On the dependent recognition of some long zinc finger proteins.

The human genome contains about 800 C2H2 zinc finger proteins (ZFPs), and most of them are composed of long arrays of zinc fingers. Standard ZFP recognition model asserts longer finger arrays should recognize longer DNA-binding sites. However, recent experimental efforts to identify in vivo ZFP binding sites contradict this assumption, with many exhibiting short motifs. Here we use ZFY, CTCF, ZIM3, and ZNF343 as examples to address three closely related questions: What are the reasons that impede current motif discovery methods? What are the functions of those seemingly unused fingers and how can we improve the motif discovery algorithms based on long ZFPs' biophysical properties? Using ZFY, we employed a variety of methods and find evidence for 'dependent recognition' where downstream fingers can recognize some previously undiscovered motifs only in the presence of an intact core site. For CTCF, high-throughput measurements revealed its upstream specificity profile depends on the strength of its core. Moreover, the binding strength of the upstream site modulates CTCF's sensitivity to different epigenetic modifications within the core, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates the epigenetic control by CTCF. Our results establish that, because of irregular motif structures, variable spacing and dependent recognition between sub-motifs, the specificities of long ZFPs are significantly underestimated, so we developed an algorithm, ModeMap, to infer the motifs and recognition models of ZIM3 and ZNF343, which facilitates high-confidence identification of specific binding sites, including repeats-derived elements. With revised concept, technique, and algorithm, we can discover the overlooked specificities and functions of those 'extra' fingers, and therefore decipher their broader roles in human biology and diseases.

Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudomodularity.

PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, Glu360Pro (Prdm9EP) and Glu360Lys (Prdm9EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9EP is slightly less hypomorphic than Prdm9EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9EP, but not Prdm9EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9EP and Prdm9EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain vs the full-length protein shows that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8.

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.

CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse.

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse models to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on PRDM9 hotspot trimethylation, double strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.

PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis.

In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9's KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.

Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage.

BACKGROUND: Genetic recombination plays an important role in evolution, facilitating the creation of new, favorable combinations of alleles and the removal of deleterious mutations by unlinking them from surrounding sequences. In most mammals, the placement of genetic crossovers is determined by the binding of PRDM9, a highly polymorphic protein with a long zinc finger array, to its cognate binding sites. It is one of over 800 genes encoding proteins with zinc finger domains in the human genome. RESULTS: 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 PRDM9(Dom2) 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. CONCLUSIONS: 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.

DNA binding specificities of the long zinc-finger recombination protein PRDM9.

BACKGROUND: Meiotic recombination ensures proper segregation of homologous chromosomes and creates genetic variation. In many organisms, recombination occurs at limited sites, termed 'hotspots', whose positions in mammals are determined by PR domain member 9 (PRDM9), a long-array zinc-finger and chromatin-modifier protein. Determining the rules governing the DNA binding of PRDM9 is a major issue in understanding how it functions. RESULTS: Mouse PRDM9 protein variants bind to hotspot DNA sequences in a manner that is specific for both PRDM9 and DNA haplotypes, and that in vitro binding parallels its in vivo biological activity. Examining four hotspots, three activated by Prdm9Cst and one activated by Prdm9Dom2, we found that all binding sites required the full array of 11 or 12 contiguous fingers, depending on the allele, and that there was little sequence similarity between the binding sites of the three Prdm9Cst activated hotspots. The binding specificity of each position in the Hlx1 binding site, activated by Prdm9Cst, was tested by mutating each nucleotide to its three alternatives. The 31 positions along the binding site varied considerably in the ability of alternative bases to support binding, which also implicates a role for additional binding to the DNA phosphate backbone. CONCLUSIONS: These results, which provide the first detailed mapping of PRDM9 binding to DNA and, to our knowledge, the most detailed analysis yet of DNA binding by a long zinc-finger array, make clear that the binding specificities of PRDM9, and possibly other long-array zinc-finger proteins, are unusually complex.

Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping.

The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.

Brief vs. extended buprenorphine detoxification in a community treatment program: engagement and short-term outcomes.

BACKGROUND: Despite evidence supporting the efficacy of buprenorphine relative to established detoxification agents such as clonidine, little research has examined: 1) how best to implement buprenorphine detoxification in outpatient settings; and 2) whether extending the length of buprenorphine detoxification improves treatment engagement and outcomes. OBJECTIVES: The current study examined the impact on 1) successful detoxification completion; 2) transition to longer-term treatment; and 3) treatment engagement of two different length opioid detoxifications using buprenorphine. METHODS: The study compared data obtained from two consecutive studies of early treatment engagement strategies. In one study (n = 364), opioid-addicted participants entered treatment through a Brief (5-day) buprenorphine detoxification. In the other study (n = 146), participants entered treatment through an Extended (i.e., 30-day) buprenorphine detoxification. RESULTS: Results indicated a greater likelihood of successful completion and of transition among participants who received the Extended as compared to the Brief detoxification. Extended detoxification participants attended more counseling sessions and submitted fewer drug-positive urine specimens during the first 30 days of treatment, inclusive of detoxification, than did Brief detoxification participants. CONCLUSIONS: Results demonstrate that longer periods of detoxification improve participant engagement in treatment and early treatment outcomes. SCIENTIFIC SIGNIFICANCE: Current findings demonstrate the feasibility of implementing an extended buprenorphine detoxification within a community-based treatment clinic.

Quantitative PCR genotyping assay for the Ts65Dn mouse model of Down syndrome.

The Ts65Dn mouse is a segmentally trisomic model for Down syndrome. Until now, Ts65Dn mice have been identified by the laborious methods of either chromosomal analysis of cultured peripheral lymphocytes or fluorescent in situ hybridization (FISH). We report here a quantitative PCR method for genotyping Ts65Dn mice, as well as a phenotypic description for visually preclassifying mice to be genotyped.