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1.  Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes 
Nucleic Acids Research  2014;42(8):e64.
Molecular stratification of tumors is essential for developing personalized therapies. Although patient stratification strategies have been successful; computational methods to accurately translate the gene-signature from high-throughput platform to a clinically adaptable low-dimensional platform are currently lacking. Here, we describe PIGExClass (platform-independent isoform-level gene-expression based classification-system), a novel computational approach to derive and then transfer gene-signatures from one analytical platform to another. We applied PIGExClass to design a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) based molecular-subtyping assay for glioblastoma multiforme (GBM), the most aggressive primary brain tumors. Unsupervised clustering of TCGA (the Cancer Genome Altas Consortium) GBM samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 19% of the samples, resulting in significant (P = 0.0103) survival differences among the refined subgroups. PIGExClass derived four-class classifier, which requires only 121 transcript-variants, assigns GBM patients’ molecular subtype with 92% accuracy. This classifier was translated to an RT-qPCR assay and validated in an independent cohort of 206 GBM samples. Our results demonstrate the efficacy of PIGExClass in the design of clinically adaptable molecular subtyping assay and have implications for developing robust diagnostic assays for cancer patient stratification.
PMCID: PMC4005667  PMID: 24503249
2.  Isoform level expression profiles provide better cancer signatures than gene level expression profiles 
Genome Medicine  2013;5(4):33.
The majority of mammalian genes generate multiple transcript variants and protein isoforms through alternative transcription and/or alternative splicing, and the dynamic changes at the transcript/isoform level between non-oncogenic and cancer cells remain largely unexplored. We hypothesized that isoform level expression profiles would be better than gene level expression profiles at discriminating between non-oncogenic and cancer cellsgene level.
We analyzed 160 Affymetrix exon-array datasets, comprising cell lines of non-oncogenic or oncogenic tissue origins. We obtained the transcript-level and gene level expression estimates, and used unsupervised and supervised clustering algorithms to study the profile similarity between the samples at both gene and isoform levels.
Hierarchical clustering, based on isoform level expressions, effectively grouped the non-oncogenic and oncogenic cell lines with a virtually perfect homogeneity-grouping rate (97.5%), regardless of the tissue origin of the cell lines. However, gene levelthis rate was much lower, being 75% at best based on the gene level expressions. Statistical analyses of the difference between cancer and non-oncogenic samples identified the existence of numerous genes with differentially expressed isoforms, which otherwise were not significant at the gene level. We also found that canonical pathways of protein ubiquitination, purine metabolism, and breast-cancer regulation by stathmin1 were significantly enriched among genes thatshow differential expression at isoform level but not at gene level.
In summary, cancer cell lines, regardless of their tissue of origin, can be effectively discriminated from non-cancer cell lines at isoform level, but not at gene level. This study suggests the existence of an isoform signature, rather than a gene signature, which could be used to distinguish cancer cells from normal cells.
PMCID: PMC3706752  PMID: 23594586
3.  IsoformEx: isoform level gene expression estimation using weighted non-negative least squares from mRNA-Seq data 
BMC Bioinformatics  2011;12:305.
mRNA-Seq technology has revolutionized the field of transcriptomics for identification and quantification of gene transcripts not only at gene level but also at isoform level. Estimating the expression levels of transcript isoforms from mRNA-Seq data is a challenging problem due to the presence of constitutive exons.
We propose a novel algorithm (IsoformEx) that employs weighted non-negative least squares estimation method to estimate the expression levels of transcript isoforms. Validations based on in silico simulation of mRNA-Seq and qRT-PCR experiments with real mRNA-Seq data showed that IsoformEx could accurately estimate transcript expression levels. In comparisons with published methods, the transcript expression levels estimated by IsoformEx showed higher correlation with known transcript expression levels from simulated mRNA-Seq data, and higher agreement with qRT-PCR measurements of specific transcripts for real mRNA-Seq data.
IsoformEx is a fast and accurate algorithm to estimate transcript expression levels and gene expression levels, which takes into account short exons and alternative exons with a weighting scheme. The software is available at
PMCID: PMC3180389  PMID: 21794104
4.  Genome-wide mapping of RNA Pol-II promoter usage in mouse tissues by ChIP-seq 
Nucleic Acids Research  2010;39(1):190-201.
Alternative promoters that are differentially used in various cellular contexts and tissue types add to the transcriptional complexity in mammalian genome. Identification of alternative promoters and the annotation of their activity in different tissues is one of the major challenges in understanding the transcriptional regulation of the mammalian genes and their isoforms. To determine the use of alternative promoters in different tissues, we performed ChIP-seq experiments using antibody against RNA Pol-II, in five adult mouse tissues (brain, liver, lung, spleen and kidney). Our analysis identified 38 639 Pol-II promoters, including 12 270 novel promoters, for both protein coding and non-coding mouse genes. Of these, 6384 promoters are tissue specific which are CpG poor and we find that only 34% of the novel promoters are located in CpG-rich regions, suggesting that novel promoters are mostly tissue specific. By identifying the Pol-II bound promoter(s) of each annotated gene in a given tissue, we found that 37% of the protein coding genes use alternative promoters in the five mouse tissues. The promoter annotations and ChIP-seq data presented here will aid ongoing efforts of characterizing gene regulatory regions in mammalian genomes.
PMCID: PMC3017616  PMID: 20843783
5.  Annotation of gene promoters by integrative data-mining of ChIP-seq Pol-II enrichment data 
BMC Bioinformatics  2010;11(Suppl 1):S65.
Use of alternative gene promoters that drive widespread cell-type, tissue-type or developmental gene regulation in mammalian genomes is a common phenomenon. Chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) or massive parallel sequencing (ChIP-seq) are enabling genome-wide identification of active promoters in different cellular conditions using antibodies against Pol-II. However, these methods produce enrichment not only near the gene promoters but also inside the genes and other genomic regions due to the non-specificity of the antibodies used in ChIP. Further, the use of these methods is limited by their high cost and strong dependence on cellular type and context.
We trained and tested different state-of-art ensemble and meta classification methods for identification of Pol-II enriched promoter and Pol-II enriched non-promoter sequences, each of length 500 bp. The classification models were trained and tested on a bench-mark dataset, using a set of 39 different feature variables that are based on chromatin modification signatures and various DNA sequence features. The best performing model was applied on seven published ChIP-seq Pol-II datasets to provide genome wide annotation of mouse gene promoters.
We present a novel algorithm based on supervised learning methods to discriminate promoter associated Pol-II enrichment from enrichment elsewhere in the genome in ChIP-chip/seq profiles. We accumulated a dataset of 11,773 promoter and 46,167 non-promoter sequences, each of length 500 bp, generated from RNA Pol-II ChIP-seq data of five tissues (Brain, Kidney, Liver, Lung and Spleen). We evaluated the classification models in building the best predictor and found that Bagging and Random Forest based approaches give the best accuracy. We implemented the algorithm on seven different published ChIP-seq datasets to provide a comprehensive set of promoter annotations for both protein-coding and non-coding genes in the mouse genome. The resulting annotations contain 13,413 (4,747) protein-coding (non-coding) genes with single promoters and 9,929 (1,858) protein-coding (non-coding) genes with two or more alternative promoters, and a significant number of unassigned novel promoters.
Our new algorithm can successfully predict the promoters from the genome wide profile of Pol-II bound regions. In addition, our algorithm performs significantly better than existing promoter prediction methods and can be applied for genome-wide predictions of Pol-II promoters.
PMCID: PMC3009539  PMID: 20122241
6.  Protein Arginine Methyltransferase 5 Suppresses the Transcription of the RB Family of Tumor Suppressors in Leukemia and Lymphoma Cells▿ †  
Molecular and Cellular Biology  2008;28(20):6262-6277.
The proper epigenetic modification of chromatin by protein arginine methyltransferases (PRMTs) is crucial for normal cell growth and health. The human SWI/SNF-associated PRMT5 is involved in the transcriptional repression of target genes by directly methylating H3R8 and H4R3. To further understand the impact of PRMT5-mediated histone methylation on cancer, we analyzed its expression in normal and transformed human B lymphocytes. Our findings reveal that PRMT5 protein levels are enhanced in various human lymphoid cancer cells, including transformed chronic lymphocytic leukemia (B-CLL) cell lines. PRMT5 overexpression is caused by the altered expression of the PRMT5-specific microRNAs 19a, 25, 32, 92, 92b, and 96 and results in the increased global symmetric methylation of H3R8 and H4R3. An evaluation of both epigenetic marks at PRMT5 target genes such as RB1 (p105), RBL1 (p107), and RBL2 (p130) showed that promoters H3R8 and H4R3 are hypermethylated, which in turn triggers pocket protein transcriptional repression. Furthermore, reducing PRMT5 expression in WaC3CD5 B-CLL cells abolishes H3R8 and H4R3 hypermethylation, restores RBL2 expression, and inhibits cancer cell proliferation. These results indicate that PRMT5 overexpression epigenetically alters the transcription of key tumor suppressor genes and suggest a causal role of the elevated symmetric methylation of H3R8 and H4R3 at the RBL2 promoter in transformed B-lymphocyte pathology.
PMCID: PMC2577430  PMID: 18694959
7.  The Protein Arginine Methyltransferase Prmt5 Is Required for Myogenesis because It Facilitates ATP-Dependent Chromatin Remodeling▿  
Molecular and Cellular Biology  2006;27(1):384-394.
Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function.
PMCID: PMC1800640  PMID: 17043109
8.  The BRG1- and hBRM-Associated Factor BAF57 Induces Apoptosis by Stimulating Expression of the Cylindromatosis Tumor Suppressor Gene†  
Molecular and Cellular Biology  2005;25(18):7953-7965.
Mutation of BRG1, hBRM, and their associated factors, INI1 and BAF57, in primary human tumors has suggested that inactivation of human SWI/SNF (hSWI/SNF) complexes may be involved in neoplastic transformation. BT549 is an invasive human breast carcinoma cell line that lacks expression of BAF57, a key hSWI/SNF subunit that mediates interaction with transcriptional activators and corepressors. In this study we investigated the role of BAF57 in suppressing tumorigenesis by establishing BT549 stable cell lines that expresses full-length BAF57 protein. BT549 clones expressing BAF57 demonstrated marked phenotypic changes, slow growth kinetics, and restoration of contact inhibition. Altered growth was found to be due in part to cell cycle arrest and induction of apoptosis. Furthermore, microarray analysis revealed that BAF57-mediated cell death was associated with up-regulation of proapoptotic genes including the tumor suppressor familial cylindromatosis (CYLD), which was found to be a direct target of BAF57 as determined by chromatin immunoprecipitation analysis. Increased expression of CYLD in BT549 cells induced apoptosis, while its suppression by small interfering RNA inhibited cell death in BAF57 expressing BT549 cells. These findings demonstrate the importance of BAF57 in cell growth regulation and provide a novel link between hSWI/SNF chromatin remodelers and apoptosis.
PMCID: PMC1234311  PMID: 16135788
9.  Human SWI/SNF-Associated PRMT5 Methylates Histone H3 Arginine 8 and Negatively Regulates Expression of ST7 and NM23 Tumor Suppressor Genes†  
Molecular and Cellular Biology  2004;24(21):9630-9645.
Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and repression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5 antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23 (NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH 3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and proliferation by controlling expression of genes involved in tumor suppression.
PMCID: PMC522266  PMID: 15485929
10.  mSin3A/Histone Deacetylase 2- and PRMT5-Containing Brg1 Complex Is Involved in Transcriptional Repression of the Myc Target Gene cad 
Molecular and Cellular Biology  2003;23(21):7475-7487.
The role of hSWI/SNF complexes in transcriptional activation is well characterized; however, little is known about their function in transcriptional repression. We have previously shown that subunits of the mSin3A/histone deacetylase 2 (HDAC2) corepressor complex copurify with hSWI/SNF complexes. Here we show that the type II arginine-specific methyltransferase PRMT5, which is involved in cyclin E repression, can be found in association with Brg1 and hBrm-based hSWI/SNF complexes. We also show that hSWI/SNF-associated PRMT5 can methylate hypoacetylated histones H3 and H4 more efficiently than hyperacetylated histones H3 and H4. Protein-protein interaction studies indicate that PRMT5 and mSin3A interact with the same hSWI/SNF subunits as those targeted by c-Myc. These observations prompted us to examine the expression profile of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (cad) and nucleolin (nuc). We found that cad repression is altered in cells that express inactive Brg1 and in cells treated with the HDAC inhibitor depsipeptide. Using chromatin immunoprecipitation assays, we found that Brg1, mSin3A, HDAC2, and PRMT5 are directly recruited to the cad promoter. These results suggest that hSWI/SNF complexes, through their ability to interact with activator and repressor proteins, control expression of genes involved in cell growth and proliferation.
PMCID: PMC207647  PMID: 14559996

Results 1-10 (10)