High resolution maps of chromatin reorganization through mouse meiosis reveal novel features of the 3D meiotic structure.

When germ cells transition from the mitotic cycle into meiotic prophase I (MPI), chromosomes condense into an array of chromatin loops that are required to promote homolog pairing and genetic recombination. To identify the changes in chromosomal conformation, we isolated nuclei on a trajectory from spermatogonia to the end of MPI. At each stage along this trajectory, we built genomic interaction maps with the highest temporal and spatial resolution to date. The changes in chromatin folding coincided with a concurrent decline in mitotic cohesion and a rise in meiotic cohesin complexes. We found that the stereotypical large-scale A and B compartmentalization was lost during meiotic prophase I alongside the loss of topological associating domains (TADs). Still, local subcompartments were detected and maintained throughout meiosis. The enhanced Micro-C resolution revealed that, despite the loss of TADs, higher frequency contact sites between two loci were detectable during meiotic prophase I coinciding with CTCF bound sites. The pattern of interactions around these CTCF sites with their neighboring loci showed that CTCF sites were often anchoring the meiotic loops. Additionally, the localization of CTCF to the meiotic axes indicated that these anchors were at the base of loops. Strikingly, even in the face of the dramatic reconfiguration of interphase chromatin into a condensed loop-array, the interactions between regulatory elements remained well preserved. This establishes a potential mechanism for how the meiotic chromatin maintains active transcription within a highly structured genome. In summary, the high temporal and spatial resolution of these data revealed previously unappreciated aspects of mammalian meiotic chromatin organization.

Cataloging Human PRDM9 Allelic Variation Using Long-Read Sequencing Reveals PRDM9 Population Specificity and Two Distinct Groupings of Related Alleles.

The PRDM9 protein determines sites of meiotic recombination in humans by directing meiotic DNA double-strand breaks to specific loci. Targeting specificity is encoded by a long array of C2H2 zinc fingers that bind to DNA. This zinc finger array is hypervariable, and the resulting alleles each have a potentially different DNA binding preference. The assessment of PRDM9 diversity is important for understanding the complexity of human population genetics, inheritance linkage patterns, and predisposition to genetic disease. Due to the repetitive nature of the PRDM9 zinc finger array, the large-scale sequencing of human PRDM9 is challenging. We, therefore, developed a long-read sequencing strategy to infer the diploid PRDM9 zinc finger array genotype in a high-throughput manner. From an unbiased study of PRDM9 allelic diversity in 720 individuals from seven human populations, we detected 69 PRDM9 alleles. Several alleles differ in frequency among human populations, and 32 alleles had not been identified by previous studies, which were heavily biased to European populations. PRDM9 alleles are distinguished by their DNA binding site preferences and fall into two major categories related to the most common PRDM9-A and PRDM9-C alleles. We also found that it is likely that inter-conversion between allele types is rare. By mapping meiotic double-strand breaks (DSBs) in the testis, we found that small variations in PRDM9 can substantially alter the meiotic recombination landscape, demonstrating that minor PRDM9 variants may play an under-appreciated role in shaping patterns of human recombination. In summary, our data greatly expands knowledge of PRDM9 diversity in humans.

CRL4 regulates recombination and synaptonemal complex aggregation in the Caenorhabditis elegans germline.

To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in Caenorhabditis elegans. Mutants of the CRL4 complex (cul-4, ddb-1, and gad-1) show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity.

Moving and stopping: Regulation of chromosome movement to promote meiotic chromosome pairing and synapsis.

Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original chromosome number. During the earliest stage of meiosis, prophase I, pairing of homologous chromosomes is achieved in preparation for their proper distribution in the coming divisions. An important question is how do homologous chromosomes find each other and establish pairing interactions. Early studies demonstrated that chromosomes are dynamic in nature and move during this early stage of meiosis. More recently, there have been several studies across different models showing the conserved nature and importance of this chromosome movement, as well as the key components involved in chromosome movement. This review will cover these major findings and also introduce unexamined areas of regulation in meiotic prophase I chromosome movement.

Prophase I: Preparing Chromosomes for Segregation in the Developing Oocyte.

Formation of an oocyte involves a specialized cell division termed meiosis. In meiotic prophase I (the initial stage of meiosis), chromosomes undergo elaborate events to ensure the proper segregation of their chromosomes into gametes. These events include processes leading to the formation of a crossover that, along with sister chromatid cohesion, forms the physical link between homologous chromosomes. Crossovers are formed as an outcome of recombination. This process initiates with programmed double-strand breaks that are repaired through the use of homologous chromosomes as a repair template. The accurate repair to form crossovers takes place in the context of the synaptonemal complex, a protein complex that links homologous chromosomes in meiotic prophase I. To allow proper execution of meiotic prophase I events, signaling processes connect different steps in recombination and synapsis. The events occurring in meiotic prophase I are a prerequisite for proper chromosome segregation in the meiotic divisions. When these processes go awry, chromosomes missegregate. These meiotic errors are thought to increase with aging and may contribute to the increase in aneuploidy observed in advanced maternal age female oocytes.

Regulating chromosomal movement by the cochaperone FKB-6 ensures timely pairing and synapsis.

In meiotic prophase I, homologous chromosome pairing is promoted through chromosome movement mediated by nuclear envelope proteins, microtubules, and dynein. After proper homologue pairing has been established, the synaptonemal complex (SC) assembles along the paired homologues, stabilizing their interaction and allowing for crossing over to occur. Previous studies have shown that perturbing chromosome movement leads to pairing defects and SC polycomplex formation. We show that FKB-6 plays a role in SC assembly and is required for timely pairing and proper double-strand break repair kinetics. FKB-6 localizes outside the nucleus, and in its absence, the microtubule network is altered. FKB-6 is required for proper movement of dynein, increasing resting time between movements. Attenuating chromosomal movement in fkb-6 mutants partially restores the defects in synapsis, in agreement with FKB-6 acting by decreasing chromosomal movement. Therefore, we suggest that FKB-6 plays a role in regulating dynein movement by preventing excess chromosome movement, which is essential for proper SC assembly and homologous chromosome pairing.

Chimeric transcripts resulting from complex duplications in chromosome Xq28.

Gene fusions have been observed in somatic alterations in cancer and in schizophrenia. However, the underlying mechanism(s) for their formation are poorly understood. We experimentally demonstrated the expression of splicing variants of in silico predicted chimeric genes F8/CSAG1 and BCAP31/TEX28 in two individuals with de novo complex genomic rearrangements of Xq28; F8/CSAG1 includes exonization of an ERVL-MaLR intronic repetitive element. We provide evidence that replicative repair may contribute to exon shuffling processes and diversify the repertoire of expressed transcripts.

The CSN/COP9 signalosome regulates synaptonemal complex assembly during meiotic prophase I of Caenorhabditis elegans.

The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

Replicative mechanisms for CNV formation are error prone.

We investigated 67 breakpoint junctions of gene copy number gains in 31 unrelated subjects. We observed a strikingly high frequency of small deletions and insertions (29%) apparently originating from polymerase slippage events, in addition to frameshifts and point mutations in homonucleotide runs (13%), at or flanking the breakpoint junctions of complex copy number variants. These single-nucleotide variants were generated concomitantly with the de novo complex genomic rearrangement (CGR) event. Our findings implicate low-fidelity, error-prone DNA polymerase activity in synthesis associated with DNA repair mechanisms as the cause of local increase in point mutation burden associated with human CGR.