RESUMEN
Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.
Asunto(s)
Dieta Alta en Grasa , Cuerpos Cetónicos/metabolismo , Células Madre/metabolismo , Ácido 3-Hidroxibutírico/sangre , Ácido 3-Hidroxibutírico/farmacología , Anciano de 80 o más Años , Animales , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Hidroximetilglutaril-CoA Sintasa/deficiencia , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Intestinos/citología , Intestinos/patología , Masculino , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Adulto JovenRESUMEN
Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.
Asunto(s)
Epigénesis Genética , Redes Reguladoras de Genes , Miocardio/citología , Animales , Diferenciación Celular , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Corazón/embriología , Humanos , Ratones , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Elucidating how chromatin influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential but unknown role during mammalian development. Here, genome-wide analysis reveals that H2AZ occupies the promoters of developmentally important genes in a manner that is remarkably similar to that of the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells, suggesting that its dynamic redistribution is necessary for cell fate transitions. Thus, H2AZ, together with PcG proteins, may establish specialized chromatin states in ES cells necessary for the proper execution of developmental gene expression programs.
Asunto(s)
Células Madre Embrionarias/citología , Histonas/química , Proteínas Represoras/química , Animales , Diferenciación Celular , Linaje de la Célula , Cromatina/metabolismo , Análisis por Conglomerados , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteínas del Grupo Polycomb , Unión Proteica , Interferencia de ARNRESUMEN
MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.
Asunto(s)
Células Madre Embrionarias/metabolismo , MicroARNs/genética , Transcripción Genética , Animales , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.
Asunto(s)
Silenciador del Gen , Corazón/fisiología , Histonas/metabolismo , Lisina/metabolismo , Regeneración/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Proliferación Celular , Citocinesis , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metilación , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Sarcómeros/metabolismoRESUMEN
The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1- ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.
Asunto(s)
Factores de Transcripción Activadores/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/inmunología , Caenorhabditis elegans/microbiología , Pseudomonas aeruginosa/inmunología , Animales , Caenorhabditis elegans/genética , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Inmunidad Innata , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Análisis de Secuencia de ARNRESUMEN
In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Regulación de la Expresión Génica , Proteínas de Homeodominio/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Femenino , Proteínas de Homeodominio/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/metabolismoRESUMEN
Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:CâT:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:CâT:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were GâT in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.
Asunto(s)
Aflatoxina B1/genética , Biomarcadores/metabolismo , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Aductos de ADN/genética , Neoplasias Hepáticas/genética , Mutación , Aflatoxina B1/toxicidad , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/patología , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Aductos de ADN/toxicidad , Femenino , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. RESULTS: We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. CONCLUSIONS: These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Ribosómico/aislamiento & purificación , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Humanos , Poli A/genética , ARN Ribosómico/genéticaRESUMEN
Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Factor C1 de la Célula Huésped/metabolismo , Animales , Proteínas de Unión al ADN/genética , Metabolismo Energético , Ratones , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas Represoras , Transcripción GenéticaRESUMEN
Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.
Asunto(s)
Daño del ADN , Guanina/análogos & derivados , Mutación , Aductos de ADN/química , ADN Polimerasa beta/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Dioxigenasas/metabolismo , Guanina/química , Secuenciación de Nucleótidos de Alto Rendimiento , Mutagénesis , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Mammalian genomes encode a large number of non-coding RNAs (ncRNAs) that greatly exceed mRNA genes. While the physiological and pathological roles of ncRNAs have been increasingly understood, the mechanisms of regulation of ncRNA expression are less clear. Here, our genomic study has shown that a significant number of long non-coding RNAs (lncRNAs, >1000 nucleotides) harbor RNA polymerase II (Pol II) engaged with the transcriptional start site. A pausing and transcriptional elongation factor for protein-coding genes, tripartite motif-containing 28 (TRIM28) regulates the transcription of a subset of lncRNAs in mammalian cells. In addition, the majority of lncRNAs in human and murine cells regulated by Pol II promoter-proximal pausing appear to function in stimulus-inducible biological pathways. Our findings suggest an important role of Pol II pausing for the transcription of mammalian lncRNA genes.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Polimerasa II/genética , ARN Largo no Codificante/genética , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Regulación de la Expresión Génica , Genómica/métodos , Células HEK293 , Humanos , Mamíferos/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/genética , Complejo Mediador/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatina/química , Proteínas Cromosómicas no Histona/genética , ADN/química , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos/genética , Fibroblastos , Complejo Mediador/genética , Ratones , Conformación de Ácido Nucleico , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Unión Proteica , CohesinasRESUMEN
Emerging evidence points to roles for tRNA modifications and tRNA abundance in cellular stress responses. While isolated instances of stress-induced tRNA degradation have been reported, we sought to assess the effects of stress on tRNA levels at a systems level. To this end, we developed a next-generation sequencing method that exploits the paucity of ribonucleoside modifications at the 3'-end of tRNAs to quantify changes in all cellular tRNA molecules. Application of this tRNA-seq method to Saccharomyces cerevisiae identified all 76 expressed unique tRNA species out of 295 coded in the yeast genome, including all isoacceptor variants, with highly precise relative (fold-change) quantification of tRNAs. In studies of stress-induced changes in tRNA levels, we found that oxidation (H2O2) and alkylation (methylmethane sulfonate, MMS) stresses induced nearly identical patterns of up- and down-regulation for 58 tRNAs. However, 18 tRNAs showed opposing changes for the stresses, which parallels our observation of signature reprogramming of tRNA modifications caused by H2O2 and MMS. Further, stress-induced degradation was limited to only a small proportion of a few tRNA species. With tRNA-seq applicable to any organism, these results suggest that translational control of stress response involves a contribution from tRNA abundance.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN de Transferencia/metabolismo , Análisis de Secuencia de ARN/métodos , Estrés Fisiológico/genética , Regulación hacia Abajo , ARN de Transferencia/química , Transcripción Reversa , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Regulación hacia ArribaRESUMEN
The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3)) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3) interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3) was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3) ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3) ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3) displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3) mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.
Asunto(s)
Diferenciación Celular/genética , Cromatina/genética , Desarrollo Embrionario/genética , Células Madre Embrionarias/citología , Histonas/genética , Animales , Asparagina/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica , Glicina/genética , Ratones , Nucleosomas/genética , Regiones Promotoras Genéticas , Serina/genéticaRESUMEN
Toxoplasma gondii is a highly successful protozoan parasite that infects all warm-blooded animals and causes severe disease in immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe, isolated strains fall predominantly into four largely clonal lineages, but in South America there is great genetic diversity and the North American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence, modulation of host-signaling pathways, growth, dissemination, and disease severity in mice and likely in humans. Most studies on Toxoplasma genetic variation have focused on either a few loci in many strains or low-resolution genome analysis of three clonal lineages. We use whole-genome sequencing to identify a large number of SNPs between 10 Toxoplasma strains from Europe and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of RNA-sequencing data of 26 Toxoplasma strains, representing global diversity, allowed us to construct a comprehensive genealogy for Toxoplasma gondii that incorporates sexual recombination. These data show that most current isolates are recent recombinants and cannot be easily grouped into a limited number of haplogroups. A complex picture emerges in which some genomic regions have not been recently exchanged between any strains, and others recently spread from one strain to many others.
Asunto(s)
Variación Genética , Genoma de Protozoos/genética , Filogenia , Recombinación Genética , Toxoplasma/genética , Animales , Cruzamientos Genéticos , Femenino , Genes Protozoarios/genética , Haplotipos/genética , Humanos , Masculino , Ratones , Polimorfismo Genético , Polimorfismo de Nucleótido Simple/genética , Selección Genética , Toxoplasmosis Animal/genética , Toxoplasmosis Animal/parasitologíaRESUMEN
Mycobacteria adapt to infection stresses by entering a reversible non-replicating persistence (NRP) with slow or no cell growth and broad antimicrobial tolerance. Hypoxia and nutrient deprivation are two well-studied stresses commonly used to model the NRP, yet little is known about the molecular differences in mycobacterial adaptation to these distinct stresses that lead to a comparable NRP phenotype. Here we performed a multisystem interrogation of the Mycobacterium bovis BCG (BCG) starvation response, which revealed a coordinated metabolic shift away from the glycolysis of nutrient-replete growth to depletion of lipid stores, lipolysis, and fatty acid ß-oxidation in NRP. This contrasts with BCG's NRP hypoxia response involving a shift to cholesterol metabolism and triglyceride storage. Our analysis reveals cryptic metabolic vulnerabilities of the starvation-induced NRP state, such as their newfound hypersensitivity to H2O2. These observations pave the way for developing precision therapeutics against these otherwise drug refractory pathogens.
Asunto(s)
Adaptación Fisiológica , Mycobacterium bovis , Mycobacterium bovis/metabolismo , Glucólisis , Reprogramación MetabólicaRESUMEN
Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.
RESUMEN
Cyclin-dependent kinase 9 (CDK9) coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release states. It is an important co-factor for transcription factors, such as MYC, that drive aberrant cell proliferation when their expression is deregulated. CDK9 modulation offers an approach for attenuating dysregulation in such transcriptional programs. As a result, numerous drug development campaigns to inhibit CDK9 kinase activity have been pursued. More recently, targeted degradation has emerged as an attractive approach. However, comprehensive evaluation of degradation versus inhibition is still critically needed to assess the biological contexts in which degradation might offer superior therapeutic benefits. We validated that CDK9 inhibition triggers a compensatory mechanism that dampens its effect on MYC expression and found that this feedback mechanism was absent when the kinase is degraded. Importantly, CDK9 degradation is more effective than its inhibition for disrupting MYC transcriptional regulatory circuitry likely through the abrogation of both enzymatic and scaffolding functions of CDK9. Highlights: - KI-CDK9d-32 is a highly potent and selective CDK9 degrader. - KI-CDK9d-32 leads to rapid downregulation of MYC protein and mRNA transcripts levels. - KI-CDK9d-32 represses canonical MYC pathways and leads to a destabilization of nucleolar homeostasis. - Multidrug resistance ABCB1 gene emerged as the strongest resistance marker for the CDK9 PROTAC degrader.
RESUMEN
Foxp3+CD4+CD25+ regulatory T (T(reg)) cells are essential for the prevention of autoimmunity. T(reg) cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T(reg) cells, is required for T(reg) development and function, and is sufficient to induce a T(reg) phenotype in conventional CD4+CD25- T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse. FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes. However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T(reg) function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress the activation of target genes on T-cell stimulation. Foxp3 suppression of its targets appears to be crucial for the normal function of T(reg) cells, because overactive variants of some target genes are known to be associated with autoimmune disease.