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1.
bioRxiv ; 2024 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-39005299

RESUMO

Background: The recently launched DNA methylation profiling platform, Illumina MethylationEPIC BeadChip Infinium microarray v2.0 (EPICv2), is highly correlated with measurements obtained from its predecessor MethylationEPIC BeadChip Infinium microarray v1.0 (EPICv1). However, the concordance between the two versions in the context of DNA methylation-based tools, including cell type deconvolution algorithms, epigenetic clocks, and inflammation and lifestyle biomarkers has not yet been investigated. To address this, we profiled DNA methylation on both EPIC versions using matched venous blood samples from individuals spanning early to late adulthood across four cohorts. Findings: Within each cohort, samples primarily clustered by the EPIC version they were measured on. High concordance between EPIC versions at the array level, but variable concordance at the individual probe level was noted. Significant differences between versions in estimates from DNA methylation-based tools were observed, irrespective of the normalization method, with some nuanced differences across cohorts and tools. Adjusting for EPIC version or calculating estimates separately for each version largely mitigated these version-specific discordances. Conclusions: Our work illustrates the importance of accounting for EPIC version differences in research scenarios, especially in meta-analyses and longitudinal studies, when samples profiled across different versions are harmonized. Alongside DNA methylation-based tools, our observations also have implications in interpretation of epigenome-wide association studies (EWAS) findings, when results obtained from one version are compared to another, particularly for probes that are poorly concordant between versions.

2.
Neurobiol Dis ; 182: 106138, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37105261

RESUMO

Frontotemporal dementia (FTD) is an early onset dementia characterized by neuropathology and behavioural changes. A common genetic cause of FTD is haploinsufficiency of the gene progranulin (GRN). Mouse models of progranulin deficiency have provided insight into progranulin neurobiology, but the description of phenotypes with preclinical relevance has been limited in the currently available heterozygous progranulin-null mice. The identification of robust and reproducible FTD-associated behavioural, neuropathological, and biochemical phenotypes in progranulin deficient mice is a critical step in the preclinical development of therapies for FTD. In this work, we report the generation of a novel, 'humanized' mouse model of progranulin deficiency that expresses a single, targeted copy of human GRN in the absence of mouse progranulin. We also report the in-depth, longitudinal characterization of humanized progranulin-deficient mice and heterozygous progranulin-null mice over 18 months. Our analysis yielded several novel progranulin-dependent physiological and behavioural phenotypes, including increased marble burying, open field hyperactivity, and thalamic microgliosis in both models. RNAseq analysis of cortical tissue revealed an overlapping profile of transcriptomic dysfunction. Further transcriptomic analysis offers new insights into progranulin neurobiology. In sum, we have identified several consistent phenotypes in two independent mouse models of progranulin deficiency that are expected to be useful endpoints in the development of therapies for progranulin-deficient FTD. Furthermore, the presence of the human progranulin gene in the humanized progranulin-deficient mice will expedite the development of clinically translatable gene therapy strategies.


Assuntos
Demência Frontotemporal , Doença de Pick , Camundongos , Humanos , Animais , Progranulinas/genética , Demência Frontotemporal/patologia , Transcriptoma , Camundongos Knockout , Mutação
3.
Genome Biol ; 24(1): 48, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36918927

RESUMO

BACKGROUND: Genomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behavior. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we use low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extraembryonic tissues at allele-specific resolution. RESULTS: We identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse reveal that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extraembryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extraembryonic imprinting. Meta-analysis of novel imprinted genes reveals multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the appearance of ZFP57 binding motifs, and the insertion of endogenous retroviral promoters. CONCLUSIONS: In summary, we provide an expanded list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.


Assuntos
Histonas , Muridae , Camundongos , Humanos , Ratos , Animais , Muridae/genética , Muridae/metabolismo , Histonas/metabolismo , Estudo de Associação Genômica Ampla , Metilação de DNA , Impressão Genômica , Alelos
4.
J Clin Invest ; 132(13)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35775490

RESUMO

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.


Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Epigênese Genética , Humanos , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/metabolismo , Infecções por Polyomavirus/genética , Neoplasias Cutâneas/patologia , Peptidase 7 Específica de Ubiquitina/metabolismo
5.
Clin Cancer Res ; 28(5): 928-938, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34907080

RESUMO

PURPOSE: Neuroendocrine prostate cancer (NEPC) is a resistance phenotype that emerges in men with metastatic castration-resistant prostate adenocarcinoma (CR-PRAD) and has important clinical implications, but is challenging to detect in practice. Herein, we report a novel tissue-informed epigenetic approach to noninvasively detect NEPC. EXPERIMENTAL DESIGN: We first performed methylated immunoprecipitation and high-throughput sequencing (MeDIP-seq) on a training set of tumors, identified differentially methylated regions between NEPC and CR-PRAD, and built a model to predict the presence of NEPC (termed NEPC Risk Score). We then performed MeDIP-seq on cell-free DNA (cfDNA) from two independent cohorts of men with NEPC or CR-PRAD and assessed the accuracy of the model to predict the presence NEPC. RESULTS: The test cohort comprised cfDNA samples from 48 men, 9 with NEPC and 39 with CR-PRAD. NEPC Risk Scores were significantly higher in men with NEPC than CR-PRAD (P = 4.3 × 10-7) and discriminated between NEPC and CR-PRAD with high accuracy (AUROC 0.96). The optimal NEPC Risk Score cutoff demonstrated 100% sensitivity and 90% specificity for detecting NEPC. The independent, multi-institutional validation cohort included cfDNA from 53 men, including 12 with NEPC and 41 with CR-PRAD. NEPC Risk Scores were significantly higher in men with NEPC than CR-PRAD (P = 7.5×10-12) and perfectly discriminated NEPC from CR-PRAD (AUROC 1.0). Applying the predefined NEPC Risk Score cutoff to the validation cohort resulted in 100% sensitivity and 95% specificity for detecting NEPC. CONCLUSIONS: Tissue-informed cfDNA methylation analysis is a promising approach for noninvasive detection of NEPC in men with advanced prostate cancer.


Assuntos
Carcinoma Neuroendócrino , Ácidos Nucleicos Livres , Tumores Neuroendócrinos , Neoplasias da Próstata , Carcinoma Neuroendócrino/genética , Ácidos Nucleicos Livres/genética , Metilação de DNA , Humanos , Masculino , Tumores Neuroendócrinos/patologia , Próstata/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo
6.
Nat Commun ; 12(1): 7308, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911936

RESUMO

Androgen receptor (AR) in prostate cancer (PCa) can drive transcriptional repression of multiple genes including MYC, and supraphysiological androgen is effective in some patients. Here, we show that this repression is independent of AR chromatin binding and driven by coactivator redistribution, and through chromatin conformation capture methods show disruption of the interaction between the MYC super-enhancer within the PCAT1 gene and the MYC promoter. Conversely, androgen deprivation in vitro and in vivo increases MYC expression. In parallel, global AR activity is suppressed by MYC overexpression, consistent with coactivator redistribution. These suppressive effects of AR and MYC are mitigated at shared AR/MYC binding sites, which also have markedly higher levels of H3K27 acetylation, indicating enrichment for functional enhancers. These findings demonstrate an intricate balance between AR and MYC, and indicate that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy.


Assuntos
Elementos Facilitadores Genéticos , Proteína Oncogênica p55(v-myc)/genética , Receptores Androgênicos/genética , Androgênios/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteína Oncogênica p55(v-myc)/metabolismo , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais
7.
Nat Cancer ; 2(1): 34-48, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33997789

RESUMO

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses, and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that is enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several Activator Protein-1 (AP-1) transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.


Assuntos
Neoplasias da Mama , Fator de Transcrição AP-1 , Animais , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Quinase 4 Dependente de Ciclina/genética , Feminino , Genes cdc , Humanos , Camundongos , Fator de Transcrição AP-1/genética
8.
FEBS J ; 288(19): 5629-5649, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33811729

RESUMO

Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Fator de Crescimento Epidérmico/genética , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Mama/crescimento & desenvolvimento , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/patologia , Células Tumorais Cultivadas
9.
Nat Commun ; 12(1): 1979, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785741

RESUMO

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Tumores Neuroendócrinos/genética , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Epigenômica/métodos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Mutação , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/terapia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Interferência de RNA , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo
10.
Ann Appl Stat ; 15(2): 880-901, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37332668

RESUMO

On the problem of scoring genes for evidence of changes in the distribution of single-cell expression, we introduce an empirical Bayesian mixture approach and evaluate its operating characteristics in a range of numerical experiments. The proposed approach leverages cell-subtype structure revealed in cluster analysis in order to boost gene-level information on expression changes. Cell clustering informs gene-level analysis through a specially-constructed prior distribution over pairs of multinomial probability vectors; this prior meshes with available model-based tools that score patterns of differential expression over multiple subtypes. We derive an explicit formula for the posterior probability that a gene has the same distribution in two cellular conditions, allowing for a gene-specific mixture over subtypes in each condition. Advantage is gained by the compositional structure of the model not only in which a host of gene-specific mixture components are allowed but also in which the mixing proportions are constrained at the whole cell level. This structure leads to a novel form of information sharing through which the cell-clustering results support gene-level scoring of differential distribution. The result, according to our numerical experiments, is improved sensitivity compared to several standard approaches for detecting distributional expression changes.

13.
Nat Genet ; 52(8): 790-799, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690948

RESUMO

Epigenetic processes govern prostate cancer (PCa) biology, as evidenced by the dependency of PCa cells on the androgen receptor (AR), a prostate master transcription factor. We generated 268 epigenomic datasets spanning two state transitions-from normal prostate epithelium to localized PCa to metastases-in specimens derived from human tissue. We discovered that reprogrammed AR sites in metastatic PCa are not created de novo; rather, they are prepopulated by the transcription factors FOXA1 and HOXB13 in normal prostate epithelium. Reprogrammed regulatory elements commissioned in metastatic disease hijack latent developmental programs, accessing sites that are implicated in prostate organogenesis. Analysis of reactivated regulatory elements enabled the identification and functional validation of previously unknown metastasis-specific enhancers at HOXB13, FOXA1 and NKX3-1. Finally, we observed that prostate lineage-specific regulatory elements were strongly associated with PCa risk heritability and somatic mutation density. Examining prostate biology through an epigenomic lens is fundamental for understanding the mechanisms underlying tumor progression.


Assuntos
Neoplasias da Próstata/genética , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Sequências Reguladoras de Ácido Nucleico/genética
14.
Nat Med ; 26(7): 1041-1043, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572266

RESUMO

Improving early cancer detection has the potential to substantially reduce cancer-related mortality. Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) is a highly sensitive assay capable of detecting early-stage tumors. We report accurate classification of patients across all stages of renal cell carcinoma (RCC) in plasma (area under the receiver operating characteristic (AUROC) curve of 0.99) and demonstrate the validity of this assay to identify patients with RCC using urine cell-free DNA (cfDNA; AUROC of 0.86).


Assuntos
Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Metilação de DNA/genética , Detecção Precoce de Câncer , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/urina , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/urina , Epigenoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
15.
Genet Med ; 22(8): 1366-1373, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32341571

RESUMO

PURPOSE: Plasma cell-free DNA (cfDNA) variant analysis is commonly used in many cancer subtypes. Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) has shown high sensitivity for cancer detection. To date, studies have not compared the sensitivity of both methods in a single cancer subtype. METHODS: cfDNA from 40 metastatic RCC (mRCC) patients was subjected to targeted panel variant analysis. For 34 of 40, cfMeDIP-seq was also performed. A separate cohort of 38 mRCC patients were used in cfMeDIP-seq analysis to train an RCC classifier. RESULTS: cfDNA variant analysis detected 21 candidate variants in 11 of 40 mRCC patients (28%), after exclusion of 2 germline variants and 6 variants reflecting clonal hematopoiesis. Among 23 patients with parallel tumor sequencing, cfDNA analysis alone identified variants in 9 patients (39%), while cfDNA analysis focused on tumor sequencing variant findings improved the sensitivity to 52%. In 34 mRCC patients undergoing cfMeDIP-seq, cfDNA variant analysis identified variants in 7 (21%), while cfMeDIP-seq detected all mRCC cases (100% sensitivity) with 88% specificity in 34 control subjects. In 5 patients with cfDNA variants and serial samples, variant frequency correlated with response to therapy. CONCLUSION: cfMeDIP-seq is significantly more sensitive for mRCC detection than cfDNA variant analysis. However, cfDNA variant analysis may be useful for monitoring response to therapy.


Assuntos
Carcinoma de Células Renais , Ácidos Nucleicos Livres , Neoplasias Renais , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Ácidos Nucleicos Livres/genética , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Plasma
16.
Genome Biol ; 20(1): 118, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164141

RESUMO

BACKGROUND: In high-throughput studies, hundreds to millions of hypotheses are typically tested. Statistical methods that control the false discovery rate (FDR) have emerged as popular and powerful tools for error rate control. While classic FDR methods use only p values as input, more modern FDR methods have been shown to increase power by incorporating complementary information as informative covariates to prioritize, weight, and group hypotheses. However, there is currently no consensus on how the modern methods compare to one another. We investigate the accuracy, applicability, and ease of use of two classic and six modern FDR-controlling methods by performing a systematic benchmark comparison using simulation studies as well as six case studies in computational biology. RESULTS: Methods that incorporate informative covariates are modestly more powerful than classic approaches, and do not underperform classic approaches, even when the covariate is completely uninformative. The majority of methods are successful at controlling the FDR, with the exception of two modern methods under certain settings. Furthermore, we find that the improvement of the modern FDR methods over the classic methods increases with the informativeness of the covariate, total number of hypothesis tests, and proportion of truly non-null hypotheses. CONCLUSIONS: Modern FDR methods that use an informative covariate provide advantages over classic FDR-controlling procedures, with the relative gain dependent on the application and informativeness of available covariates. We present our findings as a practical guide and provide recommendations to aid researchers in their choice of methods to correct for false discoveries.


Assuntos
Biologia Computacional/métodos , Biologia Computacional/normas , Simulação por Computador
17.
Biostatistics ; 20(3): 367-383, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29481604

RESUMO

With recent advances in sequencing technology, it is now feasible to measure DNA methylation at tens of millions of sites across the entire genome. In most applications, biologists are interested in detecting differentially methylated regions, composed of multiple sites with differing methylation levels among populations. However, current computational approaches for detecting such regions do not provide accurate statistical inference. A major challenge in reporting uncertainty is that a genome-wide scan is involved in detecting these regions, which needs to be accounted for. A further challenge is that sample sizes are limited due to the costs associated with the technology. We have developed a new approach that overcomes these challenges and assesses uncertainty for differentially methylated regions in a rigorous manner. Region-level statistics are obtained by fitting a generalized least squares regression model with a nested autoregressive correlated error structure for the effect of interest on transformed methylation proportions. We develop an inferential approach, based on a pooled null distribution, that can be implemented even when as few as two samples per population are available. Here, we demonstrate the advantages of our method using both experimental data and Monte Carlo simulation. We find that the new method improves the specificity and sensitivity of lists of regions and accurately controls the false discovery rate.


Assuntos
Metilação de DNA , Genômica/métodos , Modelos Estatísticos , Análise de Sequência de DNA/métodos , Animais , Simulação por Computador , Genômica/normas , Humanos , Análise de Sequência de DNA/normas , Incerteza
18.
Cell ; 174(2): 422-432.e13, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29909987

RESUMO

Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kb centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers.


Assuntos
Elementos Facilitadores Genéticos/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Acetilação , Adulto , Idoso , Antineoplásicos/farmacologia , Benzamidas , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA , Edição de Genes , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética
19.
EMBO J ; 37(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335281

RESUMO

In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever-growing catalogs require high-throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA-based functionality. We applied MPRNA to identify RNA-based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine-rich motif. These nuclear enrichment sequences are highly conserved and over-represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single-molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities.


Assuntos
RNA Longo não Codificante/genética , Núcleo Celular/genética , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência de RNA
20.
Genome Biol ; 17(1): 222, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27782827

RESUMO

The ability to quantify cellular heterogeneity is a major advantage of single-cell technologies. However, statistical methods often treat cellular heterogeneity as a nuisance. We present a novel method to characterize differences in expression in the presence of distinct expression states within and among biological conditions. We demonstrate that this framework can detect differential expression patterns under a wide range of settings. Compared to existing approaches, this method has higher power to detect subtle differences in gene expression distributions that are more complex than a mean shift, and can characterize those differences. The freely available R package scDD implements the approach.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , RNA/genética , Análise de Célula Única/estatística & dados numéricos , Software , Algoritmos , Biologia Computacional , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de RNA , Análise de Célula Única/métodos
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