Opposing roles of polycomb repressive complexes in hematopoietic stem and progenitor cells
TL;DR: It is shown that mice with loss of function mutations in PRC2 components display enhanced HSC/progenitor population activity, whereas mutations that disrupt PRC1 or pleiohomeotic repressive complex are associated with HSC or progenitor cell defects.
About: This article is published in Blood.The article was published on 2010-08-05. It has received 131 citations till now. The article focuses on the topics: Progenitor cell & Stem cell.
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TL;DR: The philosophy and design of the limma package is reviewed, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
Abstract: limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
22,147 citations
Cites methods from "Opposing roles of polycomb repressi..."
...For instance, applying genas to a microarray study looking at the relationship between polycomb repressor complex (PRC) 1 and PRC2 facilitated the discovery of the opposing roles of these two complexes (51)....
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TL;DR: This work identified a subset of tumor‐reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD‐1 and the transcription factor Tcf1 that promote tumor control in response to vaccination and checkpoint blockade immunotherapy.
764 citations
TL;DR: The robust empirical Bayes (RB) algorithm as mentioned in this paper improves the robust differential expression tests by robustifying the hyperparameter estimation procedure, which has the double benefit of reducing the chance that hypervariable genes will be spuriously identified as DE while increasing statistical power for the main body of genes.
Abstract: One of the most common analysis tasks in genomic research is to identify genes that are differentially expressed (DE) between experimental conditions. Empirical Bayes (EB) statistical tests using moderated genewise variances have been very effective for this purpose, especially when the number of biological replicate samples is small. The EB procedures can however be heavily influenced by a small number of genes with very large or very small variances. This article improves the differential expression tests by robustifying the hyperparameter estimation procedure. The robust procedure has the effect of decreasing the informativeness of the prior distribution for outlier genes while increasing its informativeness for other genes. This effect has the double benefit of reducing the chance that hypervariable genes will be spuriously identified as DE while increasing statistical power for the main body of genes. The robust EB algorithm is fast and numerically stable. The procedure allows exact small-sample null distributions for the test statistics and reduces exactly to the original EB procedure when no outlier genes are present. Simulations show that the robustified tests have similar performance to the original tests in the absence of outlier genes but have greater power and robustness when outliers are present. The article includes case studies for which the robust method correctly identifies and downweights genes associated with hidden covariates and detects more genes likely to be scientifically relevant to the experimental conditions. The new procedure is implemented in the limma software package freely available from the Bioconductor repository.
632 citations
TL;DR: Simulations show that the robustified tests have similar performance to the original tests in the absence of outlier genes but have greater power and robustness when outliers are present, and the robust method correctly identifies and downweights genes associated with hidden covariates and detects more genes likely to be scientifically relevant to the experimental conditions.
Abstract: One of the most common analysis tasks in genomic research is to identify genes that are differentially expressed (DE) between experimental conditions. Empirical Bayes (EB) statistical tests using moderated genewise variances have been very effective for this purpose, especially when the number of biological replicate samples is small. The EB procedures can however be heavily influenced by a small number of genes with very large or very small variances. This article improves the differential expression tests by robustifying the hyperparameter estimation procedure. The robust procedure has the effect of decreasing the informativeness of the prior distribution for outlier genes while increasing its informativeness for other genes. This effect has the double benefit of reducing the chance that hypervariable genes will be spuriously identified as DE while increasing statistical power for the main body of genes. The robust EB algorithm is fast and numerically stable. The procedure allows exact small-sample null distributions for the test statistics and reduces exactly to the original EB procedure when no outlier genes are present. Simulations show that the robustified tests have similar performance to the original tests in the absence of outlier genes but have greater power and robustness when outliers are present. The article includes case studies for which the robust method correctly identifies and downweights genes associated with hidden covariates and detects more genes likely to be scientifically relevant to the experimental conditions. The new procedure is implemented in the limma software package freely available from the Bioconductor repository.
489 citations
Cites background from "Opposing roles of polycomb repressi..."
...Majewski et al. (2008, 2010) studied mice with a mutation in the Suz12 gene that results in loss of function of the Suz12 protein and hence PRC2....
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TL;DR: 3-deazaneplanocin (DZNep) reduces EZH2 levels and H3K27 trimethylation, resulting in reduced cell proliferation in breast and prostate cancer cells in vitro, and synergistic effects are seen for combined treatment with DNA demethylating agents and histone deacetylation inhibitors, opening up the possibility of refined epigenetic treatments in the future.
Abstract: Control of gene expression is exerted at a number of different levels, one of which is the accessibility of genes and their controlling elements to the transcriptional machinery. Accessibility is dictated broadly by the degree of chromatin compaction, which is influenced in part by polycomb group proteins. EZH2, together with SUZ12 and EED, forms the polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3). PRC2 may recruit other polycomb complexes, DNA methyltransferases, and histone deacetylases, resulting in additional transcriptional repressive marks and chromatin compaction at key developmental loci. Overexpression of EZH2 is a marker of advanced and metastatic disease in many solid tumors, including prostate and breast cancer. Mutation of EZH2 Y641 is described in lymphoma and results in enhanced activity, whereas inactivating mutations are seen in poor prognosis myeloid neoplasms. No histone demethylating agents are currently available for treatment of patients, but 3-deazaneplanocin (DZNep) reduces EZH2 levels and H3K27 trimethylation, resulting in reduced cell proliferation in breast and prostate cancer cells in vitro. Furthermore, synergistic effects are seen for combined treatment with DNA demethylating agents and histone deacetylation inhibitors, opening up the possibility of refined epigenetic treatments in the future.
488 citations
Cites background from "Opposing roles of polycomb repressi..."
...In HSCs, however, there is evidence for opposing roles for PRC1 and PRC2 (10–13)....
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...It is currently not known whether the differential effects of PRC1 and PRC2 seen in HSC also occur in other tissues....
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...This model would predict that targets of PRC1 and PRC2 largely overlap, as has indeed been shown by RNA interference (RNAi) depletion studies in human embryonic fibroblasts (9)....
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...In addition, genes known to be repressed by CCAAT/enhancer binding protein alpha (CEBPA) or activated by homeobox A9 (HoxA9) have been found to be differentially regulated by PRC1 and PRC2 (13)....
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...BMI1 is required for HSC repopulation (10, 12), whereas reduced levels of Suz12, Ezh2, or Eed result in increased HSC-progenitor proliferation (13)....
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References
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TL;DR: The Gene Set Enrichment Analysis (GSEA) method as discussed by the authors focuses on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.
Abstract: Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
34,830 citations
TL;DR: The hierarchical model of Lonnstedt and Speed (2002) is developed into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples and the moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom.
Abstract: The problem of identifying differentially expressed genes in designed microarray experiments is considered. Lonnstedt and Speed (2002) derived an expression for the posterior odds of differential expression in a replicated two-color experiment using a simple hierarchical parametric model. The purpose of this paper is to develop the hierarchical model of Lonnstedt and Speed (2002) into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples. The model is reset in the context of general linear models with arbitrary coefficients and contrasts of interest. The approach applies equally well to both single channel and two color microarray experiments. Consistent, closed form estimators are derived for the hyperparameters in the model. The estimators proposed have robust behavior even for small numbers of arrays and allow for incomplete data arising from spot filtering or spot quality weights. The posterior odds statistic is reformulated in terms of a moderated t-statistic in which posterior residual standard deviations are used in place of ordinary standard deviations. The empirical Bayes approach is equivalent to shrinkage of the estimated sample variances towards a pooled estimate, resulting in far more stable inference when the number of arrays is small. The use of moderated t-statistics has the advantage over the posterior odds that the number of hyperparameters which need to estimated is reduced; in particular, knowledge of the non-null prior for the fold changes are not required. The moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom. The moderated t inferential approach extends to accommodate tests of composite null hypotheses through the use of moderated F-statistics. The performance of the methods is demonstrated in a simulation study. Results are presented for two publicly available data sets.
11,864 citations
TL;DR: The purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex, is reported, and it is demonstrated that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27).
Abstract: Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.
3,565 citations
"Opposing roles of polycomb repressi..." refers result in this paper
...Several lines of evidence support this prevailing model: (1) a high degree of overlap between sites bound by PRC1 and PRC2 in human and mouse cells9,10,19; (2) the failure of Drosophila E(z) mutants to recruit PRC120; and (3) a correlation between H3K27me3 levels and PRC1 recruitment and H2AK119Ub accumulation....
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TL;DR: It is shown that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation, and dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.
Abstract: The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.
2,549 citations
TL;DR: It is found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes.
2,341 citations