RESUMEN
The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.
RESUMEN
Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.
Asunto(s)
Anemia de Fanconi , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Proteínas de Ciclo Celular/genética , ADN , Emetina/uso terapéutico , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Estudio de Asociación del Genoma Completo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
The leading strand-oriented alternative PCNA clamp loader DSCC1-RFC functions in DNA replication, repair, and sister chromatid cohesion (SCC), but how it facilitates these processes is incompletely understood. Here, we confirm that loss of human DSCC1 results in reduced fork speed, increased DNA damage, and defective SCC. Genome-wide CRISPR screens in DSCC1-KO cells reveal multiple synthetically lethal interactions, enriched for DNA replication and cell cycle regulation. We show that DSCC1-KO cells require POLE3 for survival. Co-depletion of DSCC1 and POLE3, which both interact with the catalytic polymerase ε subunit, additively impair DNA replication, suggesting that these factors contribute to leading-strand DNA replication in parallel ways. An additional hit is MMS22L, which in humans forms a heterodimer with TONSL. Synthetic lethality of DSCC1 and MMS22L-TONSL likely results from detrimental SCC loss. We show that MMS22L-TONSL, like DDX11, functions in a SCC establishment pathway parallel to DSCC1-RFC. Because both DSCC1-RFC and MMS22L facilitate ESCO2 recruitment to replication forks, we suggest that distinct ESCO2 recruitment pathways promote SCC establishment following either cohesin conversion or de novo cohesin loading.
Asunto(s)
Cromátides , Replicación del ADN , Humanos , Cromátides/genética , Cromátides/metabolismo , Replicación del ADN/genética , Segregación Cromosómica/genética , Puntos de Control del Ciclo Celular , Daño del ADN/genética , ADN Polimerasa III/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , ADN Helicasas/genética , ARN Helicasas DEAD-box/metabolismo , FN-kappa B/metabolismoRESUMEN
Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alquinos/farmacología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Anemia de Fanconi/complicaciones , Anemia de Fanconi/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/complicaciones , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oligopéptidos/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.
Asunto(s)
Ciclina D/metabolismo , Fase G1/fisiología , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase S/fisiología , Proteínas ras/metabolismo , Proliferación Celular/fisiología , Células HEK293 , Células HeLa , Humanos , Multimerización de Proteína , Transducción de Señal/fisiología , Transducción Genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/farmacología , Proteínas ras/antagonistas & inhibidoresRESUMEN
Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood. We report here the occurrence of futile checkpoint responses and an unusual type of replicative stress (RS) in TSC1-/- fibroblasts exposed to low-dose genotoxins. This RS is characterized by elevated nucleotide incorporation rates despite only modest origin over-firing. Strikingly, an increased propensity for asymmetric fork progression and profuse chromosomal aberrations upon mild DNA damage confirmed that TSC loss indeed proved detrimental to stress adaptation. We conclude that low stress tolerance of TSC-/- cells manifests at the level of DNA replication control, imposing strong negative selection on genomic instability that could in turn detain TSC-mutant tumours benign.
Asunto(s)
Daño del ADN , Replicación del ADN , Doxorrubicina/toxicidad , Fibroblastos/efectos de los fármacos , Hidroxiurea/toxicidad , Sirolimus/toxicidad , Proteínas Supresoras de Tumor/deficiencia , Animales , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Aberraciones Cromosómicas/inducido químicamente , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Fibroblastos/patología , Inestabilidad Genómica/efectos de los fármacos , Ratones Noqueados , Interferencia de ARN , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Transfección , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.
Asunto(s)
Genes src/genética , Genes src/fisiología , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Acilación , Secuencia de Aminoácidos , Animales , Células COS , Proteína Tirosina Quinasa CSK , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Cisteína/metabolismo , Proteínas Fluorescentes Verdes , Lipoilación , Proteínas de la Membrana , Membranas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing 'prophasing' of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.
Asunto(s)
División Celular/genética , Heterocromatina/genética , Interfase/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/metabolismo , ADN/química , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Heterocromatina/química , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
During the last three decades, a number of B-lymphocyte specific surface antigens have been defined some of which may also show activation/differentiation specific expression. Here, we review the various signaling events and the receptor-ligand interactions for B-cell development, activation and differentiation. Our discussion and presentation include reviewing the in vivo and in vitro mechanisms. Focus is on the experiments that give us valuable insights into the B cell signaling mechanisms in vitro. Three significant pathways in B-cell development - c-Kit, FLT-3 and IL-7 signaling pathways are elucidated upon. Both antigen dependent and antigen independent mechanisms of B cell stimulation are also reviewed.