Lascarez Lagunas, L.I. et al. (2019) International Worm Meeting "Specific chromatin marks regulate the recognition of crossover position for accurate chromosome segregation."

Altendorfer, E., Lascarez Lagunas, L.I., Colaiacovo, M.

[International Worm Meeting, 2019]

A critical step of meiosis I is the repair of DNA double-strand breaks (DSBs) via crossover (CO) recombination. CO position along a chromosome is tightly regulated, since COs too close to a centromere or to a telomere can result in aneuploidy. In C. elegans, as in other organisms, COs occur mostly at the terminal thirds of the chromosomes. Moreover, only one CO occurs between each pair of homologs. This single off-centered CO leads to chromosome remodeling resulting in an asymmetric bivalent configuration required for accurate chromosome segregation in many organisms. However, the regulation of this remodeling process remains poorly understood. Here, we investigated how CO position is assessed to allow for accurate remodeling and faithful chromosome segregation. We used a single inducible DSB system in which DSBs are generated by heat shock-induced excision of a Mos1 transposon inserted at a defined genomic location. We observed that a CO at the center of a chromosome impairs chromosome remodeling in late prophase I and results in chromosome missegregation. The mechanism used in sensing the symmetry-breaking event along the bivalent may involve chromatin expansion forces which can drive changes in chromosome geometry generating the asymmetric bivalent. We assessed different chromatin marks by immunostaining in lines undergoing either a centered or an off-centered DSB/CO. We observed a decrease in H3K9me2-3 signal and an increase in H3K79me3 in late pachytene nuclei following heat-shock in centered DSB/CO lines compared to control. These results indicate there are cytologically detectable changes when a CO is induced at the center compared to at an off-centered position. ChIP/qPCR analysis revealed that H3K9me2-3 remain enriched at the terminal thirds of chromosomes even after heat shock in control lines, however their distribution changes and is observed enriched only at the center of the chromosome undergoing a centered DSB/CO. These results suggest that a centered CO may fail to be sensed as a symmetry-breaking event based on changes on the chromatin landscape. This provides a mechanism involving the distribution of specific chromatin marks for "measuring" distances between chromosome ends and the CO that may establish the asymmetric bivalent configuration.

Lascarez-Lagunas LI et al. (2014) Mol Cell Biol "LIN-35/Rb causes starvation-induced germ cell apoptosis via CED-9/Bcl2 downregulation in Caenorhabditis elegans."

Navarro RE, Silva-Garcia CG, Dinkova TD, Lascarez-Lagunas LI

[Mol Cell Biol, 2014]

Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation.

Lascarez-Lagunas LI et al. (2020) PLoS Genet "DOT-1.1-dependent H3K79 methylation promotes normal meiotic progression and meiotic checkpoint function in C. elegans."

Herruzo E, San-Segundo PA, Lascarez-Lagunas LI, Colaiacovo MP, Grishok A

[PLoS Genet, 2020]

Epigenetic modifiers are emerging as important regulators of the genome. However, how they regulate specific processes during meiosis is not well understood. Methylation of H3K79 by the histone methyltransferase Dot1 has been shown to be involved in the maintenance of genomic stability in various organisms. In S. cerevisiae, Dot1 modulates the meiotic checkpoint response triggered by synapsis and/or recombination defects by promoting Hop1-dependent Mek1 activation and Hop1 distribution along unsynapsed meiotic chromosomes, at least in part, by regulating Pch2 localization. However, how this protein regulates meiosis in metazoans is unknown. Here, we describe the effects of H3K79me depletion via analysis of dot-1.1 or zfp-1 mutants during meiosis in Caenorhabditis elegans. We observed decreased fertility and increased embryonic lethality in dot-1.1 mutants suggesting meiotic dysfunction. We show that DOT-1.1 plays a role in the regulation of pairing, synapsis and recombination in the worm. Furthermore, we demonstrate that DOT-1.1 is an important regulator of mechanisms surveilling chromosome synapsis during meiosis. In sum, our results reveal that regulation of H3K79me plays an important role in coordinating events during meiosis in C. elegans.

Lascarez-Lagunas LI et al. (2022) Curr Biol "ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice"

Faull P, Ferrandiz N, Zetka M, Lascarez-Lagunas LI, Berson E, Todisco E, Martinez-Perez E, Crawley O, Smolikove S, Montoya A, Quinn JN, Eaford D, Nadarajan S, Martinez-Garcia M, Colaiacovo MP, Thakkar T, Labella S, Barroso C, Koury E

[Curr Biol, 2022]

DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C. elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3-9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C. elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (γ-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.

Lascarez-Lagunas LI et al. (2023) PLoS Genet "Chromatin landscape, DSB levels, and cKU-70/80 contribute to patterning of meiotic DSB processing along chromosomes in C. elegans."

Nadarajan S, Diaz-Pacheco BN, Colaiacovo MP, Lascarez-Lagunas LI, Martinez-Garcia M, Berson E

[PLoS Genet, 2023]

Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans.

Cevallos Porta, Diego et al. (2015) International Worm Meeting "ced-9/Bcl-2 gene regulation during starvation-induced apoptosis in the germline."

Cevallos Porta, Diego, Navarro, Rosa, Recillas-Targa, Felix

[International Worm Meeting, 2015]

Apoptosis in the adult hermaphrodite's gonad occurs in a basal level to maintain homeostasis and oocyte quality (Gummieny et al., 1999 and Andux and Ellis 2008). Different types of stress further induce germ cell apoptosis. Oxidative, osmotic and heat shock stresses induce germ cell apoptosis in EGL-1 and p53 independent pathways that act through the MAPKK via (Salinas et al., 2006). During starvation, there is an increase in germ cell apoptosis due to a reduction on the expression of the anti-apoptotic protein CED-9/Bcl-2. ced-9/Bcl-2 down-regulation during starvation is not observed in a lin-35(n745) mutant background suggesting that LIN-35/RB regulates ced-9/Bcl-2 expression during starvation (Lascarez-Lagunas et al., 2014). LIN-35/Rb lacks a DNA binding domain and it forms different protein complexes to regulate transcription. Our aim is to investigate how ced-9/Bcl-2 expression is down-regulated during starvation.To address this question, we are making a transcriptional reporter that consists of ced-9/Bcl-2 promoter fused to Luc2 (firefly luciferese gene) and ced-9/Bcl-2's 3'UTR. This construct will be inserted into C. elegans genome using the MosCI method. We will also determine ced-9/Bcl2 regulatory sequences needed to reduce the expression during starvation by making specific deletions of its promoter in this reporter strain.We have found, using previously published ChIP databases, some proteins that associate with ced-9/Bcl-2 promoter which also interact directly or indirectly with LIN-35/pRB in the Wormbase interaction network. By RNAi, we are testing whether any of these proteins are required for physiological and/or starvation-induced apoptosis.