RESUMO
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.
Assuntos
Dieta Hiperlipídica , Corpos Cetônicos/metabolismo , Células-Tronco/metabolismo , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/farmacologia , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidroximetilglutaril-CoA Sintase/deficiência , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Adulto JovemRESUMO
Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Miócitos Cardíacos/citologia , RNA Longo não Codificante , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Redes Reguladoras de Genes , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RatosRESUMO
The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.
Assuntos
Infecções por HIV , Proteostase , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Infecções por HIV/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Resposta a Proteínas não DobradasRESUMO
Targeted mutagenesis mediated by nucleotide base deaminase-T7 RNA polymerase fusions has recently emerged as a novel and broadly useful strategy to power genetic diversification in the context of in vivo directed evolution campaigns. Here, we expand the utility of this approach by introducing a highly active adenosine deaminase-T7 RNA polymerase fusion protein (eMutaT7AâG), resulting in higher mutation frequencies to enable more rapid directed evolution. We also assess the benefits and potential downsides of using this more active mutator. We go on to show in Escherichia coli that adenosine deaminase-bearing mutators (MutaT7AâG or eMutaT7AâG) can be employed in tandem with a cytidine deaminase-bearing mutator (MutaT7CâT) to introduce all possible transition mutations simultaneously. We illustrate the efficacy of this in vivo mutagenesis approach by exploring mutational routes to antibacterial drug resistance. This work sets the stage for general application of optimized MutaT7 tools able to induce all types of transition mutations during in vivo directed evolution campaigns across diverse organisms.
Assuntos
Mutagênese , Adenosina Desaminase/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Técnicas GenéticasRESUMO
BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.
Assuntos
Cálcio , Miocárdio , Proteínas de Ligação a RNA , Proteínas de Peixe-Zebra , Animais , Humanos , Cálcio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Inativação Gênica , Coração/fisiologia , Histonas/metabolismo , Lisina/metabolismo , Regeneração/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Proliferação de Células , Citocinese , Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metilação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismoRESUMO
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.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Pseudomonas aeruginosa/imunologia , Animais , Caenorhabditis elegans/genética , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Imunidade Inata , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Análise de Sequência de RNARESUMO
The threat of viral pandemics demands a comprehensive understanding of evolution at the host-pathogen interface. Here, we show that the accessibility of adaptive mutations in influenza nucleoprotein at fever-like temperatures is mediated by host chaperones. Particularly noteworthy, we observe that the Pro283 nucleoprotein variant, which (1) is conserved across human influenza strains, (2) confers resistance to the Myxovirus resistance protein A (MxA) restriction factor, and (3) critically contributed to adaptation to humans in the 1918 pandemic influenza strain, is rendered unfit by heat shock factor 1 inhibition-mediated host chaperone depletion at febrile temperatures. This fitness loss is due to biophysical defects that chaperones are unavailable to address when heat shock factor 1 is inhibited. Thus, influenza subverts host chaperones to uncouple the biophysically deleterious consequences of viral protein variants from the benefits of immune escape. In summary, host proteostasis plays a central role in shaping influenza adaptation, with implications for the evolution of other viruses, for viral host switching, and for antiviral drug development.
Assuntos
Adaptação Fisiológica , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Sistema Imunitário/virologia , Imunidade Inata , Chaperonas Moleculares/metabolismo , Orthomyxoviridae/imunologia , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Análise Mutacional de DNA , Cães , Humanos , Células Madin Darby de Rim Canino , Modelos Biológicos , Proteínas de Resistência a Myxovirus/metabolismo , Nucleoproteínas/química , Estrutura Secundária de Proteína , Temperatura , Proteínas Virais/químicaRESUMO
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.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Ribossômico/isolamento & purificação , Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Humanos , Poli A/genética , RNA Ribossômico/genéticaRESUMO
We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the "A12-resin", that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12. The resulting data provide insight into how cells respond to acute Zn(II) deficiency. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology.
Assuntos
Proteína S100A12/química , Zinco/isolamento & purificação , Células Cultivadas , Células HEK293 , Humanos , Íons/química , Íons/isolamento & purificação , Íons/metabolismo , Proteína S100A12/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.
Assuntos
Evolução Molecular Direcionada/métodos , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/genética , Doxorrubicina/farmacologia , Resistência a Medicamentos/genética , Células HEK293 , Humanos , Integrases/genética , Leucina-tRNA Ligase/genética , Mutagênese , Peptídeo Hidrolases/genética , Estudo de Prova de Conceito , Engenharia de Proteínas , Fatores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
Macrophages display flexible activation states that range between pro-inflammatory (classical activation) and anti-inflammatory (alternative activation). These macrophage polarization states contribute to a variety of organismal phenotypes such as tissue remodeling and susceptibility to infectious and inflammatory diseases. Several macrophage- or immune-related genes have been shown to modulate infectious and inflammatory disease pathogenesis. However, the potential role that differences in macrophage activation phenotypes play in modulating differences in susceptibility to infectious and inflammatory disease is just emerging. We integrated transcriptional profiling and linkage analyses to determine the genetic basis for the differential murine macrophage response to inflammatory stimuli and to infection with the obligate intracellular parasite Toxoplasma gondii. We show that specific transcriptional programs, defined by distinct genomic loci, modulate macrophage activation phenotypes. In addition, we show that the difference between AJ and C57BL/6J macrophages in controlling Toxoplasma growth after stimulation with interferon gamma and tumor necrosis factor alpha mapped to chromosome 3, proximal to the Guanylate binding protein (Gbp) locus that is known to modulate the murine macrophage response to Toxoplasma. Using an shRNA-knockdown strategy, we show that the transcript levels of an RNA helicase, Ddx1, regulates strain differences in the amount of nitric oxide produced by macrophage after stimulation with interferon gamma and tumor necrosis factor. Our results provide a template for discovering candidate genes that modulate macrophage-mediated complex traits.
Assuntos
RNA Helicases DEAD-box/genética , Inflamação/genética , Ativação de Macrófagos/genética , Toxoplasmose/genética , Transcrição Gênica , Animais , Estudos de Associação Genética , Ligação Genética , Inflamação/microbiologia , Inflamação/patologia , Interferon gama/administração & dosagem , Interferon gama/genética , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Toxoplasma/patogenicidade , Toxoplasmose/microbiologia , Toxoplasmose/patologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/genéticaRESUMO
Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity.
Assuntos
Processamento Alternativo , Citidina Desaminase/genética , Macrófagos/metabolismo , Camundongos/genética , Edição de RNA , Desaminase APOBEC-1 , Animais , Citosina/metabolismo , Ligação Genética , Variação Genética , Genoma , Interferon gama/metabolismo , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , Locos de Características Quantitativas , Isoformas de RNA/genética , Toxoplasma/fisiologia , Uracila/metabolismoRESUMO
RATIONALE: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. OBJECTIVE: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. METHODS AND RESULTS: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart. CONCLUSIONS: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.
Assuntos
Miócitos Cardíacos/metabolismo , Regeneração/fisiologia , Transcrição Gênica , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular/fisiologia , Ciclo Celular , Desdiferenciação Celular/genética , Diferenciação Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Ventrículos do Coração/citologia , Interleucina-13/farmacologia , Interleucina-13/fisiologia , Subunidade alfa1 de Receptor de Interleucina-13/antagonistas & inibidores , Subunidade alfa1 de Receptor de Interleucina-13/genética , Subunidade alfa de Receptor de Interleucina-4/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-4/genética , Camundongos , Desenvolvimento Muscular , Miócitos Cardíacos/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/fisiologia , Fator de Transcrição STAT6/fisiologia , Alinhamento de Sequência , Fatores de Transcrição/fisiologia , TranscriptomaRESUMO
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
Assuntos
Dano ao DNA , Reparo do DNA , Técnicas Genéticas , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Reparo de Erro de Pareamento de DNA , Citometria de Fluxo , Genes Reporter , Guanina/análogos & derivados , Guanina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutagênese , Plasmídeos/genética , Análise de Sequência de RNA , Transcrição Gênica , TransfecçãoRESUMO
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.
Assuntos
Diferenciação Celular/genética , Cromatina/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Histonas/genética , Animais , Asparagina/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicina/genética , Camundongos , Nucleossomos/genética , Regiões Promotoras Genéticas , Serina/genéticaRESUMO
MOTIVATION: Gene expression profiling using RNA-seq is a powerful technique for screening RNA species' landscapes and their dynamics in an unbiased way. While several advanced methods exist for differential expression analysis of RNA-seq data, proper tools to anal.yze RNA-seq time-course have not been proposed. RESULTS: In this study, we use RNA-seq to measure gene expression during the early human T helper 17 (Th17) cell differentiation and T-: cell activation (Th0). To quantify Th17-: specific gene expression dynamics, we present a novel statistical methodology, DyNB, for analyzing time-course RNA-seq data. We use non-parametric Gaussian processes to model temporal correlation in gene expression and combine that with negative binomial likelihood for the count data. To account for experiment-: specific biases in gene expression dynamics, such as differences in cell differentiation efficiencies, we propose a method to rescale the dynamics between replicated measurements. We develop an MCMC sampling method to make inference of differential expression dynamics between conditions. DyNB identifies several known and novel genes involved in Th17 differentiation. Analysis of differentiation efficiencies revealed consistent patterns in gene expression dynamics between different cultures. We use qRT-PCR to validate differential expression and differentiation efficiencies for selected genes. Comparison of the results with those obtained via traditional timepoint-: wise analysis shows that time-course analysis together with time rescaling between cultures identifies differentially expressed genes which would not otherwise be detected. AVAILABILITY: An implementation of the proposed computational methods will be available at http://research.ics.aalto.fi/csb/software/
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Células Th17/metabolismo , Humanos , Recém-Nascido , Distribuição Normal , Células Th17/citologiaRESUMO
Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNß production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.
Assuntos
Interações Hospedeiro-Parasita/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Toxoplasma/patogenicidade , Animais , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Transdução de Sinais/genéticaRESUMO
Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.
RESUMO
Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Owing to unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.