RESUMEN
An arrayed CRISPR screen is a high-throughput functional genomic screening method, which typically uses 384 well plates and has different gene knockouts in different wells. Despite various computational workflows, there is currently no systematic way to find what is a good workflow for arrayed CRISPR screening data analysis. To guide this choice, we developed a statistical simulation model that mimics the data generating process of arrayed CRISPR screening experiments. Our model is flexible and can simulate effects on phenotypic readouts of various experimental factors, such as the effect size of gene editing, as well as biological and technical variations. With two examples, we showed that the simulation model can assist making principled choice of normalization and hit calling method for the arrayed CRISPR data analysis. This simulation model is implemented in an R package and can be downloaded from Github.
Asunto(s)
Sistemas CRISPR-Cas , Modelos Estadísticos , Edición Génica/métodos , Humanos , Simulación por Computador , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.
Asunto(s)
Proteoma , Proteómica , Animales , Proteoma/metabolismo , Centrosoma/metabolismo , Centro Organizador de los Microtúbulos , Microtúbulos , MamíferosRESUMEN
Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/fisiología , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Línea Celular , Chlamydomonas/clasificación , Cristalografía por Rayos X , Células HEK293 , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Filogenia , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)-based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole-cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Linfocitos/metabolismo , Animales , Autoantígenos/metabolismo , Pollos , Células HEK293 , Homeostasis , Humanos , Células Jurkat , Linfocitos/citología , ProteómicaRESUMEN
Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen , Oligonucleótidos Antisentido , Oligonucleótidos , Interferencia de ARN , Transcripción Genética , Perfilación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Oligonucleótidos Antisentido/química , Proteínas/genética , ARN Largo no Codificante/metabolismoRESUMEN
Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis.
Asunto(s)
Replicación del ADN , ADN/química , Inestabilidad Genómica , Conformación de Ácido Nucleico , Separasa/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , ADN/genética , ADN/metabolismo , Células HeLa , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Genéticos , Unión Proteica , Interferencia de ARN , Separasa/genética , CohesinasRESUMEN
Cohesin is an evolutionarily conserved protein complex that plays a role in many biological processes: it ensures faithful chromosome segregation, regulates gene expression and preserves genome stability. In mammalian cells, the mitotic cohesin complex consists of two structural maintenance of chromosome proteins, SMC1A and SMC3, the kleisin protein RAD21 and a fourth subunit either STAG1 or STAG2. Meiotic paralogs in mammals were reported for SMC1A, RAD21 and STAG1/STAG2 and are called SMC1B, REC8 and STAG3 respectively. It is believed that SMC1B is only a meiotic-specific cohesin member, required for sister chromatid pairing and for preventing telomere shortening. Here we show that SMC1B is also expressed in somatic mammalian cells and is a member of a mitotic cohesin complex. In addition, SMC1B safeguards genome stability following irradiation whereas its ablation has no effect on chromosome segregation. Finally, unexpectedly SMC1B depletion impairs gene transcription, particularly at genes mapping to clusters such as HOX and PCDHB. Genome-wide analyses show that cluster genes changing in expression are enriched for cohesin-SMC1B binding.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Mitosis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , CohesinasRESUMEN
In addition to its role in sister chromatid cohesion, genome stability and integrity, the cohesin complex is involved in gene transcription. Mutations in core cohesin subunits SMC1A, SMC3 and RAD21, or their regulators NIPBL and HDAC8, cause Cornelia de Lange syndrome (CdLS). Recent evidence reveals that gene expression dysregulation could be the underlying mechanism for CdLS. These findings raise intriguing questions regarding the potential role of cohesin-mediated transcriptional control and pathogenesis. Here, we identified numerous dysregulated genes occupied by cohesin by combining the transcriptome of CdLS cell lines carrying mutations in SMC1A gene and ChIP-Seq data. Genome-wide analyses show that genes changing in expression are enriched for cohesin-binding. In addition, our results indicate that mutant cohesin impairs both RNA polymerase II (Pol II) transcription initiation at promoters and elongation in the gene body. These findings highlight the pivotal role of cohesin in transcriptional regulation and provide an explanation for the typical gene dysregulation observed in CdLS patients.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Síndrome de Cornelia de Lange/genética , Regulación de la Expresión Génica , Mutación/genética , ARN Polimerasa II/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Transcripción GenéticaRESUMEN
UNLABELLED: The ETS family of transcription factors has been repeatedly implicated in tumorigenesis. In prostate cancer, ETS family members, such as ERG, ETV1, ETV4, and ETV5, are frequently overexpressed due to chromosomal translocations, but the molecular mechanisms by which they promote prostate tumorigenesis remain largely undefined. Here, we show that ETS family members, such as ERG and ETV1, directly repress the expression of the checkpoint kinase 1 (CHK1), a key DNA damage response cell-cycle regulator essential for the maintenance of genome integrity. Critically, we find that ERG expression correlates with CHK1 downregulation in human patients and demonstrate that Chk1 heterozygosity promotes the progression of high-grade prostatic intraepithelial neoplasia into prostatic invasive carcinoma in Pten(+) (/-) mice. Importantly, CHK1 downregulation sensitizes prostate tumor cells to etoposide but not to docetaxel treatment. Thus, we identify CHK1 as a key functional target of the ETS proto-oncogenic family with important therapeutic implications. SIGNIFICANCE: Genetic translocation and aberrant expression of ETS family members is a common event in different types of human tumors. Here, we show that through the transcriptional repression of CHK1, ETS factors may favor DNA damage accumulation and consequent genetic instability in proliferating cells. Importantly, our findings provide a rationale for testing DNA replication inhibitor agents in ETS-positive TP53-proficient tumors.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Daño del ADN , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Proteínas Quinasas/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Regulador Transcripcional ERG , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by constitutional aneuploidies. Mutations in BUB1B and CEP57 genes, which are involved in mitotic spindle and microtubule stabilization, respectively, are responsible for a subset of patients with MVA. To date, CEP57 mutations have been reported only in four probands. We report on a girl with this disorder due to c.915-925dup11 mutation in CEP57, which predicts p.Leu309ProfsX9 and review the literature in order to facilitate genotype-phenotype correlation. Rhizomelic shortening of the upper limbs, skull anomalies with conserved head circumference, and absence of tumor development could be features suggesting a need for molecular screening of the CEP57 gene in patients with this disorder.
Asunto(s)
Trastornos de los Cromosomas/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Proteínas Nucleares/genética , Aneuploidia , Preescolar , Trastornos de los Cromosomas/diagnóstico , Consanguinidad , Análisis Mutacional de ADN , Facies , Femenino , Humanos , Mosaicismo , Linaje , FenotipoRESUMEN
Cornelia de Lange syndrome (CdLS) is a clinically and genetically heterogeneous developmental disorder. Clinical features include growth retardation, intellectual disability, limb defects, typical facial dysmorphism, and other systemic involvement. The increased understanding of the genetic basis of CdLS has led to diagnostic improvement and expansion of the phenotype. Mutations in five genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8), all regulators or structural components of cohesin, have been identified. Approximately 60% of CdLS cases are due to NIPBL mutations, 5% caused by mutations in SMC1A, RAD21, and HDAC8 and one proband was found to carry a mutation in SMC3. To date, 311 CdLS-causing mutations are known including missense, nonsense, small deletions and insertions, splice site mutations, and genomic rearrangements. Phenotypic variability is seen both intra- and intergenically. This article reviews the spectrum of CdLS mutations with a particular emphasis on their correlation to the clinical phenotype.