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The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.
Assuntos
Doença de Alzheimer , Amiloide , Encéfalo , Líquido Cefalorraquidiano , Líquido Extracelular , Ritmo Gama , Sistema Glinfático , Animais , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/prevenção & controle , Amiloide/metabolismo , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Líquido Cefalorraquidiano/metabolismo , Modelos Animais de Doenças , Líquido Extracelular/metabolismo , Sistema Glinfático/fisiologia , Interneurônios/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Estimulação ElétricaRESUMO
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Assuntos
Anticorpos Neutralizantes/metabolismo , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , SARS-CoV-2/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Cultivadas , Epitopos , Humanos , Ativação Linfocitária , Polissorbatos , RNA Viral/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Esqualeno , Vacinação , Vacinas de mRNARESUMO
Psychiatric disorders are highly heritable yet polygenic, potentially involving hundreds of risk genes. Genome-wide association studies have identified hundreds of genomic susceptibility loci with susceptibility to psychiatric disorders; however, the contribution of these loci to the underlying psychopathology and etiology remains elusive. Here we generated deep human brain proteomics data by quantifying 11,608 proteins across 268 subjects using 11-plex tandem mass tag coupled with two-dimensional liquid chromatography-tandem mass spectrometry. Our analysis revealed 788 cis-acting protein quantitative trait loci associated with the expression of 883 proteins at a genome-wide false discovery rate <5%. In contrast to expression at the transcript level and complex diseases that are found to be mainly influenced by noncoding variants, we found protein expression level tends to be regulated by non-synonymous variants. We also provided evidence of 76 shared regulatory signals between gene expression and protein abundance. Mediation analysis revealed that for most (88%) of the colocalized genes, the expression levels of their corresponding proteins are regulated by cis-pQTLs via gene transcription. Using summary data-based Mendelian randomization analysis, we identified 4 proteins and 19 genes that are causally associated with schizophrenia. We further integrated multiple omics data with network analysis to prioritize candidate genes for schizophrenia risk loci. Collectively, our findings underscore the potential of proteome-wide linkage analysis in gaining mechanistic insights into the pathogenesis of psychiatric disorders.
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Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line-specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi.
Assuntos
Proteínas Argonautas/fisiologia , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Células Germinativas/metabolismo , Heterocromatina/fisiologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/metabolismo , Northern Blotting , Western Blotting , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Primers do DNA/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Heterocromatina/metabolismo , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.
Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Endodesoxirribonucleases , Técnicas de Inativação de Genes , Humanos , Técnicas de Inativação de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Linhagem Celular Tumoral , Genes Essenciais , Células HEK293 , Epistasia Genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.
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Diferenciação Celular , Interleucina-12 , Linfócitos T Reguladores , Humanos , Interleucina-12/imunologia , Diferenciação Celular/imunologia , Linfócitos T Reguladores/imunologia , Fator de Transcrição STAT4/imunologia , Fator de Transcrição STAT4/genética , Receptores de Interleucina-12/imunologia , Receptores de Interleucina-12/genética , Feminino , MasculinoRESUMO
Sample-wise deconvolution methods estimate cell-type proportions and gene expressions in bulk tissue samples, yet their performance and biological applications remain unexplored, particularly in human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk tissue RNA sequencing (RNA-seq), single-cell/nuclei (sc/sn) RNA-seq, and immunohistochemistry. A total of 1,130,767 nuclei per cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expressions. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk tissue or single-cell eQTLs did alone. Differential gene expressions associated with Alzheimer's disease, schizophrenia, and brain development were also examined using the deconvoluted data. Our findings, which were replicated in bulk tissue and single-cell data, provided insights into the biological applications of deconvoluted data in multiple brain disorders.
Assuntos
Encéfalo , Análise de Célula Única , Transcriptoma , Humanos , Encéfalo/metabolismo , Análise de Célula Única/métodos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Perfilação da Expressão Gênica/métodos , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Estudo de Associação Genômica Ampla/métodos , Análise de Sequência de RNA/métodos , AdultoRESUMO
Acute cellular stress is known to induce a global reduction in mRNA translation through suppression of cap dependent translation. Selective translation in response to acute stress has been shown to play important roles in regulating the stress response. However, accurately profiling translational changes transcriptome-wide in response to acute cellular stress has been challenging. Commonly used data normalization methods operate on the assumption that any systematic shifts are experimental artifacts. Consequently, if applied to profiling acute cellular stress-induced mRNA translation changes, these methods are expected to produce biased estimates. To address this issue, we designed, produced, and evaluated a panel of 16 oligomers to serve as external standards for ribosome profiling studies. Using Sodium Arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, we applied spike-in oligomers as external standards. We found our spike-in oligomers to display a strong linear correlation between the observed and the expected quantification, with small ratio compression at the lower concentration range. Using the expected fold changes constructed from spike-in controls, we found in our dataset that TMM normalization, a popular global scaling normalization approach, produced 87.5% false positives at a significant cutoff that is expected to produce only 10% false positive discoveries. In addition, TMM normalization produced a systematic shift of fold change by 3.25 fold. These results highlight the consequences of applying global scaling approaches to conditions that clearly violate their key assumptions. In contrast, we found RUVg normalization using spike-in oligomers as control genes recapitulated the expected stress induced global reduction of translation and resulted in little, if any, systematic shifts in the expected fold change. Our results clearly demonstrated the utility of our spike-in oligomers, both for constructing expected results as controls and for data normalization.
Assuntos
Ribossomos , Transcriptoma , Ribossomos/genética , Ribossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Linhagem Celular , Estresse Oxidativo , Biossíntese de ProteínasRESUMO
Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.
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Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in â¼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.
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Background: The impact of genetic variants on gene expression has been intensely studied at the transcription level, yielding in valuable insights into the association between genes and the risk of complex disorders, such as schizophrenia (SCZ). However, the downstream impact of these variants and the molecular mechanisms connecting transcription variation to disease risk are not well understood. Results: We quantitated ribosome occupancy in prefrontal cortex samples of the BrainGVEX cohort. Together with transcriptomics and proteomics data from the same cohort, we performed cis-Quantitative Trait Locus (QTL) mapping and identified 3,253 expression QTLs (eQTLs), 1,344 ribosome occupancy QTLs (rQTLs), and 657 protein QTLs (pQTLs) out of 7,458 genes quantitated in all three omics types from 185 samples. Of the eQTLs identified, only 34% have their effects propagated to the protein level. Further analysis on the effect size of prefrontal cortex eQTLs identified from an independent dataset showed clear post-transcriptional attenuation of eQTL effects. To investigate the biological relevance of the attenuated eQTLs, we identified 70 expression-specific QTLs (esQTLs), 51 ribosome-occupancy-specific QTLs (rsQTLs), and 107 protein-specific QTLs (psQTLs). Five of these omics-specific QTLs showed strong colocalization with SCZ GWAS signals, three of them are esQTLs. The limited number of GWAS colocalization discoveries from omics-specific QTLs and the apparent prevalence of eQTL attenuation prompted us to take a complementary approach to investigate the functional relevance of attenuated eQTLs. Using S-PrediXcan we identified 74 SCZ risk genes, 34% of which were novel, and 67% of these risk genes were replicated in a MR-Egger test. Notably, 52 out of 74 risk genes were identified using eQTL data and 70% of these SCZ-risk-gene-driving eQTLs show little to no evidence of driving corresponding variations at the protein level. Conclusion: The effect of eQTLs on gene expression in the prefrontal cortex is commonly attenuated post-transcriptionally. Many of the attenuated eQTLs still correlate with SCZ GWAS signal. Further investigation is needed to elucidate a mechanistic link between attenuated eQTLs and SCZ disease risk.
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Sample-wise deconvolution methods have been developed to estimate cell-type proportions and gene expressions in bulk-tissue samples. However, the performance of these methods and their biological applications has not been evaluated, particularly on human brain transcriptomic data. Here, nine deconvolution methods were evaluated with sample-matched data from bulk-tissue RNAseq, single-cell/nuclei (sc/sn) RNAseq, and immunohistochemistry. A total of 1,130,767 nuclei/cells from 149 adult postmortem brains and 72 organoid samples were used. The results showed the best performance of dtangle for estimating cell proportions and bMIND for estimating sample-wise cell-type gene expression. For eight brain cell types, 25,273 cell-type eQTLs were identified with deconvoluted expressions (decon-eQTLs). The results showed that decon-eQTLs explained more schizophrenia GWAS heritability than bulk-tissue or single-cell eQTLs alone. Differential gene expression associated with multiple phenotypes were also examined using the deconvoluted data. Our findings, which were replicated in bulk-tissue RNAseq and sc/snRNAseq data, provided new insights into the biological applications of deconvoluted data.
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BACKGROUND: Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is silenced in some cells as a consequence of juxtaposition with heterochromatin. High levels of variation in PEV phenotypes are commonly observed in reporter stocks. To ascertain whether background mutations are the major contributors to this variation, we asked how much of the variation is determined by genetic variants segregating in the population, examining both the level and pattern of expression using the fruit fly, Drosophila melanogaster, as the model. RESULTS: Using selective breeding of a fourth chromosome PEV reporter line, 39C-12, we isolated two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in the degree of variegation: 90% of the variation between the two inbred lines in the degree of silencing can be explained by genotype. Further analyses suggest that this result reflects the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter scored across the whole fly. Further, the pattern of variegation becomes almost identical within each inbred line; significant pigment enrichment in the same quadrant of the eye was found for both A1 and D1 lines despite different degrees of expression. CONCLUSIONS: The results indicate that background genetic variants play the major role in determining the variable degrees of PEV commonly observed in laboratory stocks. Interestingly, not only does the degree of variegation become consistent in inbred lines, the patterns of variegation also appear similar. Combining these observations with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV in which the genetic background drives the overall level of silencing, working with the cell lineage-specific regulatory environment to determine the on/off probability at the reporter locus in each cell. This model acknowledges cell type-specific events in the context of broader genetic impacts on heterochromatin formation.
Assuntos
Efeitos da Posição Cromossômica , Drosophila melanogaster/genética , Animais , Linhagem da Célula , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Genes Reporter , Patrimônio Genético , Variação Genética , Proteínas de Choque Térmico HSP70/genética , Heterocromatina/metabolismo , Modelos Genéticos , Fenótipo , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Differences in gene regulation between human and closely related species influence phenotypes that are distinctly human. While gene regulation is a multi-step process, the majority of research concerning divergence in gene regulation among primates has focused on transcription. RESULTS: To gain a comprehensive view of gene regulation, we surveyed genome-wide ribosome occupancy, which reflects levels of protein translation, in lymphoblastoid cell lines derived from human, chimpanzee, and rhesus macaque. We further integrated messenger RNA and protein level measurements collected from matching cell lines. We find that, in addition to transcriptional regulation, the major factor determining protein level divergence between human and closely related species is post-translational buffering. Inter-species divergence in transcription is generally propagated to the level of protein translation. In contrast, gene expression divergence is often attenuated post-translationally, potentially mediated through post-translational modifications. CONCLUSIONS: Results from our analysis indicate that post-translational buffering is a conserved mechanism that led to relaxation of selective constraint on transcript levels in humans.
Assuntos
Regulação da Expressão Gênica/genética , Macaca mulatta/genética , Pan troglodytes/genética , Biossíntese de Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Animais , Linhagem Celular , Humanos , RNA Mensageiro/genética , Ribossomos/genética , Transcrição Gênica/genéticaRESUMO
Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy.
Assuntos
Separação Celular/métodos , Genes Reporter , Desenvolvimento Muscular , Músculo Esquelético/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Feminino , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Mesoderma/citologia , Camundongos Endogâmicos mdx , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX7/metabolismo , Células-Tronco Pluripotentes/citologia , Regeneração , Transdução de Sinais , Transplante de Células-Tronco , Fatores de Tempo , Transcriptoma/genéticaRESUMO
Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.
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Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4α. This study reveals that P1-HNF4α, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4α, driven by an alternative promoter (P2-HNF4α), is induced in HNF4α-positive human hepatocellular carcinoma (HCC). P2-HNF4α represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4α. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4α in HCC, and demonstrate that forced expression of BMAL1 in HNF4α-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4α-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.
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Fatores de Transcrição ARNTL/metabolismo , Carcinoma Hepatocelular/metabolismo , Fator 4 Nuclear de Hepatócito/fisiologia , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição ARNTL/genética , Transporte Ativo do Núcleo Celular , Carcinoma Hepatocelular/patologia , Relógios Circadianos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas/patologia , Isoformas de Proteínas/fisiologiaRESUMO
Accurate annotation of protein coding regions is essential for understanding how genetic information is translated into function. We describe riboHMM, a new method that uses ribosome footprint data to accurately infer translated sequences. Applying riboHMM to human lymphoblastoid cell lines, we identified 7273 novel coding sequences, including 2442 translated upstream open reading frames. We observed an enrichment of footprints at inferred initiation sites after drug-induced arrest of translation initiation, validating many of the novel coding sequences. The novel proteins exhibit significant selective constraint in the inferred reading frames, suggesting that many are functional. Moreover, ~40% of bicistronic transcripts showed negative correlation in the translation levels of their two coding sequences, suggesting a potential regulatory role for these novel regions. Despite known limitations of mass spectrometry to detect protein expressed at low level, we estimated a 14% validation rate. Our work significantly expands the set of known coding regions in humans.
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Biologia Molecular/métodos , Fases de Leitura Aberta , Biossíntese de Proteínas , Ribossomos/metabolismo , Linhagem Celular , Humanos , Linfócitos/fisiologiaRESUMO
The phenotypic consequences of expression quantitative trait loci (eQTLs) are presumably due to their effects on protein expression levels. Yet the impact of genetic variation, including eQTLs, on protein levels remains poorly understood. To address this, we mapped genetic variants that are associated with eQTLs, ribosome occupancy (rQTLs), or protein abundance (pQTLs). We found that most QTLs are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, eQTLs tend to have significantly reduced effect sizes on protein levels, which suggests that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we identified a class of cis QTLs that affect protein abundance with little or no effect on messenger RNA or ribosome levels, which suggests that they may arise from differences in posttranslational regulation.
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Regulação da Expressão Gênica , Variação Genética , Biossíntese de Proteínas/genética , Locos de Características Quantitativas , RNA Mensageiro/genética , Transcrição Gênica , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Linhagem Celular , Éxons , Humanos , Fenótipo , Ribossomos/metabolismoRESUMO
Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.