RESUMO
In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume.
Assuntos
Doença de Huntington , Animais , Humanos , Camundongos , Caspase 6/genética , Caspase 6/metabolismo , Corpo Estriado/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Oligonucleotídeos Antissenso/farmacologia , FenótipoRESUMO
Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5'-end by an intron that is retained in the transcript longer ('slow' intron) leads to overall higher exon skipping efficiency than when the 5'-end flanking intron is 'fast'. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5'-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3'-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies.
Assuntos
Distrofia Muscular de Duchenne , Oligonucleotídeos Antissenso , Humanos , Oligonucleotídeos Antissenso/genética , Íntrons , Distrofina/genética , Éxons , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapiaRESUMO
While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.
Assuntos
Distrofina/genética , Genoma/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Adulto , Sequência de Bases , Éxons/genética , Genética , Humanos , Masculino , Sequenciamento Completo do Genoma/métodos , Adulto JovemRESUMO
The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.
Assuntos
Células Dendríticas/metabolismo , Vesículas Extracelulares/genética , Transcriptoma , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Colecalciferol/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Corantes Fluorescentes/química , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Microscopia Eletrônica , Nanopartículas/química , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/isolamento & purificação , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Análise de Sequência de RNARESUMO
DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.
Assuntos
Técnicas de Química Combinatória , DNA/química , Bibliotecas de Moléculas Pequenas/química , Trombina/análise , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Estrutura MolecularRESUMO
The dystrophin protein encoding DMD gene is the longest human gene. The 2.2 Mb long human dystrophin transcript takes 16 hours to be transcribed and is co-transcriptionally spliced. It contains long introns (24 over 10kb long, 5 over 100kb long) and the heterogeneity in intron size makes it an ideal transcript to study different aspects of the human splicing process. Splicing is a complex process and much is unknown regarding the splicing of long introns in human genes. Here, we used ultra-deep transcript sequencing to characterize splicing of the dystrophin transcripts in 3 different human skeletal muscle cell lines, and explored the order of intron removal and multi-step splicing. Coverage and read pair analyses showed that around 40% of the introns were not always removed sequentially. Additionally, for the first time, we report that non-consecutive intron removal resulted in 3 or more joined exons which are flanked by unspliced introns and we defined these joined exons as an exon block. Lastly, computational and experimental data revealed that, for the majority of dystrophin introns, multistep splicing events are used to splice out a single intron. Overall, our data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.
Assuntos
Distrofina/genética , Sítios de Splice de RNA , Splicing de RNA , Linhagem Celular , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de RNA/métodosRESUMO
Many disease-associated variants affect gene expression levels (expression quantitative trait loci, eQTLs) and expression profiling using next generation sequencing (NGS) technology is a powerful way to detect these eQTLs. We analyzed 94 total blood samples from healthy volunteers with DeepSAGE to gain specific insight into how genetic variants affect the expression of genes and lengths of 3'-untranslated regions (3'-UTRs). We detected previously unknown cis-eQTL effects for GWAS hits in disease- and physiology-associated traits. Apart from cis-eQTLs that are typically easily identifiable using microarrays or RNA-sequencing, DeepSAGE also revealed many cis-eQTLs for antisense and other non-coding transcripts, often in genomic regions containing retrotransposon-derived elements. We also identified and confirmed SNPs that affect the usage of alternative polyadenylation sites, thereby potentially influencing the stability of messenger RNAs (mRNA). We then combined the power of RNA-sequencing with DeepSAGE by performing a meta-analysis of three datasets, leading to the identification of many more cis-eQTLs. Our results indicate that DeepSAGE data is useful for eQTL mapping of known and unknown transcripts, and for identifying SNPs that affect alternative polyadenylation. Because of the inherent differences between DeepSAGE and RNA-sequencing, our complementary, integrative approach leads to greater insight into the molecular consequences of many disease-associated variants.
Assuntos
Regulação da Expressão Gênica/genética , Poliadenilação/genética , Locos de Características Quantitativas/genética , Retroelementos/genética , Regiões 3' não Traduzidas/genética , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Understanding the molecular basis of domestication can provide insights into the processes of rapid evolution and crop improvement. Here we demonstrated the processes of carrot domestication and identified genes under selection based on transcriptome analyses. RESULTS: The root transcriptomes of widely differing cultivated and wild carrots were sequenced. A method accounting for sequencing errors was introduced to optimize SNP (single nucleotide polymorphism) discovery. 11,369 SNPs were identified. Of these, 622 (out of 1000 tested SNPs) were validated and used to genotype a large set of cultivated carrot, wild carrot and other wild Daucus carota subspecies, primarily of European origin. Phylogenetic analysis indicated that eastern carrot may originate from Western Asia and western carrot may be selected from eastern carrot. Different wild D. carota subspecies may have contributed to the domestication of cultivated carrot. Genetic diversity was significantly reduced in western cultivars, probably through bottlenecks and selection. However, a high proportion of genetic diversity (more than 85% of the genetic diversity in wild populations) is currently retained in western cultivars. Model simulation indicated high and asymmetric gene flow from wild to cultivated carrots, spontaneously and/or by introgression breeding. Nevertheless, high genetic differentiation exists between cultivated and wild carrots (Fst = 0.295) showing the strong effects of selection. Expression patterns differed radically for some genes between cultivated and wild carrot roots which may be related to changes in root traits. The up-regulation of water-channel-protein gene expression in cultivars might be involved in changing water content and transport in roots. The activated expression of carotenoid-binding-protein genes in cultivars could be related to the high carotenoid accumulation in roots. The silencing of allergen-protein-like genes in cultivated carrot roots suggested strong human selection to reduce allergy. These results suggest that regulatory changes of gene expressions may have played a predominant role in domestication. CONCLUSIONS: Western carrots may originate from eastern carrots. The reduction in genetic diversity in western cultivars due to domestication bottleneck/selection may have been offset by introgression from wild carrot. Differential gene expression patterns between cultivated and wild carrot roots may be a signature of strong selection for favorable cultivation traits.
Assuntos
Daucus carota/genética , Perfilação da Expressão Gênica , Raízes de Plantas/genética , Evolução Molecular , Genes de Plantas/genética , Marcadores Genéticos/genética , Genótipo , Polimorfismo de Nucleotídeo Único/genética , Seleção GenéticaRESUMO
Alternative polyadenylation (APA) at the 3' UTR of transcripts contributes to the cell transcriptome. APA is suppressed by the nuclear RNA-binding protein PABPN1. Aging-associated reduced PABPN1 levels in skeletal muscles lead to muscle wasting. Muscle weakness in oculopharyngeal muscular dystrophy (OPMD) is caused by short alanine expansion in PABPN1 exon1. The expanded PABPN1 forms nuclear aggregates, an OPMD hallmark. Whether the expanded PABPN1 affects APA and how it contributes to muscle pathology is unresolved. To investigate these questions, we developed a procedure including RNA library preparation and a simple pipeline calculating the APA-shift ratio as a readout for PABPN1 activity. Comparing APA-shift results to previously published PAS utilization and APA-shift results, we validated this procedure. The procedure was then applied on the OPMD cell model and on RNA from OPMD muscles. APA-shift was genome-wide in the mouse OPMD model, primarily affecting muscle transcripts. In OPMD individuals, APA-shift was enriched with muscle transcripts. In an OPMD cell model APA-shift was not significant. APA-shift correlated with reduced expression levels of a subset of PABPN1 isoforms, whereas the expression of the expanded PABPN1 did not correlate with APA-shift. PABPN1 activity is not affected by the expression of expanded PABPN1, but rather by reduced PABPN1 expression levels. In muscles, PABPN1 activity initially affects muscle transcripts. We suggest that muscle weakness in OPMD is caused by PABPN1 loss-of-function leading to APA-shift that primarily affects in muscle transcripts.
Assuntos
Distrofia Muscular Oculofaríngea , Animais , Camundongos , Modelos Animais de Doenças , Debilidade Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/genética , Poliadenilação/genética , RNA/metabolismoRESUMO
The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Humanos , RNA Guia de Sistemas CRISPR-Cas/genética , Células K562 , Sistemas CRISPR-Cas/genética , Células HEK293 , Genoma Humano/genética , Diferenciação Celular/genética , Miócitos Cardíacos/metabolismo , RNA não Traduzido/genética , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Biblioteca GênicaRESUMO
Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.
Assuntos
Proteínas de Ciclo Celular , Proteínas Nucleares , DNA , Metilação de DNA , Isoformas de Proteínas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismoRESUMO
The measles, mumps, and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized, placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. Using single-cell RNA-Seq, we found that MMR caused transcriptomic changes in CD14+ monocytes and NK cells, but most profoundly in γδ T cells. Monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was markedly enhanced by MMR vaccination, with higher production of TNF and IFN-γ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.
Assuntos
Caxumba , Rubéola (Sarampo Alemão) , Criança , Adulto , Humanos , Lactente , Caxumba/prevenção & controle , Vacina contra Sarampo-Caxumba-Rubéola , Rubéola (Sarampo Alemão)/prevenção & controle , Reprogramação Metabólica , Imunidade Treinada , Vacinação , Anticorpos AntiviraisRESUMO
Terminal osseous dysplasia (TOD) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with onset in female infancy. After performing X-exome capture and sequencing, we identified a mutation at the last nucleotide of exon 31 of the FLNA gene as the most likely cause of the disease. The variant c.5217G>A was found in six unrelated cases (three families and three sporadic cases) and was not found in 400 control X chromosomes, pilot data from the 1000 Genomes Project, or the FLNA gene variant database. In the families, the variant segregated with the disease, and it was transmitted four times from a mildly affected mother to a more seriously affected daughter. We show that, because of nonrandom X chromosome inactivation, the mutant allele was not expressed in patient fibroblasts. RNA expression of the mutant allele was detected only in cultured fibroma cells obtained from 15-year-old surgically removed material. The variant activates a cryptic splice site, removing the last 48 nucleotides from exon 31. At the protein level, this results in a loss of 16 amino acids (p.Val1724_Thr1739del), predicted to remove a sequence at the surface of filamin repeat 15. Our data show that TOD is caused by this single recurrent mutation in the FLNA gene.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Neoplasias Ósseas/genética , Proteínas Contráteis/genética , Fibroma/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas dos Microfilamentos/genética , Transtornos da Pigmentação/genética , Adulto , Doenças do Desenvolvimento Ósseo/complicações , Neoplasias Ósseas/complicações , Pré-Escolar , Feminino , Fibroma/complicações , Filaminas , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Recidiva Local de Neoplasia , Linhagem , Transtornos da Pigmentação/complicações , Pigmentação da PeleRESUMO
Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad's intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, 'accelerated-evolution' approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad's intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.
Assuntos
Adenoviridae/genética , DNA Polimerase Dirigida por DNA/genética , Evolução Molecular Direcionada/métodos , Vírus Oncolíticos/genética , Proteínas Virais/genética , Adenoviridae/enzimologia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/química , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Mutação , Splicing de RNA , Replicação ViralRESUMO
Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.
Assuntos
Hibridização Genômica Comparativa/métodos , Análise Mutacional de DNA/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , alfa-Globinas/genética , Talassemia alfa , Globinas beta/genética , Talassemia beta , Pontos de Quebra do Cromossomo , Primers do DNA , Éxons , Rearranjo Gênico , Humanos , Reação em Cadeia da Polimerase , Deleção de Sequência , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Talassemia beta/diagnóstico , Talassemia beta/genéticaRESUMO
Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3'-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10,000 genes expressed at levels >2 transcripts per million (approximately 0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3'-UTR usage.
Assuntos
Perfilação da Expressão Gênica/métodos , Mioblastos/metabolismo , Análise de Sequência de RNA , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Camundongos , Modelos Biológicos , Desenvolvimento Muscular/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Alinhamento de Sequência , Sítio de Iniciação de TranscriçãoRESUMO
Despite high levels of homology, transcription coactivators p300 and CREB binding protein (CBP) are both indispensable during embryogenesis. They are largely known to regulate the same genes. To identify genes preferentially regulated by p300 or CBP, we performed an extensive genome-wide survey using the ChIP-seq on cell-cycle synchronized cells. We found that 57% of the tags were within genes or proximal promoters, with an overall preference for binding to transcription start and end sites. The heterogeneous binding patterns possibly reflect the divergent roles of CBP and p300 in transcriptional regulation. Most of the 16 103 genes were bound by both CBP and p300. However, after stimulation 89 and 1944 genes were preferentially bound by CBP or p300, respectively. Target genes were found to be primarily involved in the regulation of metabolic and developmental processes, and transcription, with CBP showing a stronger preference than p300 for genes active in negative regulation of transcription. Analysis of transcription factor binding sites suggest that CBP and p300 have many partners in common, but AP-1 and Serum Response Factor (SRF) appear to be more prominent in CBP-specific sequences, whereas AP-2 and SP1 are enriched in p300-specific targets. Taken together, our findings further elucidate the distinct roles of coactivators p300 and CBP in transcriptional regulation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequência Consenso , Genoma Humano , Humanos , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , DNA , Recombinação HomólogaRESUMO
Iron accumulation in microglia has been observed in Alzheimer's disease and other neurodegenerative disorders and is thought to contribute to disease progression through various mechanisms, including neuroinflammation. To study this interaction, we treated human induced pluripotent stem cell-derived microglia (iPSC-MG) with iron, in combination with inflammatory stimuli such as interferon gamma (IFN-γ) and amyloid ß. Both IFN-γ and iron treatment increased labile iron levels, but only iron treatment led to a consistent increase of ferritin levels, reflecting long-term iron storage. Therefore, in iPSC-MG, ferritin appeared to be regulated by iron revels rather than inflammation. Further investigation showed that while IFN-γ induced pro-inflammatory activation, iron treatment dampened both classic pro- and anti-inflammatory activation on a transcriptomic level. Notably, iron-loaded microglia showed strong upregulation of cellular stress response pathways, the NRF2 pathway, and other oxidative stress pathways. Functionally, iPSC-MG exhibited altered phagocytosis and impaired mitochondrial metabolism following iron treatment. Collectively, these data suggest that in MG, in contrast to current hypotheses, iron treatment does not result in pro-inflammatory activation, but rather dampens it and induces oxidative stress.
Assuntos
Células-Tronco Pluripotentes Induzidas , Microglia , Peptídeos beta-Amiloides/metabolismo , Ferritinas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Ferro/metabolismo , Microglia/metabolismo , Estresse OxidativoRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation of the D4Z4 repeat resulting in misexpression of the D4Z4-encoded DUX4 gene in skeletal muscle. One of the key genetic requirements for the stable production of full-length DUX4 mRNA in skeletal muscle is a functional polyadenylation signal (ATTAAA) in exon three of DUX4 that is used in somatic cells. Base editors hold great promise to treat DNA lesions underlying genetic diseases through their ability to carry out specific and rapid nucleotide mutagenesis even in postmitotic cells such as skeletal muscle. In this study, we present a simple and straightforward strategy for mutagenesis of the somatic DUX4 polyadenylation signal by adenine base editing in immortalized myoblasts derived from independent FSHD-affected individuals. We show that mutating this critical cis-regulatory element results in downregulation of DUX4 mRNA and its direct transcriptional target genes. Our findings identify the somatic DUX4 polyadenylation signal as a therapeutic target and represent the first step toward clinical application of the CRISPR-Cas9 base editing platform for FSHD gene therapy.