DNA binding sites. Following binding, DNA–protein complexes are then isolated on streptavidin beads and the DNA extracted for deep sequencing. Two replicate Affinity-seq samples were sequenced at 100-bp reads using the Illumina HiSeq 2500. Alignments to the mm9 mouse genome were obtained utilizing BWA v1.2.3 with default parameters and reads which failed to align to unique positions in the genome were discarded. Peaks were called individually for the two replicates with MACS2 at a p value threshold of 0.01 utilizing a control dataset obtained by sequencing the input DNA and subsequently compared, leading ultimately to combining the two replicates for definitive analysis.
Project description:Mammalian genetic recombination is concentrated at hotspots, specialized 1-2 Kb sites separated by long stretches of DNA lacking recombination. Mammalian hotspot locations depend on PRDM9, a zinc finger protein that binds at hotspots and uses its SET domain to locally trimethylate histone H3K4. Here we find that PRDM9 also locally trimethylates H3K36 at hotspots. Using ChIP-seq and immunoprecipitation data for H3K36me3 in murine spermatocytes, we show that H3K4me3 and H3K36me3 coincide only at hotspots in germ cells, and that this H3K4me3/H3K36me3-double-positive signature is almost entirely dependent on PRDM9. We performed ChIP-seq with an antibody against H3K36me3, using chromatin extracted from murine spermatocytes, and compared it to previously generated ChIP-seq data for H3K4me3 in the same cell type. ---------------------------------- This dataset represents the H3K36 component only
Project description:PRDM9 is a histone methyltransferase expressed in meiotic germ cells that determines the location of genetic recombination hotspots through binding of its allele-specific DNA binding domain. Here we characterize the genome-wide chromatin modification for two human PRDM9 alleles (A and C) in human cell lines. HEK293 cells were transfected with both alleles and an empty vector control. Resulting chromatin was subjected to H3K4me3 ChIP followed by high-throughput sequencing. We find that different PRDM9 allele largely modified chromatin in entirely different genomic regions in somatic cells determined by the protein's zinc-finger DNA binding domains. Many of the allele-specific peaks overlap sites of meiotic double-strand breaks found in vivo in human germ cells suggesting that transient expression of PRDM9 in somatic cells can reflect binding in vivo. Identify PRDM9-dependent H3K4me3 sites by comparing modified chromatin after expression of different human PRDM9 alleles in HEK293 cells.
Project description:Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. The DNA double strand breaks (DSBs) that initiate meiotic recombination are directed by sequence-specific DNA binding of the PRDM9 protein. Gradual elimination of PRDM9 binding sites by gene conversion is thought to result in the hotspot erosion while mutations affecting DNA binding specificity of PRDM9 will create the new sets of hotspots. To better understand evolutionary turnover of recombination hotspots we mapped DSB hotspots in six inbred mouse strains representing all four major subspecies of Mus musculus and in their F1 hybrids. We found that hotspot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hotspots that become active in the hybrids. As crossovers are disfavored at such hotspots, we propose that sequence divergence generated by hotspot turnover creates impediments for recombination in hybrids, potentially leading to reduction in fertility and eventually, speciation. Overall design: Detection of meiotic DNA double strand breaks in testis of 26 mouse strains and F1 hybrids
Project description:PRDM9, a histone methyltransferase, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we compared activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced. Comparing these two strains, we find that haplotype differences at hotspots leads to qualitative and quantitative changes in PRDM9 binding and activity. Most variants affecting PRDM9Cst binding arose and were fixed in M.m castaneus, suppressing hotspot activity. Furthermore, M.m castaneus x M.m domesticus F1 hybrids exhibit novel hotspots, representing sites of historic evolutionary erosion. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion. Identify position of meiotic H3K4me3 from various sub-species of mice and F1 hybrids from crosses between subspecies. In addition, perform ChIP-seq analysis on the meiosis-specific methyltransferase PRDM9.
Project description: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 non-canonical recruitment of PRDM9 to sites which are devoid of both, recombination activity and the PRDM9-binding consensus motif. These sites include transcription promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset of non-canonical sites also reveals DSB-independent interactions between PRDM9 and genomic sites, which include 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. Overall design: PRDM9-, H3K4me3-, H3K36me3-, DMC1-ChIPseq experiments were performed on testis from different mouse lines
Project description:Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It predominantly occurs at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three domains which are typically different families of proteins involved in regulation of transcription in other proteins, yet, the role of PRDM9 in gene expression control has not been evaluated. Here we analyze the germline transcriptome of the Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the activity of PRDM9 lacking effect of the KRAB, SSXRD and post-SET zinc finger domains deletion mutants in a cell expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in a cell culture expression systemin vitro, mice lacking the KRAB domain mutant mice domain show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that these domains that are classically involved in gene regulation do not serve that role in PRDM9, but instead are involved in PRDM9 function has likely diverged from the typical function of a transcription factor and adapted the new role in setting the proper chromatin environment for initiation and completion of homologous recombination. Overall design: Testes mRNA-seq, miRNA-seq, Chip-seq were generated using next generation sequencing;
Project description:The programmed formation of hundreds DNA double strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA binding specificity of its zinc finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9 binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA binding specificities, we reveal severalthe basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset are selected for DSB formation. Overall design: H3K4me3-, DMC1-ChIPseq experiments were performed on testis from different mouse lines
Project description:Here we characterize the genome-wide chromatin modification by PRDM9, a histone H3 lysine 4 methyltransferase. In order to detect PRDM9 binding sites we created coisogenic strains of mice differing only in the zinc finger array of PRDM9. One strain is C57BL/6J, which carries the Prdm9Dom2 allele, the other strain was created using genomic replacement and named B6.PRDM9Cst (also called KI), and contains the Prdm9Cst allele originally found in CAST/EiJ mice. Many H3K4me3 positions are common between strains and represent other methyltransferase activity (such as promoters), sites that are unique to one mouse strain likely represent the binding position of that allele of PRDM9. Identify PRDM9-dependent H3K4me3 sites by comparing modified chromatin from mice coisogenic for Prdm9.
Project description:Genetic recombination occurs during meiosis, the key developmental program of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyl-transferase, PRDM9, the product of the only known speciation gene in mammals. PRDM9 is thought to determine the preferred recombination sites - recombination hotspots - through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyze the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F1 hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the remarkable exception of the pseudoautosomal region – the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice and as in wild-type, these hotspots are found at H3K4 trimethylation marks. However, in the absence of PRDM9, the majority of recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene promoter regions and other functional genomic elements. Overall design: Detection of meiotic double strand breaks in mouse testis and comparison with H3K4me3 marks.
Project description:Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes – normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions. Detection of meiotic double strand breaks in mice with a hypomorphic Spo11 allele.