An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation

et al. (2008) An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation. PLoS ONE 3(3): e1882. doi:10.1371/journal.pone.0001882 An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation Maria E. Figueroa 0 Mark Reimers 0 Reid F. Thompson 0 Kenny Ye 0 Yushan Li 0 Rebecca R. Selzer 0 Jakob Fridriksson 0 Elisabeth Paietta 0 Peter Wiernik 0 Roland D. Green 0 John M. Greally 0 Ari 0 Melnick 0 Eshel Ben-Jacob, Tel Aviv University, Israel 0 1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Biostatistics, Virginia Commonwealth University , Richmond , Virginia, United States of America, 3 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York, United States of America , 5 Roche NimbleGen , Inc. Madison, Wisconsin, United States of America, 6 Our Lady of Mercy Comprehensive Cancer Center, Bronx, New York, United States of America, 7 Department of Medicine, Albert Einstein College of Medicine , Bronx, New York , United States of America The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes. Gene expression, cytosine methylation and histone H3 lysine 9 (H3K9) acetylation were measured using highdensity oligonucleotide microarrays in primary human acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) specimens. We found that DNA methylation and H3K9 acetylation distinguished these leukemias of distinct cell lineage, as expected, but that an integrative analysis combining the information from each platform revealed hundreds of additional differentially expressed genes that were missed by gene expression arrays alone. This integrated analysis also enhanced the detection and statistical significance of biological pathways dysregulated in AML and ALL. Integrative epigenomic studies are thus feasible using clinical samples and provide superior detection of aberrant transcriptional programming than singleplatform microarray studies. - Funding: Maria E. Figueroa is supported by an ASH Fellow Scholar Award. Ari M. Melnick is supported by NCI R01 CA104348, the Chemotherapy Foundation, the Sam Waxman Cancer Research Foundation, and the G&P Foundation and is a Leukemia and Lymphoma Society Scholar. John M. Greally is supported by a grant from the National Institutes of Health (NIH) (R01 HD044078). Reid F. Thompson is supported by NIH MSTP Training Grant GM007288. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Regulation of gene expression involves multi-layered mechanisms in which epigenetic modifications such as DNA methylation and histone tail modifications play a major role[1,2]. Posttranslational modifications of histones at specific residues help to determine chromatin structure and therefore accessibility to gene promoters and regulatory regions. Amongst these marks, acetylation of lysine 9 on histone H3 (H3K9 acetylation) has been linked to gene activation and active transcription[3,4]. Cytosine methylation at promoter regions, on the other hand, is associated with gene silencing[5]. Epigenetic regulation of gene expression has additional complexities; not only is the presence of specific epigenetic marks important but their localization and density also seem to play a crucial role[68]. Disruption of epigenetic regulation during malignant transformation can profoundly alter a cellular phenotype, resulting in aberrant cellular proliferation and survival. Epigenetic dysregulation is currently recognized as one of the hallmarks of cancer [9,10]. DNA methylation at promoter regions of key negative cell cycle regulators and DNA repair genes leads to their abnormal epigenetic silencing in many neoplasms[5,1114]. However, it is not clear whether this aberrant DNA methylation pattern is sufficient to determine gene silencing, or whether it is in fact part of a more complex process involving chromatin remodeling factors and changes in histone modifications[15,16]. Gene expression profiling studies have been performed with the aim of dissecting the molecular subtypes of several neoplasms, in an effort to predict accurately tumor behavior and to identify important oncogenic genes and biological pathways. These studies have revealed the presence of unique gene expression signatures distinguishing specific subgroups of cancers and have served to improve our understanding of the biology of these diseases (e.g. [1720]). However, only part of the cellular information is contained at the messenger RNA level, and transcriptional activity is dependent on multiple factors. Among these factors are epigenetic marks, such as cytosine methylation and histone tail modifications, which help to determine and regulate chromatin structure and function including gene expression. t(9;22)(q34;q11.2), add(16)(q21), 220, +mar negative by RT-PCR for negative by RT-PCR for BCR/ABL, AML1/ETO, BCR/ABL, AML1/ETO, CBFbeta/MYH11, MLL-TD CBFbeta/MYH11, MLL-TD and FLT3-ITD and FLT3-ITD Therefore, while gene expression studies using DNA microarrays have had a great impact in the study of cancer, it is important to recognize that there are limitations associated with this technique. Firstly, gene expression microarrays capture a snapshot of the cells transcriptome, detecting genes being actively transcribed at the time of RNA extraction, but they do not capture any information concerning the genes regulatory states and consequently their potential for transcriptional changes in response to stimuli. For example, a locus such as the O6methylguanine DNA methyltransferase (MGMT) gene is not prognostically useful in terms of its basal expression state [21] but the cytosine methylation status of its promoter provides an excellent indicator of how well gliomas will respond when treated by alkylating agents [22]. We hypothesize that biologically significant changes in expression can be missed by expression arrays due to technical limitations, but might be captured by epigenomic studies by identifying genes at which promoter cytosine methylation or H3K9 acetylation differ and testing them with highly-quantitative techniques. In order to test these hypotheses, we carried out genome-wide studies for DNA methylation and H3K9 acetylation as well as gene expression microarrays in patient (...truncated)