Biology subjects

Yanowitz, J. L.

Publications and source records attributed to Yanowitz, J. L..

6 recordsLinked to original sources

bioRxiv2022

Proteasomal subunit depletions differentially affect germline integrity in C. elegans

The 26S proteasome is a multi-subunit protein complex that is canonically known for its ability to degrade proteins in cells and maintain protein homeostasis. Non-canonical or non-proteolytic roles of proteasomal subunits exist, but remain less well studied. We provide characterization of germline-specific functions of different 19S RP proteasome subunits in C. elegans using RNAi specifically from the L4 stage and through generation of endogenously tagged 19S RP lid subunit strains. We show functions for the 19S RP in regulation of proliferation and maintenance of integrity of mitotic zone nuclei, in polymerization of the synaptonemal complex (SC) onto meiotic chromosomes and in the timing of SC subunit redistribution to the short arm of the bivalent, and in turnover of XND-1 proteins at late pachytene. Furthermore, we report that certain 19S RP subunits are required for proper germ line localization of WEE-1.3, a major meiotic kinase. Additionally, endogenous fluorescent labeling revealed that the two isoforms of the essential 19S RP proteasome subunit RPN-6.1 are expressed in a tissue-specific manner in the hermaphrodite. Also, we demonstrate that the 19S RP subunits RPN-6.1 and RPN-7 are crucial for the nuclear localization of the lid subunits RPN-8 and RPN-9 in oocytes, potentially introducing C. elegans germ line as model to study proteasome assembly real-time. Collectively, our data support the premise that certain 19S RP proteasome subunits are playing tissue-specific roles, especially in the germ line. We propose C. elegans as a versatile multicellular model to study the diverse proteolytic and non-proteolytic roles that proteasome subunits play in vivo.

developmental biology↗

bioRxiv2021

ANALYSIS OF CHD-7 DEFECTIVE DAUER NEMATODES IMPLICATES COLLAGEN MISREGULATION IN CHARGE SYNDROME FEATURES.

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, and stress response. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the TGF-{beta} pathway. Notably, DAF-12 promoted chd-7 expression, which is necessary to repress daf-9 for execution of the dauer program. Transcriptomic analysis comparing chd-7-defective and normal dauers showed enrichment of collagen genes, consistent with a conserved role for the TGF-{beta} pathway in formation of the extracellular matrix. To validate a conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino mediated knockdown of Chd7 led to a reduction in col2a1 mRNA levels. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by over-expression of col2a1 mRNA. We suggest that pathogenic features of CHARGE syndrome caused by Chd7 mutations, such as craniofacial malformations, result from the reduction of collagen levels, implying that the extracellular matrix might represent a critical target of Chd7 in CHARGE development. SIGNIFICANCE STATEMENTCHARGE Syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7). Unfortunately, the cellular events that lead to CHARGE syndrome are still poorly understood. In C. elegans, we identified chd-7 in a screen for suppressors of dauer formation, an alternative larval stage that develops in response to sensory signals of a harsh environment. We found that chd-7 regulates expression of collagens, which constitute the worms cuticle, a specialized extracellular matrix. In frogs embryos, we show that Chd7 inhibition leads to poor Col2a1, which is necessary and sufficient to exhibit CHARGE features. These studies establish C. elegans as an amenable animal model to study the etiology of the developmental defects associated with pathogenic Chd7.

genetics↗

bioRxiv2020

Poly(ADP-ribose) glycohydrolase promotes formation and homology-directed repair of meiotic DNA double-strand breaks independent of its catalytic activity

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in promoting the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

developmental biology↗

bioRxiv2019

ATM and ATR Influence Meiotic Crossover Formation Through Antagonistic and Overlapping Functions in C. elegans

During meiosis, formation of double-strand breaks (DSBs) and repair by homologous recombination between homologs creates crossovers (COs) that facilitate chromosome segregation. CO formation is tightly regulated to ensure the integrity of this process. The DNA damage response kinases, Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) have emerged as key regulators of CO formation in yeast, flies, and mice, influencing DSB formation, repair pathway choice, and cell cycle progression. The molecular networks that ATM and ATR influence during meiosis are still being resolved in other organisms. Here we show that Caenorhabditis elegans ATM and ATR homologs, ATM-1 and ATL-1 respectively, act at multiple steps in CO formation to ultimately ensure that COs are formed on all chromosomes. We show a role for ATM-1 in regulating the choice of repair template, biasing use of the homologous chromosome instead of the sister chromatid. Our data suggests a model in which ATM-1 and ATL-1 have antagonistic roles in very early repair processing, but are redundantly required for accumulation of the RAD-51 recombinase at DSB sites. We propose that these features of ATM-1 and ATL-1 ensure both CO formation on all chromosomes and accurate repair of additional DSBs.\n\nArticle SummaryCrossovers formed during meiosis connect homologs and properly align them for cell division. The central importance of crossovers is underscored by the existence of extensive regulatory processes that ensures the proper execution of these events. This paper explores the evolutionary conserved roles of the central DNA damage response kinases, ATM and ATR, in crossover formation. The authors show that these kinases function together as rheostats to promote timely formation of crossovers on all chromosomes but to limit extensive DNA damage. This work provides a platform for identifying conserved meiotic targets of ATM and ATR that affect fertility across species.

genetics↗

bioRxiv2019

GCNA preserves genome integrity and fertility across species

The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While these cells employ a number of known repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ Cell Nuclear Acidic Peptidase (GCNA) as a highly conserved regulator of genome stability in flies, worms, zebrafish, and humans. GCNA contains a long acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, GCNA mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, most of GCNAs function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.\n\nHighlightsGCNA ensures genomic stability in germ cells and early embryos across species\n\nGCNA limits replication stress and DNA double stranded breaks\n\nGCNA restricts DNA-Protein Crosslinks within germ cells and early embryos\n\nThe IDR and SprT domains of GCNA govern distinct aspects of genome integrity\n\nGraphic Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC=\"FIGDIR/small/570804_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@1928d82org.highwire.dtl.DTLVardef@885ff2org.highwire.dtl.DTLVardef@15322c9org.highwire.dtl.DTLVardef@110c615_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2018

Meiotic Double-Strand Break Proteins Influence Repair Pathway Utilization

Double-strand breaks (DSBs) are among the most deleterious lesions DNA can endure. Yet, DSBs are programmed at the onset of meiosis and are required to facilitate appropriate reduction of ploidy in daughter cells. Repair of these break is tightly controlled to favor homologous recombination (HR), the only repair pathway that can form crossovers.\n\nHowever, little is known about how the activities of alternative repair pathways are regulated at these stages. We discovered an unexpected synthetic interaction between the DSB machinery and strand-exchange proteins. Depleting the C. elegans DSB-promoting factors HIM-5 and DSB-2 suppresses the formation of chromosome fusions that arise in the absence of RAD-51 or other strand-exchange mediators. Our investigations reveal that non-homologous and theta-mediated end joining (c-NHEJ and TMEJ, respectively) and single strand annealing (SSA) function redundantly to repair DSBs when HR is compromised and that HIM-5 influences the utilization of TMEJ and SSA.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC=\"FIGDIR/small/376582_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@6e6a89org.highwire.dtl.DTLVardef@1145d1borg.highwire.dtl.DTLVardef@1eccd82org.highwire.dtl.DTLVardef@1a82e7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗