Publications
25 results found
Belan O, Barroso C, Kaczmarczyk A, et al., 2021, Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins, Molecular Cell, Vol: 81, Pages: 1058-1073.e7, ISSN: 1097-2765
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a “chaperone” to promote 3′ to 5′ filament growth via highly dynamic engagement with 5′ filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Roelens B, Barroso C, Montoya A, et al., 2019, Spatial Regulation of Polo-Like Kinase Activity During <i>Caenorhabditis elegans</i> Meiosis by the Nucleoplasmic HAL-2/HAL-3 Complex, GENETICS, Vol: 213, Pages: 79-96, ISSN: 0016-6731
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- Citations: 7
Beltran T, Barroso C, Birkle TY, et al., 2019, Comparative epigenomics reveals that RNA polymerase II pausing and chromatin domain organization control nematode piRNA biogenesis, Developmental Cell, Vol: 48, Pages: 1-18, ISSN: 1534-5807
Piwi-interacting RNAs (piRNAs) are important for genome regulation across metazoans, but their biogenesis evolves rapidly. In Caenorhabditis elegans, piRNA loci are clustered within two 3-Mb regions on chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce an ∼28 nt primary transcript. We used comparative epigenomics across nematodes to gain insight into the origin, evolution, and mechanism of nematode piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA transcription. We describe two alternative modes of piRNA organization in nematodes: in C. elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, while in other species, typified by Pristionchus pacificus, piRNAs are found within introns of active genes. Additionally, we discover that piRNA production depends on sequence signals associated with RNA polymerase II pausing. We show that pausing signals synergize with chromatin to control piRNA transcription.
Gorlitz F, Guldbrand S, Runcorn T, et al., 2018, easySLM-STED: stimulated emission depletion microscopy with aberration correction, extended field of view and multiple beam scanning, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X
We demonstrate a simplified set‐up for STED microscopy with a straightforward alignment procedure that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams to provide phase modulation of the beam profiles and correction of optical aberrations. We show that this approach can be used to extend the field of view for STED microscopy by correcting chromatic aberration that otherwise leads to walk‐off between the focused excitation and depletion beams. We further show how this arrangement can be adapted to increase the imaging speed through multibeam excitation and depletion. Fine adjustments to the alignment can be accomplished using the SLM only, conferring the potential for automation.
Link J, Paouneskou D, Velkova M, et al., 2018, Transient and partial nuclear lamina disruption promotes chromosome movement in early meiotic prophase, Developmental Cell, Vol: 45, Pages: 212-225, ISSN: 1534-5807
Meiotic chromosome movement is important for the pairwise alignment of homologous chromosomes, which is required for correct chromosome segregation. Movement is driven by cytoplasmic forces, transmitted to chromosome ends by nuclear membrane-spanning proteins. In animal cells, lamins form a prominent scaffold at the nuclear periphery, yet the role lamins play in meiotic chromosome movement is unclear. We show that chromosome movement correlates with reduced lamin association with the nuclear rim, which requires lamin phosphorylation at sites analogous to those that open lamina network crosslinks in mitosis. Failure to remodel the lamina results in delayed meiotic entry, altered chromatin organization, unpaired or interlocked chromosomes, and slowed chromosome movement. The remodeling kinases are delivered to lamins via chromosome ends coupled to the nuclear envelope, potentially enabling crosstalk between the lamina and chromosomal events. Thus, opening the lamina network plays a role in modulating contacts between chromosomes and the nuclear periphery during meiosis.
Ferrandiz N, Barroso C, Telecan O, et al., 2018, Spatiotemporal regulation of Aurora B recruitment ensures release of cohesion during <i>C-elegans</i> oocyte meiosis, NATURE COMMUNICATIONS, Vol: 9, ISSN: 2041-1723
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- Citations: 39
Hillers KJ, Jantsch V, Martinez-Perez E, et al., 2017, Meiosis., WormBook, Vol: 2017, Pages: 1-43
Sexual reproduction requires the production of haploid gametes (sperm and egg) with only one copy of each chromosome; fertilization then restores the diploid chromosome content in the next generation. This reduction in genetic content is accomplished during a specialized cell division called meiosis, in which two rounds of chromosome segregation follow a single round of DNA replication. In preparation for the first meiotic division, homologous chromosomes pair and synapse, creating a context that promotes formation of crossover recombination events. These crossovers, in conjunction with sister chromatid cohesion, serve to connect the two homologs and facilitate their segregation to opposite poles during the first meiotic division. During the second meiotic division, which is similar to mitosis, sister chromatids separate; the resultant products are haploid cells that become gametes. In Caenorhabditis elegans (and most other eukaryotes) homologous pairing and recombination are required for proper chromosome inheritance during meiosis; accordingly, the events of meiosis are tightly coordinated to ensure the proper execution of these events. In this chapter, we review the seminal events of meiosis: pairing of homologous chromosomes, the changes in chromosome structure that chromosomes undergo during meiosis, the events of meiotic recombination, the differentiation of homologous chromosome pairs into structures optimized for proper chromosome segregation at Meiosis I, and the ultimate segregation of chromosomes during the meiotic divisions. We also review the regulatory processes that ensure the coordinated execution of these meiotic events during prophase I.
Gao J, Barroso C, Zhang P, et al., 2016, N-terminal acetylation promotes synaptonemal complex assembly in C. elegans, Genes and Development, Vol: 30, Pages: 2404-2416, ISSN: 0890-9369
N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis.
Nadarajan S, Mohideen F, Tzur YB, et al., 2016, The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis, eLife, Vol: 5, Pages: 1-26, ISSN: 2050-084X
Asymmetric disassembly of the synaptonemal complex (SC) is crucial for propermeiotic chromosome segregation. However, the signaling mechanisms that directly regulate thisprocess are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2,functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegansgermline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central regioncomponent, is a potential target for MPK-1-mediated phosphorylation and that constitutivelyphosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referredto as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical fortimely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO)promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAPkinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accuratechromosome segregation.
Crawley O, Barroso C, Testori S, et al., 2016, Cohesin-interacting protein WAPL-1 regulates meiotic chromosome structure and cohesion by antagonizing specific cohesin complexes, eLife, Vol: 5, ISSN: 2050-084X
Wapl induces cohesin dissociation from DNA throughout the mitotic cell cycle, modulating sister chromatid cohesion and higher-order chromatin structure. Cohesin complexes containing meiosis-specific kleisin subunits govern most aspects of meiotic chromosome function, but whether Wapl regulates these complexes remains unknown. We show that during C. elegans oogenesis WAPL-1 antagonizes binding of cohesin containing COH-3/4 kleisins, but not REC-8, demonstrating that sensitivity to WAPL-1 is dictated by kleisin identity. By restricting the amount of chromosome-associated COH-3/4 cohesin, WAPL-1 controls chromosome structure throughout meiotic prophase. In the absence of REC-8, WAPL-1 inhibits COH-3/4-mediated cohesion, which requires crossover-fated events formed during meiotic recombination. Thus, WAPL-1 promotes functional specialization of meiotic cohesin: WAPL-1-sensitive COH-3/4 complexes modulate higher-order chromosome structure, while WAPL-1-refractory REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-independent mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes.
Silva N, Ferrandiz N, Barroso C, et al., 2014, The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression, Developmental Cell, Vol: 31, Pages: 503-511, ISSN: 1534-5807
Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, andpoorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint.
Sato-Carlton A, Li X, Crawley O, et al., 2014, Protein Phosphatase 4 Promotes Chromosome Pairing and Synapsis, and Contributes to Maintaining Crossover Competence with Increasing Age, PLOS GENETICS, Vol: 10, ISSN: 1553-7404
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- Citations: 15
Schvarzstein M, Pattabiraman D, Libuda DE, et al., 2014, DNA Helicase HIM-6/BLM Both Promotes MutSγ-Dependent Crossovers and Antagonizes MutSγ-Independent Interhomolog Associations During <i>Caenorhabditis elegans</i> Meiosis, GENETICS, Vol: 198, Pages: 193-+, ISSN: 0016-6731
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- Citations: 21
Weick E-M, Sarkies P, Silva N, et al., 2014, PRDE-1 is a nuclear factor essential for the biogenesis of Ruby motif-dependent piRNAs in C-elegans, Genes and Development, Vol: 28, Pages: 783-796, ISSN: 0890-9369
Piwi-interacting RNAs (piRNA) are small regulatory RNAs with essential roles in maintaining genome integrity in animals and protists. Most Caenorhabditis elegans piRNAs are transcribed from two genomic clusters that likely contain thousands of individual transcription units; however, their biogenesis is not understood. Here we identify and characterize prde-1 (piRNA silencing-defective) as the first essential C. elegans piRNA biogenesis gene. Analysis of prde-1 provides the first direct evidence that piRNA precursors are 28- to 29-nucleotide (nt) RNAs initiating 2 nt upstream of mature piRNAs. PRDE-1 is a nuclear germline-expressed protein that localizes to chromosome IV. PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an 8-nt upstream motif, the Ruby motif. The expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. We exploited this finding to determine the targets of the motif-independent class of piRNAs. Together, our data provide new insights into both the biogenesis and function of piRNAs in gene regulation.
Silva N, Adamo A, Santonicola P, et al., 2013, Pro-crossover factors regulate damage-dependent apoptosis in the <i>Caenorhabditis elegans</i> germ line, CELL DEATH AND DIFFERENTIATION, Vol: 20, Pages: 1209-1218, ISSN: 1350-9047
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- Citations: 11
Labrador L, Barroso C, Lightfoot J, et al., 2013, Chromosome Movements Promoted by the Mitochondrial Protein SPD-3 Are Required for Homology Search during <i>Caenorhabditis elegans</i> Meiosis, PLOS GENETICS, Vol: 9, ISSN: 1553-7404
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- Citations: 25
Aragon L, Martinez-Perez E, Merkenschlager M, 2013, Condensin, cohesin and the control of chromatin states, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 23, Pages: 204-211, ISSN: 0959-437X
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- Citations: 35
Zhang W, Miley N, Zastrow MS, et al., 2012, HAL-2 Promotes Homologous Pairing during <i>Caenorhabditis elegans</i> Meiosis by Antagonizing Inhibitory Effects of Synaptonemal Complex Precursors, PLOS GENETICS, Vol: 8, ISSN: 1553-7404
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- Citations: 27
Lightfoot J, Testori S, Barroso C, et al., 2011, Loading of Meiotic Cohesin by SCC-2 Is Required for Early Processing of DSBs and for the DNA Damage Checkpoint, CURRENT BIOLOGY, Vol: 21, Pages: 1421-1430, ISSN: 0960-9822
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- Citations: 43
Adamo A, Collis SJ, Adelman CA, et al., 2010, Preventing Nonhomologous End Joining Suppresses DNA Repair Defects of Fanconi Anemia, MOLECULAR CELL, Vol: 39, Pages: 25-35, ISSN: 1097-2765
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- Citations: 235
Ward JD, Muzzini DM, Petalcorin MIR, et al., 2010, Overlapping Mechanisms Promote Postsynaptic RAD-51 Filament Disassembly during Meiotic Double-Strand Break Repair, MOLECULAR CELL, Vol: 37, Pages: 259-272, ISSN: 1097-2765
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- Citations: 71
Martinez-Perez E, Colaiacovo MP, 2009, Distribution of meiotic recombination events: talking to your neighbors, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 19, Pages: 105-112, ISSN: 0959-437X
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- Citations: 57
Martinez-Perez E, 2009, Meiosis in cereal crops: the grasses are back., Genome Dyn, Vol: 5, Pages: 26-42, ISSN: 1660-9263
A major goal of breeding programs is to increase and manipulate the genetic diversity of crops. The incorporation of beneficial genes from wild relatives into crops is achieved by producing hybrid plants in which meiotic recombination events occur between the two genomes. Furthering our understanding of meiosis in the cereals could enable the manipulation of homolog pairing and recombination, significantly enhancing the efficiency of breeding programs. The main obstacle to the genetic analysis of meiosis in cereal crops has been the complex organization of most cereal genomes, many of which are polyploid. However, the recent sequencing of the rice genome, the use of insertional mutagenesis and reverse genetics approaches has opened the door for the genetic and genomic analysis of cereal meiosis. The goal of this review is to show how these new resources, as well as the use of three-dimensional microscopy, are rapidly providing insights into the mechanisms that control pairing, recombination and segregation of homologous chromosomes during meiosis in four major cereal crops: wheat, rice, maize and rye.
Martinez-Perez E, Schvarzstein M, Barroso C, et al., 2008, Crossovers trigger a remodeling of meiotic chromosome axis composition that is linked to two-step loss of sister chromatid cohesion, GENES & DEVELOPMENT, Vol: 22, Pages: 2886-2901, ISSN: 0890-9369
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- Citations: 109
Martinez-Perez E, Moore G, 2008, To check or not to check? The application of meiotic studies to plant breeding, CURRENT OPINION IN PLANT BIOLOGY, Vol: 11, Pages: 222-227, ISSN: 1369-5266
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- Citations: 26
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