The role of APC/CFZR-1 in the functionality of the gonad of Caenorhabditis elegans

[ES] Una correcta coordinación entre la división celular y la diferenciación es imprescindible durante el desarrollo de los organismos. Para mantener dicha coordinación, la maquinaria del ciclo celular determina durante la fase G1 si una célula se diferencia o continua dividiéndose. Diversos componentes del ciclo celular activos en G1 son capaces de actuar directamente sobre factores implicados en diferenciación. Entre dichos componentes de ciclo celular destaca la ubiquitín E3 ligasa APC/CCdh1 por su papel emergente en la degradación de diversos factores de diferenciación. Caenorhabditis elegans constituye un sistema excelente para el estudio de los posibles papeles de APC/CCdh1/FZR-1 más allá del ciclo celular. Resultados previos de nuestro laboratorio demostraron que APC/CFZR-1 promueve para su degradación a la metiltransferasa de histonas (HMT) MES-4. En esta tesis, describimos que MES-3, una subunidad de otra HMT, el complejo Poycomb, también es diana de APC/CFZR-1. La incapacidad de la línea germinal para degradar MES-3 mediante la ruta descrita en esta tesis resulta en una caída drástica de los niveles de fertilidad en este nematodo. Hemos observado que dicha bajada en los niveles de fertilidad se contrarrestan en presencia de una versión de MES-4 que no se degrada vía APC/CFZR-1. Concluimos que la regulación post-traduccional y simultánea de MES-3 y MES-4 por parte de APC/CFZR-1 constituye un importante mecanismo para asegurar el mantenimiento de la funcionalidad de la línea germinal. Por otro lado, hemos explorado el papel de APC/CFZR-1 durante el desarrollo de C. elegans. Hemos obtenido el primer alelo completamente nulo de fzr-1 descrito hasta la fecha. APC/CFZR-1 participa en el desarrollo de la gónada somática, órgano encargado de sustentar a la línea germinal. Concretamente, APC/CFZR-1 es necesario para la producción de la Distal Tip Cell (DTC), un nicho celular que mantiene un reservorio de células germinales y lidera el desarrollo de la gónada. En ausencia de FZR-1, las células destinadas a convertirse en DTC, adquieren el destino celular “precursor de SS” propio de la célula hermana. En cambio, un alelo activo constitutivamente de fzr-1 genera la producción de extra DTCs. Ambos resultados sugieren que APC/CFZR-1 es parte de un balance durante la toma de decisión DTC-SS en la gónada somática

Divulgación en tiempos de pandemia: el poder de las webs

Trabajo presentado en el simposio Micromundo Aprendizaje-Servicio para el descubrimiento de antibiótico

The role of APC/CFZR-1 in the functionality of the gonad of Caenorhabditis elegans

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Salamanca.--2020-11-13Proper coordination between the cell division and the differentiation is essential during the development of organisms. The cell-cycle machinery determines during the G1 phase, whether a cell differentiates or continues dividing to achieve this coordination. Many components of the cell cycle active in G1 can act directly on differentiation factors. Among these cell-cycle components highlights the E3 ubiquitin ligase APC/CCdh1 due to its emergent role in the degradation of several differentiation factors. Caenorhabditis elegans constitutes and excellent system to study the possible roles of APC/CCdh1/FZR-1 beyond cell cycle. Previous results from our laboratory demonstrated that APC/CFZR-1 sends to degradation the histone methyltransferase (HMT) MES-4. In this thesis, we have discovered that MES3, a subunit of HMT Polycomb, is also targeted by APC/CFZR-1 . We have observed that the low fertility levels associated with a version of MES-3 not recognizable by APC/CFZR-1 are counteracted in combination with a version of MES-4 not degraded through APC/CFZR-1 . The post-translational and simultaneous regulation of MES-4 and MES-3 through APC/CFZR-1 constitutes a critical mechanism to ensure germline functionality. On the other hand, we have explored the role of APC/CFZR-1 during C. elegans development. We have obtained the first null-allele of fzr-1 described so far. APC/CFZR-1 participates in the development of the somatic gonad, an organ that supports germline development. Specifically, APC/CFZR-1 is necessary for the production of the Distal tip Cell (DTC), a stem-cell niche that maintains a pool of germ cells and leads the outgrowth of the gonad, Absence of FZR-1 makes that cells committed to being DTCs, acquire the SS precursor fate of sister cells. However, an allele of fzr-1 supposed to be constitutively active yields extra DTCs. Both results suggest that APC/CFZR-1 is part of a balance during the fate choice decision 'DTC-SS' in the somatic gonad.Peer reviewe

The Nuclear Envelope in Ageing and Progeria

Development from embryo to adult, organismal homeostasis and ageing are consecutive processes that rely on several functions of the nuclear envelope (NE). The NE compartmentalises the eukaryotic cells and provides physical stability to the genetic material in the nucleus. It provides spatiotemporal regulation of gene expression by controlling nuclear import and hence access of transcription factors to target genes as well as organisation of the genome into open and closed compartments. In addition, positioning of chromatin relative to the NE is important for DNA replication and repair and thereby also for genome stability. We discuss here the relevance of the NE in two classes of age-related human diseases. Firstly, we focus on the progeria syndromes Hutchinson–Gilford (HGPS) and Nestor–Guillermo (NGPS), which are caused by mutations in the LMNA and BANF1 genes, respectively. Both genes encode ubiquitously expressed components of the nuclear lamina that underlines the nuclear membranes. HGPS and NGPS patients manifest symptoms of accelerated ageing and cells from affected individuals show similar defects as cells from healthy old donors, including signs of increased DNA damage and epigenetic alternations. Secondly, we describe how several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis and Huntington’s disease, are related with defects in nucleocytoplasmic transport. A common feature of this class of diseases is the accumulation of nuclear pore proteins and other transport factors in inclusions. Importantly, genetic manipulations of the nucleocytoplasmic transport machinery can alleviate disease-related phenotypes in cell and animal models, paving the way for potential therapeutic interventions.We also wish to acknowledge funding from the Spanish State Research Agency (doi: 10.13039/501100011033; PID2019-105069GB-I00 and CEX2020-001088-M), the Regional Government of Andalusia (P20_00873) and the European Regional Development Fund “Una manera de hacer Europa”

The cell cycle regulator APC/CFZR-1 controls the decision between the SS and DTC cell fate in Caenorhabditis elegans

Resumen del trabajo presentado en el 24th International C. elegans Conference, celebrado en Glasgow (Escocia), del 24 al 28 de junio de 2023Proper coordination between cell division and differentiation is essential during the development of organisms. Many components of the regulatory network involved in the cell cycle control can act directly on differentiation factors. Among these cell-cycle components highlights the E3 ubiquitin ligase APC/CCdh1/Fzr1. We have explored the role of APC/CFZR-1 during C. elegans development. We have obtained a complete loss-of-function allele of fzr-1. From the analysis of this mutant, we have found that APC/CFZR-1 participates in the development of the somatic gonad, an organ that supports germline development. Specifically, APC/CFZR-1 is necessary to produce the Distal Tip Cell (DTC), a stem-cell niche that maintains a pool of germ cells and leads the outgrowth of the gonad. The absence of FZR-1 makes cells committed to being DTCs, acquire the SS precursor fate of sister cells. Moreover, an allele of fzr-1 constitutively active yields extra DTCs. Both results suggest that APC/CFZR-1 is part of a balance during the fate choice decision ¿DTC-SS¿ in the somatic gonad. Forced expression of the transcriptional factors involved in determining the DTC fate, LIN-32 and HLH-12 suppressed the requirement of APC/CFZR-1 during DTC determination, suggesting the involvement of a transcriptional regulator as a target of APC/CFZR-1. To search for this target, we carried out an RNAi search, looking for putative targets of APC/CFZR-1 involved in this developmental decision. We will discuss obtained candidates and their proposed roles in the process

P99/B30 GLD-1 and APC/CFZR-1 are involved in the somatic conversion of germ cells

Resumendel trabajo presentado en el European Worm Meeting (EWM), celebrado en Viena (Austria), del 27 al 30 de julio de 2022In hermaphrodite gonads, the loss of GLD-1 activity results in female germ cells failing to progress through the meiotic prophase properly and subsequently re-entering the mitotic cell cycle (Francis et al. 1995). We wondered whether the antimitotic activity of GLD-1 was helped by some of the described G1/S cell cycle repressors, and therefore we analyzed gonads silenced for gld-1 together lin-35 (Retinoblastoma), cki-1, and cki-2 (CDK inhibitors) and fzr-1 (one of the coactivators of APC/C). Strikingly, we have found that gonads silenced for gld-1 and fzr-1 seem composed mainly of mitotic cells. Furthermore, co-silencing gld-1 and fzr-1 bypass the requirement of the Notch pathway for the maintenance of germ cells. We also showed that the gld-1 fzr-1 silenced gonads do not express meiotic determinants like rme-2. Using somatic cell markers like an unc-119::GFP fusion, it was found that a number of the mitotic cells within a gld-1 tumor were not undifferentiated totipotent cells but rather were differentiating as somatic cells, analogous to those in a human teratoma (Ciosk et al. 2006). Strikingly, we have found that in gld-1 gonads, the absence of fzr-1 resulted in the abrogation of such trans-differentiation. We have found that two chromatin regulators that played an essential role in the maintenance of the distal germline, MES-4, and MES-3, are targeted by APC/CFZR1 for degradation. mes-4 and mes-3 alleles unable to be targeted by APC/CFZR1 invade the pachytene region of the gonad. These two alleles together do not recapitulate the effects of fzr-1 silencing in the abrogation of trans-differentiation in gld-1 gonads, although its activity was required for that effect. Alternative explanations for the role of FZR-1 in the differentiation of the gonad will be discussed. Ciosk R, DePalma M, Priess JR (2006) Translational regulators maintain totipotency in the Caenorhabditis elegans germline. Science 311, 851¿853. Francis R, Barton MK, Kimble JE, Schedl TB (1995). gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans. Genetics, 139, 579-60

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

Resumen del trabajo presentado en European Worm Meeting, celebrado en Vienna (Austria) del 27 al 30 de julio de 2022.Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also implemented FLP-mediated recombination to identify the transcriptome in a tissue of interest. We have generated FLP-controlled Dam::RPB-6 and UPRT lines for RNA Polymerase DamID (RAPID) and thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue (SLAM-ITseq), respectively. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes

C. elegans as a Nestor Guillermo Progeria Syndrome Model

Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022.Variety of tissues, leading to growth retardation, severe skeletal defects and scoliosis. The syndrome is caused by a single amino acid substitution (A12T) in BAF1 (Barrier to Autointegration Factor 1). BAF1 is a highly conserved chromatin binding protein implicated in nuclear envelope (NE) breakdown, assembly and repair as well as chromatin compaction. Its NE localization is interdependent of lamins and LEM-domain proteins (LAP2, emerin, and MAN1) and contributes to chromatin organization although BAF1 is also present in the nucleoplasm. We have modified the baf-1 locus in Caenorhabditis elegans to mimic the human NGPS mutation (baf-1(G12T)) to elucidate why a mutation in an essential protein expressed throughout development triggers the appearance of symptoms ~2 years after birth. We report that NE levels of lamin/LMN-1 and emerin/EMR-1 are reduced in baf-1(G12T) mutants, whereas errors in chromosome segregation are increased. The baf-1(G12T) mutation reduces fertility and lifespan and accelerates age-dependent nuclear morphology deterioration. Moreover, we found that baf-1(G12T) mutants are hypersensitive to NE perturbations, particularly to modifications affecting lamin/LMN-1. Like other progerias, NGPS-derived fibroblasts feature malformed nuclei. Importantly, a set of genes whose depletion alleviates the nuclear associated defects was unveiled by CRISPR-mediated gene knockout in NGPS fibroblasts. When orthologs were silenced by RNAi in C. elegans, several reduced the embryonic lethality of sensitized baf-1(G12T) mutants. This represents a first and encouraging list of candidate genes to be further explored for the development of NGPS therapies

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in Caenorhabditis elegans

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.This project was funded by the Spanish State Research Agency, the European Union, and the European Regional Development Fund (BFU2016-79313-P, CEX2020-001088-M, and PID2019-105069GB-I00; doi:10.13039/501100011033).Peer reviewe