We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R2 of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.

We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.

Although mantle cell lymphoma (MCL) frequently harbors inactivated ataxia telangiectasia mutated (ATM) and p53 alleles, little is known about the molecular phenotypes caused by these genetic changes. We identified point mutations and genomic deletions in these genes in a series of cyclin D1-positive MCL cases and correlated genotype with gene expression profiles and overall survival. Mutated and͞or deleted ATM and p53 alleles were found in 56% (40͞72) and 26% (21͞82) of the cases examined, respectively. Although MCL patients with inactive p53 alleles showed a significant reduction in median overall survival, aberrant ATM status did not predict for survival. Nevertheless, specific gene expression signatures indicative of the mutation and genomic deletion status of each gene were identified that were different from wild-type cases. These signatures were comprised of a select group of genes related to apoptosis, stress responses, and cell cycle regulation that are relevant to ATM or p53 function. Importantly, we found the molecular signatures are different between cases with mutations and deletions, because the latter are characterized by loss of genes colocalized in the same chromosome region of ATM or p53. This information on molecular phenotypes may provide new areas of investigation for ATM function or may be exploited by designing specific therapies for MCL cases with p53 aberrations.cancer ͉ cell cycle ͉ genetics ͉ microarray ͉ signature M antle cell lymphoma (MCL) is an aggressive tumor that accounts for Ϸ6% of all non-Hodgkin lymphoma cases in the U.S., with higher rates in North America (1, 2). Although the median survival of MCL patients is only 3 years, some individuals survive Ͼ10 years from the time of diagnosis (2, 3). There is considerable interest in defining the molecular basis for this clinical heterogeneity to develop better prognostic markers and more effective therapies.MCL corresponds to B cells of the mantle zone of the lymphoid follicles that have acquired distinctive alterations in genes related to cell cycle control and apoptosis (4). The hallmark of these genetic alterations is the t(11;14)(q13;q32) translocation that juxtaposes the IGH locus near the CCND1 gene, resulting in the overexpression of cyclin D1 (5). A subset of MCL cases acquire p53 mutations, and these patients have a significantly shortened median survival relative to cases with wild-type p53 (6-8). Interestingly, the ataxia telangiectasia mutated (ATM) gene, whose product regulates some p53-dependent apoptosis pathways, is mutated or deleted in 25-40% of MCL cases (reviewed in refs. 9 and 10). Although preliminary studies suggest that ATM mutation status does not have a significant impact on patient survival (7, 11), they may have lacked the statistical power to identify more subtle effects on survival, such as the effect of functional subsets of mutations.We determined the ATM and p53 genotypes in a large cohort of MCL cases with previous gene expression profiles to further elucidate the relationship between molecular phe...

Although much is known about genetic variation in human and African great ape (chimpanzee, bonobo, and gorilla) genomes, substantially less is known about variation in gene-expression profiles within and among these species. This information is necessary for defining transcriptional regulatory networks that contribute to complex phenotypes unique to humans or the African great apes. We took a systematic approach to this problem by investigating gene-expression profiles in well-defined cell populations from humans, bonobos, and gorillas. By comparing these profiles from 18 human and 21 African great ape primary fibroblast cell lines, we found that gene-expression patterns could predict the species, but not the age, of the fibroblast donor. Several differentially expressed genes among human and African great ape fibroblasts involved the extracellular matrix, metabolic pathways, signal transduction, stress responses, as well as inherited overgrowth and neurological disorders. These gene-expression patterns could represent molecular adaptations that influenced the development of species-specific traits in humans and the African great apes.

Glucocorticoid (GC) treatment for the management of autoimmune and inflammatory diseases is associated with decreased bone formation and increased risk for fracture. In MC3T3-E1 cell cultures, 0·1 -1 µM dexamethasone (DEX) arrests development of the osteoblast phenotype when administration commences at a commitment stage around the time of confluency. To gain new insights into GC-induced osteoporosis, we performed microarray-based gene expression analysis of GC-arrested MC3T3-E1 cultures, 2·5 days after the administration of DEX. Of the .12 000 transcripts interrogated, 74 were up-regulated and 17 were down-regulated by at least 2·5-fold (P¯0·05). Some of these genes, such as Mmp13, Serum/GC-regulated kinase and Tieg, have previously been reported as GC-responsive. Others are shown here for the first time to respond to GCs. DEX strongly repressed Krox20/Egr2 at both the mRNA and the protein level. This is especially significant because mice lacking this transcription factor develop osteoporosis. The data also suggest that the bone morphogenetic protein (BMP) pathway, which is involved in regulating bone mass, and other pathways that influence BMP signaling, are abrogated by GCs: (i) DEX increased the mRNA levels of the BMP antagonists Follistatin and Dan; (ii) DEX increased the levels of p21 Rasgap3 and Ptpn16/MKP-1 mRNAs, negative regulators of the MAP kinase pathway; and (iii) DEX decreased Cox mRNA levels. DEX also increased thrombospondin mRNA levels, which negatively regulate bone mass in vivo, as well as the adipocytic marker Fkbp51. These and other observations disclose novel gene targets, whose regulation by GCs in osteoblasts may shed light on and provide new therapeutic approaches to osteoporosis.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

To gain a better understanding of the mechanism of action of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer. (Cancer Res 2005; 65(9): 3837-45)

Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5′ untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5′ UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5′ UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.