RESUMO
The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.
Assuntos
Diferenciação Celular/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Fatores de Transcrição SOXB1/imunologia , Microambiente Tumoral/imunologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Microambiente Tumoral/genéticaRESUMO
The c-Myc protooncogene places a demand on glucose uptake to drive glucose-dependent biosynthetic pathways. To meet this demand, c-Myc protein (Myc henceforth) drives the expression of glucose transporters, glycolytic enzymes, and represses the expression of thioredoxin interacting protein (TXNIP), which is a potent negative regulator of glucose uptake. A Mychigh/TXNIPlow gene signature is clinically significant as it correlates with poor clinical prognosis in triple-negative breast cancer (TNBC) but not in other subtypes of breast cancer, suggesting a functional relationship between Myc and TXNIP. To better understand how TXNIP contributes to the aggressive behavior of TNBC, we generated TXNIP null MDA-MB-231 (231:TKO) cells for our study. We show that TXNIP loss drives a transcriptional program that resembles those driven by Myc and increases global Myc genome occupancy. TXNIP loss allows Myc to invade the promoters and enhancers of target genes that are potentially relevant to cell transformation. Together, these findings suggest that TXNIP is a broad repressor of Myc genomic binding. The increase in Myc genomic binding in the 231:TKO cells expands the Myc-dependent transcriptome we identified in parental MDA-MB-231 cells. This expansion of Myc-dependent transcription following TXNIP loss occurs without an apparent increase in Myc's intrinsic capacity to activate transcription and without increasing Myc levels. Together, our findings suggest that TXNIP loss mimics Myc overexpression, connecting Myc genomic binding and transcriptional programs to the nutrient and progrowth signals that control TXNIP expression.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genômica , Glucose/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.
Assuntos
Neoplasias da Mama , Neoplasias Ovarianas , Animais , Proteína BRCA1/genética , Teorema de Bayes , Neoplasias da Mama/genética , Feminino , Humanos , Mamíferos , Mutação de Sentido Incorreto/genética , Neoplasias Ovarianas/genética , Domínios ProteicosRESUMO
The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common ß-chain (ßc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade ßc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Endocitose , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/genética , Camundongos , Transcriptoma , Células Tumorais CultivadasRESUMO
Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Glioblastoma/metabolismo , Proteínas Nucleares/metabolismo , Fatores Sexuais , Fatores de Transcrição/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/fisiologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Caracteres Sexuais , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy-resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.
Assuntos
Neoplasias do Endométrio/genética , Receptor alfa de Estrogênio/genética , Perfilação da Expressão Gênica/métodos , Mutação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias do Endométrio/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , HumanosRESUMO
The majority of clinical cancer specimens are preserved as formalin-fixed paraffin-embedded (FFPE) samples. For clinical molecular tests to have wide-reaching impact, they must be applicable to FFPE material. Accurate quantitative measurements of RNA derived from FFPE specimens is challenging because of low yields and high amounts of degradation. Here, we present FFPEcap-seq, a method specifically designed for sequencing capped 5' ends of RNA derived from FFPE samples. FFPEcap-seq combines enzymatic enrichment of 5' capped RNAs with template switching to create sequencing libraries. We find that FFPEcap-seq can faithfully capture mRNA expression levels in FFPE specimens while also detecting enhancer RNAs that arise from distal regulatory regions. FFPEcap-seq is a fast and straightforward method for making high-quality 5' end RNA-seq libraries from FFPE-derived RNA.
Assuntos
Formaldeído , Inclusão em Parafina , Capuzes de RNA , Análise de Sequência de RNA/métodos , Fixação de Tecidos , Elementos Facilitadores Genéticos , HumanosRESUMO
The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of this enhancer cooperativity. We generated genetic deletions of four estrogen receptor α (ER) bound enhancers that regulate two genes and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences, we found that the genomic location of a full ERE strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing permissible chromatin environment can also be needed for estrogen-driven gene regulation to occur.
Assuntos
Elementos Facilitadores Genéticos/genética , Receptor alfa de Estrogênio/genética , Motivos de Nucleotídeos/genética , Transcrição Gênica , Acetilação , Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a 5-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: classical and basal-like. The classical subtype is characterised by a more favourable prognosis and better response to chemotherapy than the basal-like subtype. The classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor hepatocyte nuclear factor 4 α (HNF4α). The objective of this study is to evaluate the role of HNF4α, SIX4 and SIX1 in regulating the growth and molecular subtype of PDAC. DESIGN: We manipulate the expression of HNF4α, SIX4 and SIX1 in multiple in vitro and in vivo PDAC models. We determine the consequences of manipulating these genes on PDAC growth, differentiation and molecular subtype using functional assays, gene expression analysis and cross-species comparisons with human datasets. RESULTS: We show that HNF4α restrains tumour growth and drives tumour cells toward an epithelial identity. Gene expression analysis of murine models and human tumours shows that HNF4α activates expression of genes associated with the classical subtype. HNF4α also directly represses SIX4 and SIX1, two mesodermal/neuronal lineage specifiers expressed in the basal-like subtype. Finally, SIX4 and SIX1 drive proliferation and regulate differentiation in HNF4α-negative PDAC. CONCLUSION: Our data show that HNF4α regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the classical gene expression programme and repression of SIX4 and SIX1, which may represent novel dependencies of the basal-like subtype.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transativadores/genética , Neoplasias PancreáticasRESUMO
GIGGLE is a genomics search engine that identifies and ranks the significance of genomic loci shared between query features and thousands of genome interval files. GIGGLE (https://github.com/ryanlayer/giggle) scales to billions of intervals and is over three orders of magnitude faster than existing methods. Its speed extends the accessibility and utility of resources such as ENCODE, Roadmap Epigenomics, and GTEx by facilitating data integration and hypothesis generation.
Assuntos
Neoplasias da Mama/genética , Genoma Humano , Genômica/métodos , Ferramenta de Busca/métodos , Análise de Sequência de DNA/métodos , Software , Bases de Dados Genéticas , Feminino , Humanos , InternetRESUMO
Ribosome profiling, an application of nucleic acid sequencing for monitoring ribosome activity, has revolutionized our understanding of protein translation dynamics. This technique has been available for a decade, yet the current state and standardization of publicly available computational tools for these data is bleak. We introduce XPRESSyourself, an analytical toolkit that eliminates barriers and bottlenecks associated with this specialized data type by filling gaps in the computational toolset for both experts and non-experts of ribosome profiling. XPRESSyourself automates and standardizes analysis procedures, decreasing time-to-discovery and increasing reproducibility. This toolkit acts as a reference implementation of current best practices in ribosome profiling analysis. We demonstrate this toolkit's performance on publicly available ribosome profiling data by rapidly identifying hypothetical mechanisms related to neurodegenerative phenotypes and neuroprotective mechanisms of the small-molecule ISRIB during acute cellular stress. XPRESSyourself brings robust, rapid analysis of ribosome-profiling data to a broad and ever-expanding audience and will lead to more reproducible and accessible measurements of translation regulation. XPRESSyourself software is perpetually open-source under the GPL-3.0 license and is hosted at https://github.com/XPRESSyourself, where users can access additional documentation and report software issues.
Assuntos
Biologia Computacional/métodos , RNA/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos , Software , Bases de Dados Genéticas , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Internet , Biossíntese de Proteínas/genética , Reprodutibilidade dos TestesRESUMO
Most human transcription factors bind a small subset of potential genomic sites and often use different subsets in different cell types. To identify mechanisms that govern cell-type-specific transcription factor binding, we used an integrative approach to study estrogen receptor α (ER). We found that ER exhibits two distinct modes of binding. Shared sites, bound in multiple cell types, are characterized by high-affinity estrogen response elements (EREs), inaccessible chromatin, and a lack of DNA methylation, while cell-specific sites are characterized by a lack of EREs, co-occurrence with other transcription factors, and cell-type-specific chromatin accessibility and DNA methylation. These observations enabled accurate quantitative models of ER binding that suggest tethering of ER to one-third of cell-specific sites. The distinct properties of cell-specific binding were also observed with glucocorticoid receptor and for ER in primary mouse tissues, representing an elegant genomic encoding scheme for generating cell-type-specific gene regulation.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Metilação de DNA , Estradiol/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/efeitos dos fármacos , Interferência de RNA , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Termodinâmica , Fatores de Transcrição/genética , TransfecçãoRESUMO
Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type-specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites.
Assuntos
Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Células Hep G2 , Histonas/metabolismo , Humanos , Células K562 , Especificidade de Órgãos/genética , Ligação Proteica , Elementos de Resposta , Análise de Sequência de DNARESUMO
Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
Assuntos
DNA/genética , Enciclopédias como Assunto , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Alelos , Linhagem Celular , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Genômica , Humanos , Células K562 , Especificidade de Órgãos , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Mapas de Interação de Proteínas , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Seleção Genética/genética , Sítio de Iniciação de TranscriçãoRESUMO
Back pain is a major contributor to disability and has significant socioeconomic impacts worldwide. The degenerative intervertebral disc (IVD) has been hypothesized to contribute to back pain, but a better understanding of the interactions between the degenerative IVD and nociceptive neurons innervating the disc and treatment strategies that directly target these interactions is needed to improve our understanding and treatment of back pain. We investigated degenerative IVD-induced changes to dorsal root ganglion (DRG) neuron activity and utilized CRISPR epigenome editing as a neuromodulation strategy. By exposing DRG neurons to degenerative IVD-conditioned media under both normal and pathological IVD pH levels, we demonstrate that degenerative IVDs trigger interleukin (IL)-6-induced increases in neuron activity to thermal stimuli, which is directly mediated by AKAP and enhanced by acidic pH. Utilizing this novel information on AKAP-mediated increases in nociceptive neuron activity, we developed lentiviral CRISPR epigenome editing vectors that modulate endogenous expression of AKAP150 by targeted promoter histone methylation. When delivered to DRG neurons, these epigenome-modifying vectors abolished degenerative IVD-induced DRG-elevated neuron activity while preserving non-pathologic neuron activity. This work elucidates the potential for CRISPR epigenome editing as a targeted gene-based pain neuromodulation strategy.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Gânglios Espinais/citologia , Edição de Genes , Degeneração do Disco Intervertebral/genética , Neurônios/metabolismo , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Concentração de Íons de Hidrogênio , Interleucina-6/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Estimulação Física , Regiões Promotoras Genéticas , RatosRESUMO
Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.
Assuntos
Perfilação da Expressão Gênica/métodos , Genes , Splicing de RNA , RNA/análise , Linhagem Celular Tumoral , Genoma Humano , Humanos , RNA/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , TranscriptomaRESUMO
As studies of DNA methylation increase in scope, it has become evident that methylation has a complex relationship with gene expression, plays an important role in defining cell types, and is disrupted in many diseases. We describe large-scale single-base resolution DNA methylation profiling on a diverse collection of 82 human cell lines and tissues using reduced representation bisulfite sequencing (RRBS). Analysis integrating RNA-seq and ChIP-seq data illuminates the functional role of this dynamic mark. Loci that are hypermethylated across cancer types are enriched for sites bound by NANOG in embryonic stem cells, which supports and expands the model of a stem/progenitor cell signature in cancer. CpGs that are hypomethylated across cancer types are concentrated in megabase-scale domains that occur near the telomeres and centromeres of chromosomes, are depleted of genes, and are enriched for cancer-specific EZH2 binding and H3K27me3 (repressive chromatin). In noncancer samples, there are cell-type specific methylation signatures preserved in primary cell lines and tissues as well as methylation differences induced by cell culture. The relationship between methylation and expression is context-dependent, and we find that CpG-rich enhancers bound by EP300 in the bodies of expressed genes are unmethylated despite the dense gene-body methylation surrounding them. Non-CpG cytosine methylation occurs in human somatic tissue, is particularly prevalent in brain tissue, and is reproducible across many individuals. This study provides an atlas of DNA methylation across diverse and well-characterized samples and enables new discoveries about DNA methylation and its role in gene regulation and disease.
Assuntos
Ilhas de CpG , Metilação de DNA , Linhagem Celular Tumoral , Cromatina , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNA , Sulfitos/metabolismoRESUMO
Endogenous estrogens that are synthesized in the body impact gene regulation by activating estrogen receptors in diverse cell types. Exogenous compounds that have estrogenic properties can also be found circulating in the blood in both children and adults. The genome-wide impact of these environmental estrogens on gene regulation is unclear. To obtain an integrated view of gene regulation in response to environmental and endogenous estrogens on a genome-wide scale, we performed ChIP-seq to identify estrogen receptor 1 (ESR1; previously estrogen receptor α) binding sites, and RNA-seq in endometrial cancer cells exposed to bisphenol A (BPA; found in plastics), genistein (GEN; found in soybean), or 17ß-estradiol (E2; an endogenous estrogen). GEN and BPA treatment induces thousands of ESR1 binding sites and >50 gene expression changes, representing a subset of E2-induced gene regulation changes. Genes affected by E2 were highly enriched for ribosome-associated proteins; however, GEN and BPA failed to regulate most ribosome-associated proteins and instead enriched for transporters of carboxylic acids. Treatment-dependent changes in gene expression were associated with treatment-dependent ESR1 binding sites, with the exception that many genes up-regulated by E2 harbored a BPA-induced ESR1 binding site but failed to show any expression change after BPA treatment. GEN and BPA exhibited a similar relationship to E2 in the breast cancer line T-47D, where cell type specificity played a much larger role than treatment specificity. Overall, both environmental estrogens clearly regulate gene expression through ESR1 on a genome-wide scale, although with lower potency resulting in less ESR1 binding sites and less gene expression changes compared to the endogenous estrogen, E2.
Assuntos
Compostos Benzidrílicos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios não Esteroides/farmacologia , Genisteína/farmacologia , Fenóis/farmacologia , Fitoestrógenos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Estradiol/metabolismo , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Genoma Humano , Humanos , Especificidade de Órgãos , Análise de Sequência de RNA , Transcrição Gênica , Regulação para CimaRESUMO
RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (â¼1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/química , Biblioteca Gênica , RNA Mensageiro/química , Transposases/química , Linhagem Celular Tumoral , Elementos de DNA Transponíveis/genética , DNA Complementar/biossíntese , DNA Complementar/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Humanos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Análise de Sequência de DNA/métodos , Uracila-DNA Glicosidase/químicaRESUMO
CTCF is a ubiquitously expressed regulator of fundamental genomic processes including transcription, intra- and interchromosomal interactions, and chromatin structure. Because of its critical role in genome function, CTCF binding patterns have long been assumed to be largely invariant across different cellular environments. Here we analyze genome-wide occupancy patterns of CTCF by ChIP-seq in 19 diverse human cell types, including normal primary cells and immortal lines. We observed highly reproducible yet surprisingly plastic genomic binding landscapes, indicative of strong cell-selective regulation of CTCF occupancy. Comparison with massively parallel bisulfite sequencing data indicates that 41% of variable CTCF binding is linked to differential DNA methylation, concentrated at two critical positions within the CTCF recognition sequence. Unexpectedly, CTCF binding patterns were markedly different in normal versus immortal cells, with the latter showing widespread disruption of CTCF binding associated with increased methylation. Strikingly, this disruption is accompanied by up-regulation of CTCF expression, with the result that both normal and immortal cells maintain the same average number of CTCF occupancy sites genome-wide. These results reveal a tight linkage between DNA methylation and the global occupancy patterns of a major sequence-specific regulatory factor.