RESUMO
Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or a highly related FET/ETS chimera. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma cells using a well-validated 'knock-down/rescue' model of EWS/FLI function in combination with next generation sequencing assays to evaluate how the chromatin landscape changes with loss, and recovery, of EWS/FLI expression. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. This EWS/FLI binding signature is associated with establishment of topologically-associated domain (TAD) boundaries, compartment activation, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. In addition, EWS/FLI co-localizes with the loop-extrusion factor cohesin to promote chromatin loops and TAD boundaries. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators of three-dimensional reprogramming of chromatin.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing , Linhagem Celular Tumoral , Criança , Cromatina/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMO
BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.
Assuntos
Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Humanos , Osteossarcoma/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Perfilação da Expressão Gênica , Neoplasias Ósseas/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Microambiente Tumoral/genéticaRESUMO
Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.
Assuntos
Receptor Nuclear Órfão DAX-1/genética , Proteínas de Ligação a DNA/genética , Proteínas dos Microfilamentos/genética , Repetições de Microssatélites/genética , Proteína EWS de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Sarcoma de Ewing/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Proteínas dos Microfilamentos/metabolismo , Domínios Proteicos/genética , Proteína EWS de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Sarcoma de Ewing/metabolismo , TransativadoresRESUMO
Single nucleotide polymorphisms (SNPs) in one-carbon metabolism genes and lifestyle factors (alcohol drinking and breast folate) may be determinants of whole-genome methylation in the breast. DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip in 81 normal breast tissues from women undergoing reduction mammoplasty and no history of cancer. ANCOVA, adjusting for age, race and BMI, was used to identify differentially-methylated (DM) CpGs. Gene expression, by the Affymetrix GeneChip Human Transcriptome Array 2.0, was correlated with DM. Biological networks of DM genes were assigned using Ingenuity Pathway Analysis. Fifty-seven CpG sites were DM in association with eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P <5.0 x 10-5); 56% of the DM CpGs were associated with FTHFD SNPs, including DM within FTHFD. Gene expression was negatively correlated with FTHFD methylation (r=-0.25, P=0.017). Four DM CpGs identified by SNPs in MTRR, MTHFR, and FTHFD were significantly associated with alcohol consumption and/or breast folate. The top biological network of DM CpGs was associated with Energy Production, Molecular Transportation, and Nucleic Acid Metabolism. This is the first comprehensive study of the association between SNPs in one-carbon metabolism genes and genome-wide DNA methylation in normal breast tissues. These SNPs, especially FTHFD, as well as alcohol intake and folate exposure, appear to affect DM in breast tissues of healthy women. The finding that SNPs in FTHFD and MTR are associated with their own methylation is novel and highlights a role for these SNPs as cis-methylation quantitative trait loci.
RESUMO
The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers' peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers' blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. © 2015 Wiley Periodicals, Inc.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fumar/genética , Células Cultivadas , Estudos Transversais , DNA/sangue , Detecção Precoce de Câncer , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Fumaça/efeitos adversos , Nicotiana/efeitos adversosRESUMO
Mapping the chromosomal locations of transcription factors, nucleosomes, histone modifications, chromatin remodeling enzymes, chaperones, and polymerases is one of the key tasks of modern biology, as evidenced by the Encyclopedia of DNA Elements (ENCODE) Project. To this end, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is the standard methodology. Mapping such protein-DNA interactions in vivo using ChIP-seq presents multiple challenges not only in sample preparation and sequencing but also for computational analysis. Here, we present step-by-step guidelines for the computational analysis of ChIP-seq data. We address all the major steps in the analysis of ChIP-seq data: sequencing depth selection, quality checking, mapping, data normalization, assessment of reproducibility, peak calling, differential binding analysis, controlling the false discovery rate, peak annotation, visualization, and motif analysis. At each step in our guidelines we discuss some of the software tools most frequently used. We also highlight the challenges and problems associated with each step in ChIP-seq data analysis. We present a concise workflow for the analysis of ChIP-seq data in Figure 1 that complements and expands on the recommendations of the ENCODE and modENCODE projects. Each step in the workflow is described in detail in the following sections.
Assuntos
Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reprodutibilidade dos TestesRESUMO
Recently, much attention has been given to elucidate how long-range gene regulation comes into play and how histone modifications and distal transcription factor binding contribute toward this mechanism. Androgen receptor (AR), a key regulator of prostate cancer, has been shown to regulate its target genes via distal enhancers, leading to the hypothesis of global long-range gene regulation. However, despite numerous flows of newly generated data, the precise mechanism with respect to AR-mediated long-range gene regulation is still largely unknown. In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data. We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block. Importantly, genes in this class were enriched in cancer-related pathways and were downregulated in clinical metastatic versus localized prostate cancer. Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking. Under such a mechanism, H3K4me2, AR and FoxA1 within the same CTCF block combinatorially regulate a subset of distally located androgen-responsive genes involved in prostate carcinogenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Androgênios/farmacologia , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Genoma Humano , Células HeLa , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/metabolismo , Humanos , Células Jurkat , Masculino , Metástase Neoplásica , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5' portion of the FET gene with a 3' partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the in vitro potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas.
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Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-ð¼4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-ð¼4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatinmarks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-ð¼4 helix. Our studies revealed DBD-ð¼4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.
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Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1's in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1's recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.
RESUMO
We report a universal strategy for 2D chromatin sequencing, to increase uniform data analyses and sharing across labs, and to facilitate highly quantitative comparisons across experimental conditions. Within our system, we provide wetlab and drylab tools for researchers to establish and analyze protein-genome binding data with PerCell ChIP-seq. Our methodology is virtually no cost and flexible, enabling rapid, quantitative, internally normalized chromatin sequencing to catalyze project development in a variety of systems, including in vivo zebrafish epigenomics and cancer cell epigenomics. While we highlight utility in these key areas, our methodology is flexible enough such that rapid comparisons of cellular spike-in versus non spike-in are possible, and generalizability to nuclease-based 2D chromatin sequencing would also be possible within the framework of our pipeline. Through the use of well-defined cellular ratios containing orthologous species' chromatin, we enable cross-species comparative epigenomics and highly quantitative low-cost chromatin sequencing with utility across a range of disciplines.
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Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS-FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS-FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.
Assuntos
Sarcoma de Ewing , Criança , Humanos , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genéticaRESUMO
Regulation of gene expression has been shown to involve not only the binding of transcription factor at target gene promoters but also the characterization of histone around which DNA is wrapped around. Some histone modification, for example di-methylated histone H3 at lysine 4 (H3K4me2), has been shown to bind to promoters and activate target genes. However, no clear pattern has been shown to predict human promoters. This paper proposed a novel quantitative approach to characterize patterns of promoter regions and predict novel and alternative promoters. We utilized high-throughput data generated using chromatin immunoprecipitation methods followed by massively parallel sequencing (ChIP-seq) technology on RNA Polymerase II (Pol-II) and H3K4me2. Common patterns of promoter regions are modeled using a mixture model involving double-exponential and uniform distributions. The fitted model obtained were then used to search for regions displaying similar patterns over the entire genome to find novel and alternative promoters. Regions with high correlations with the common patterns are identified as putative novel promoters. We used this proposed algorithm, RNA-seq data and several transcripts databases to find alternative promoters in MCF7 (normal breast cancer) cell line. We found 7,235 high-confidence regions that display the identified promoter patterns. Of these, 4,167 regions (58%) can be mapped to RefSeq regions. 2,444 regions are in a gene body or overlap with transcripts (non-coding RNAs, ESTs, and transcripts that are predicted by RNA-seq data). Some of these maybe potential alternative promoters. We also found 193 regions that map to enhancer regions (represented by androgen and estrogen receptor binding sites) and other regulatory regions such as CTCF (CCCTC binding factor) and CpG island. Around 5% (431 regions) of these correlated regions do not overlap with any transcripts or regulatory regions suggesting that these might be potential new promoters or markers for other annotation which are currently undiscovered.
Assuntos
Cromatina/metabolismo , Genoma Humano , Modelos Genéticos , Algoritmos , Imunoprecipitação da Cromatina , Análise por Conglomerados , Histonas/genética , Histonas/metabolismo , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
SUMMARY: Differential Identification using Mixtures Ensemble (DIME) is a package for identification of biologically significant differential binding sites between two conditions using ChIP-seq data. It considers a collection of finite mixture models combined with a false discovery rate (FDR) criterion to find statistically significant regions. This leads to a more reliable assessment of differential binding sites based on a statistical approach. In addition to ChIP-seq, DIME is also applicable to data from other high-throughput platforms. AVAILABILITY AND IMPLEMENTATION: DIME is implemented as an R-package, which is available at http://www.stat.osu.edu/~statgen/SOFTWARE/DIME. It may also be downloaded from http://cran.r-project.org/web/packages/DIME/.
Assuntos
Imunoprecipitação da Cromatina/métodos , Software , Algoritmos , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Humanos , Modelos Estatísticos , Análise de Sequência de DNARESUMO
Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine-specific demethylase-1 (LSD1) inhibitors, which are currently in early-phase clinical trials. Single-agent-targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR-Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP-2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches, including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509. IMPLICATIONS: These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents that prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.
Assuntos
Neoplasias Ósseas , Sarcoma de Ewing , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Resistência a Medicamentos , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
Paediatric cancers commonly harbour quiet mutational landscapes and are instead characterized by single driver events such as the mutation of critical chromatin regulators, expression of oncohistones, or expression of oncogenic fusion proteins. These events ultimately promote malignancy through disruption of normal gene regulation and development. The driver protein in Ewing sarcoma, EWS/FLI, is an oncogenic fusion and transcription factor that reshapes the enhancer landscape, resulting in widespread transcriptional dysregulation. Lysine-specific demethylase 1 (LSD1) is a critical functional partner for EWS/FLI as inhibition of LSD1 reverses the transcriptional activity of EWS/FLI. However, how LSD1 participates in fusion-directed epigenomic regulation and aberrant gene activation is unknown. We now show EWS/FLI causes dynamic rearrangement of LSD1 and we uncover a role for LSD1 in gene activation through colocalization at EWS/FLI binding sites throughout the genome. LSD1 is integral to the establishment of Ewing sarcoma super-enhancers at GGAA-microsatellites, which ubiquitously overlap non-microsatellite loci bound by EWS/FLI. Together, we show that EWS/FLI induces widespread changes to LSD1 distribution in a process that impacts the enhancer landscape throughout the genome.
Assuntos
Cromatina , Lisina , Linhagem Celular Tumoral , Criança , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismoRESUMO
Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.
Assuntos
Oncogenes , Criança , Humanos , Fagocitose , Sarcoma de EwingRESUMO
Ewing sarcoma is a pediatric bone cancer defined by a chromosomal translocation fusing one of the FET family members to an ETS transcription factor. There have been seven reported chromosomal translocations, with the most recent reported over a decade ago. We now report a novel FET/ETS translocation involving FUS and ETV4 detected in a patient with Ewing sarcoma. Here, we characterized FUS/ETV4 by performing genomic localization and transcriptional regulatory studies on numerous FET/ETS fusions in a Ewing sarcoma cellular model. Through this comparative analysis, we demonstrate significant similarities across these fusions, and in doing so, validate FUS/ETV4 as a bona fide Ewing sarcoma translocation. This study presents the first genomic comparison of Ewing sarcoma-associated translocations and reveals that the FET/ETS fusions share highly similar, but not identical, genomic localization and transcriptional regulation patterns. These data strengthen the notion that FET/ETS fusions are key drivers of, and thus pathognomonic for, Ewing sarcoma. IMPLICATIONS: Identification and initial characterization of the novel Ewing sarcoma fusion, FUS/ETV4, expands the family of Ewing fusions and extends the diagnostic possibilities for this aggressive tumor of adolescents and young adults.
Assuntos
Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genética , Translocação Genética/genética , Humanos , Recém-Nascido , Proteínas de Fusão Oncogênica/genética , Sarcoma de Ewing/patologiaRESUMO
MOTIVATION: Antibody-based Chromatin Immunoprecipitation assay followed by high-throughput sequencing technology (ChIP-seq) is a relatively new method to study the binding patterns of specific protein molecules over the entire genome. ChIP-seq technology allows scientist to get more comprehensive results in shorter time. Here, we present a non-linear normalization algorithm and a mixture modeling method for comparing ChIP-seq data from multiple samples and characterizing genes based on their RNA polymerase II (Pol II) binding patterns. RESULTS: We apply a two-step non-linear normalization method based on locally weighted regression (LOESS) approach to compare ChIP-seq data across multiple samples and model the difference using an Exponential-Normal(K) mixture model. Fitted model is used to identify genes associated with differential binding sites based on local false discovery rate (fdr). These genes are then standardized and hierarchically clustered to characterize their Pol II binding patterns. As a case study, we apply the analysis procedure comparing normal breast cancer (MCF7) to tamoxifen-resistant (OHT) cell line. We find enriched regions that are associated with cancer (P < 0.0001). Our findings also imply that there may be a dysregulation of cell cycle and gene expression control pathways in the tamoxifen-resistant cells. These results show that the non-linear normalization method can be used to analyze ChIP-seq data across multiple samples. AVAILABILITY: Data are available at http://www.bmi.osu.edu/~khuang/Data/ChIP/RNAPII/.
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Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , RNA Polimerase II/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Evasion of immune surveillance is one of the hallmarks of cancer. Although the adaptive immune system has been targeted via checkpoint inhibition, many patients do not sustain durable remissions due to the heterogeneity of the tumor microenvironment, so additional strategies are needed. The innate immune system has its own set of checkpoints, and tumors have co-opted this system by expressing surface receptors that inhibit phagocytosis. One of these receptors, CD47, also known as the "don't eat me" signal, has been found to be overexpressed by most cancer histologies and has been successfully targeted by antibodies blocking the receptor or its ligand, signal regulatory protein α (SIRPα). By enabling phagocytosis via antigen-presenting cells, interruption of CD47-SIRPα binding leads to earlier downstream activation of the adaptive immune system. Recent and ongoing clinical trials are demonstrating the safety and efficacy of CD47 blockade in combination with monoclonal antibodies, chemotherapy, or checkpoint inhibitors for adult cancer histologies. The aim of this review is to highlight the current literature and research on CD47, provide an impetus for investigation of its blockade in pediatric cancer histologies, and provide a rationale for new combination therapies in these patients.