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Supplementary Figures 1-10, Tables 1-5 from CpG Island Hypermethylation in Human Astrocytomas

The base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, we investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC were depleted substantially with up to 5-fold reduction compared with normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to more than 30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1. Furthermore, immunohistochemical analysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. The data therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis. Cancer Res; 71(24); 7360–5. ©2011 AACR.

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We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

Objective Disease-associated, differentially hypermethylated regions have been reported in rheumatoid arthritis (RA), but no DNA methyltransferase inhibitors have been evaluated in either RA or any animal models of RA. The present study was conducted to evaluate the therapeutic potential of 5'-azacytidine (5'-azaC), a DNA methyltransferase inhibitor, and explore the cellular and gene regulatory networks involved in the context of autoimmune arthritis. Methods A disease-associated genome-wide DNA methylation profile was explored by methylated CpG island recovery assay-chromatin immunoprecipitation (ChIP) in arthritic B cells. Mice with proteoglycan-induced arthritis (PGIA) were treated with 5'-azaC. The effect of 5'-azaC on the pathogenesis of PGIA was explored by measuring serum IgM and IgG1 antibody levels using enzyme-linked immunosorbent assay, investigating the efficiency of class-switch recombination (CSR) and Aicda gene expression using real-time quantitative polymerase chain reaction, monitoring germinal center (GC) formation by immunohistochemistry, and determining alterations in B cell subpopulations by flow cytometry. The 5'-azaC-induced regulation of the Aicda gene was explored using RNA interference, ChIP, and luciferase assays. Results We explored arthritis-associated hypermethylated regions in mouse B cells and demonstrated that DNA demethylation had a beneficial effect on autoimmune arthritis. The 5'-azaC-mediated demethylation of the epigenetically inactivated Ahr gene resulted in suppressed expression of the Aicda gene, reduced CSR, and compromised GC formation. Ultimately, this process led to diminished IgG1 antibody production and amelioration of autoimmune arthritis in mice. Conclusion DNA hypermethylation plays a leading role in the pathogenesis of autoimmune arthritis and its targeted inhibition has therapeutic potential in arthritis management.

DNA methylation is a decisive regulator of gene expression. Differentially methylated promoters were described in rheumatoid arthritis (RA), but we do not know how these epimutations can trigger a proinflammatory cytokine milieu. B cell-focused DNA methylome studies identified a group of genes that had undergone disease-associated changes in a murine model of RA. An arthritis-specific epimutation (hypomethylation) was detected in the promoter region of the Zbtb38 gene, which encodes a transcriptional repressor. Gene expression studies revealed that hypomethylation of the Zbtb38 promoter was accompanied by disease-specific repressor expression, and two anti-inflammatory factors interleukin 1 receptor 2 gene (IL1r2) and interleukin-1 receptor antagonist (IL1rn) were among the downregulated genes. We hypothesized that Zbtb38 repressor could induce downregulated expression of these anti-inflammatory genes and that this could significantly contribute to arthritis pathogenesis. Our studies demonstrate that Zbtb38 forms a molecular bridge between an arthritis-associated epimutation (DNA hypomethylation in Zbtb38 promoter) and transcriptional silencing of the IL1r2 gene in B cells. In this way, disease-associated DNA hypomethylation can support autoimmune arthritis by interfering with an anti-inflammatory pathway.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders characterized by the chronic and progressive inflammation of various organs, most notably the synovia of joints leading to joint destruction, a shorter life expectancy, and reduced quality of life. Although we have substantial information about the pathophysiology of the disease with various groups of immune cells and soluble mediators identified to participate in the pathogenesis, several aspects of the altered immune functions and regulation in RA remain controversial. Animal models are especially useful in such scenarios. Recently research focused on IL-17 and IL-17 producing cells in various inflammatory diseases such as in RA and in different rodent models of RA. These studies provided occasionally contradictory results with IL-17 being more prominent in some of the models than in others; the findings of such experimental setups were sometimes inconclusive compared to the human data. The aim of this review is to summarize briefly the recent advancements on the role of IL-17, particularly in the different rodent models of RA.

Objective The regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis. Methods Peptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis. Results Following desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups. Conclusion This is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints. Electronic supplementary material The online version of this article (10.1186/s12974-018-1364-5) contains supplementary material, which is available to authorized users.

DNA methylation patterns are significantly reorganized during carcinogenesis, which is accompanied by altered gene expression, reduced plasticity of the transcriptional landscape, possible reactivation of transposable elements, and chromosomal instability. A number of approaches have been developed for cataloguing DNA methylation changes within the epigenome at high resolution. These techniques rely on several principles, including methylation-sensitive restriction enzymes, sodium bisulfite conversion of genomic DNA, and affinity capture with either DNA methylation-specific antibodies or proteins that bind to methylated DNA. Originally, these techniques were employed in conjunction with microarray platforms; however, high-throughput sequencing methodologies are now routinely being used. Here, we summarize and discuss several of these technologies in detail.

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most frequent cytosine modifications in mammalian DNA. Various approaches have been developed and employed to analyze DNA cytosine modification patterns in genomic DNA. These techniques are based on common principles, including methylation-sensitive restriction enzymes, sodium bisulfite conversion of genomic DNA, and affinity capture with either antibodies or proteins that bind specifically to methylated CpG sequences. For many applications, high-throughput sequencing approaches have replaced microarray platforms. These methods are useful to analyze the entire genome at various levels of resolution or can be designed to focus on the most critical genomic loci. In this chapter, we summarize and discuss several of these technologies in detail.

Objective To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors. Methods Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription–PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice. Results A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan–aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice. Conclusion Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management.

Proteoglycan (PG)-induced arthritis (PGIA) is a murine model of rheumatoid arthritis. Arthritis-prone BALB/c mice are 100% susceptible, whereas the major histocompatibility complex-matched DBA/2 strain is completely resistant to PGIA. To reduce the size of the disease-suppressive loci for sequencing and to find causative genes of arthritis, we created a set of BALB/c.DBA/ 2-congenic/subcongenic strains carrying DBA/2 genomic intervals overlapping the entire Pgia26 locus on chromosome 3 (chr3) and Pgia23/Pgia12 loci on chr19 in the arthritis-susceptible BALB/c background. Upon immunization of these subcongenic strains and their wild-type (BALB/c) littermates, we identified a major Pgia26a sublocus on chr3 that suppressed disease onset, incidence and severity via controlling the complex trait of T-cell responses. The region was reduced to 3 Mbp (11.8 Mbp with flanking regions) in size and contained gene(s) influencing the production of a number of proinflammatory cytokines. Additionally, two independent loci (Pgia26b and Pgia26c) suppressed the clinical scores of arthritis. The Pgia23 locus (~3 Mbp in size) on chr19 reduced arthritis susceptibility and onset, and the Pgia12 locus (6 Mbp) associated with low arthritis severity. Thus, we have reached the critical sizes of arthritis-associated genomic loci on mouse chr3 and chr19, which are ready for high-throughput sequencing of genomic DNA.

Summary Objective To study temporomandibular joint (TMJ) involvement in an autoimmune murine model of rheumatoid arthritis (RA), a disease characterized by inflammatory destruction of the synovial joints. Although TMJ dysfunction is frequently found in RA, TMJ involvement in RA remains unclear, and TMJ pathology has not been studied in systemic autoimmune animal models of RA. Methods Proteoglycan (PG) aggrecan-induced arthritis (PGIA) was generated in genetically susceptible BALB/c mice. TMJs and joint tissues/cartilage were harvested for histological and immunohistochemical analyses and RNA isolation for quantitative polymerase chain-reaction. Serum cytokine levels were measured in mice with acute or chronic arthritis, and in non-arthritic control animals. Results Despite the development of destructive synovitis in the limbs, little or no synovial inflammation was found in the TMJs of mice with PGIA. However, the TMJs of arthritic mice showed evidence of aggrecanase- and matrix metalloproteinase-mediated loss of glycosaminoglycan-containing aggrecan, and in the most severe cases, structural damage of cartilage. Serum levels of pro-inflammatory cytokines, including interleukin (IL)-1β, were elevated in arthritic animals. Expression of the IL-1β gene was also high in the inflamed limbs, but essentially normal in the TMJs. Local expression of genes encoding matrix-degrading enzymes (aggrecanases and stromelysin) was upregulated to a similar degree in both the limbs and the TMJs. Conclusion We propose that constantly elevated levels of catabolic cytokines, such as IL-1β, in the circulation (released from inflamed joints) create a pro-inflammatory milieu within the TMJ, causing local upregulation of proteolytic enzymes and subsequent loss of aggrecan from cartilage.

Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.—Nguyen, A., Rauch, T. A., Pfeifer, G. P., Hu, V. W. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain.

De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX , LHX , PAX , DLX , and Engrailed , were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7 - and HOXA9 -associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type–dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms. (Cancer Res 2006; 66(16): 7939-47)

DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters.

Germline mutations in the BRCA1 tumor suppressor gene contribute to familial breast and ovarian tumor formation. Sporadic breast and ovarian cancer, however, which accounts for more than 90% of total cases and virtually lacks BRCA1 mutations, exhibits reduced expression of the BRCA1 gene. The magnitude of this reduction correlates with disease progression. In this report we have identified an imperfect palindrome sequence for binding of the 53BP1-containing complex, -40TTCCGTGG CAACGGAA-25, within the BRCA1 minimal promoter. Over expression of 53BP1 activates a luciferase reporter driven by the wild type BRCA1 minimal promoter, but not by the BRCA1 minimal promoter with mutated palindrome sequence. Depletion of endogenous 53BP1 by siRNA suppresses activity of the BRCA1 minimal promoter. In vitro and in vivo DNA-protein interaction studies demonstrate that this palindrome sequence binds to the 53BP1-containing complex. These findings establish a positive regulation of the BRCA1 promoter by 53BP1. Disruption of this regulation in cancer cells may provide a molecular mechanistic basis for sporadic breast and ovarian tumor formation.

Matrilin-1 is a non-collagenous protein, which functions in the organization of the extracellular matrix by forming collagen-dependent and -independent filamentous networks. It is secreted primarily by chondrocytes in a characteristic spatial, temporal and developmental stage-specific pattern during skeletogenesis. As a first step to define the tissue- and site-specific regulatory regions of the chicken matrilin-1 gene in vivo, we generated transgenic mice harboring various promoter and intronic fragments fused to the LacZ reporter gene. Histological analysis of the transgene expression pattern during ontogenic development revealed specific X-gal staining in most primordial elements of endochondral bones of transgenic mouse lines carrying either the long promoter between -2011 and +67 or the intronic fragment with a short promoter between -338 and +1819. The cartilage-specific activity of the latter transgene, however, was accompanied with variable ectopic expression pattern in neural and other tissues depending on the site of integration. The presence of both promoter upstream and intronic elements was necessary for the high level transgene activity in all chondrogenic tissues and for the extraskeletal transgene expression pattern resembling the most to that of the chicken matrilin-1 gene, e.g. expression in the eye, and lack of expression in the diminishing notochord and nucleus pulposus. The activity of the transgenes was restricted to the columnar proliferating and pre-hypertrophic chondrocytes visualized by BrdU incorporation and distribution of phosphorylated Sox9, respectively. DNA elements between -2011 and -338 also mediated ectopic LacZ expression in cells of neural crest origin. These results suggest that an interplay of modularly arranged cartilage- and neural crest-specific DNA elements control the expression of the matrilin-1 gene. The dispersal of cartilage-specific elements in the promoter upstream and intronic regions shows similarity to the transcriptional regulation of the Col11a2 gene.

The mature mRNA always carries nucleotide sequences that faithfully mirror the protein product according to the rules of the genetic code. However, in the chromosome, the nucleotide sequence that represents a certain protein is interrupted by additional sequences. Therefore, most eukaryotic genes are longer than their final mRNA products. The human genome project revealed that only a tiny portion of sequences serves as protein-coding region and almost one quarter of the genome is occupied by non-coding intervening sequences. The elimination of these non-coding regions from the precursor RNA in a process termed splicing must be extremely precise, because even a single nucleotide mistake may cause a fatal error. At present, two types of intervening sequences have been identified in protein-coding genes. One of them, the U2-dependent or major-class is prevalent and represents 99% of known sequences. The other one, the so-called U12-dependent or minor-class of introns, occurs in much lesser amounts in the genome. The basic problem of nuclear splicing concerns i/ the molecular mechanisms, which ensure that the coding regions are correctly recognized and spliced together; ii/ the principles and mechanisms that guarantee the high fidelity of the splicing system; iii/ the differences in the excision mechanisms of the two classes of introns. We are going to present models explaining how intervening sequences are accurately removed and the coding regions correctly juxtaposed. The two splicing mechanisms will also be compared.

Alterations in DNA methylation patterns occur in every type of human cancer and are considered a hallmark of malignant transformation. Most notable is the cancer-associated hypermethylation of CpG-rich sequences, the so-called CpG islands, which are often found near the 5' ends and promoters of genes. This CpG island methylation represents a positive signal that can be used to distinguish malignant tissue from normal tissue. Thus, characterization of CpG island hypermethylation has become a valuable tool for cancer detection and diagnosis. There are several methods used for detection of gene-specific DNA methylation. However, besides looking at individual genes, an even greater potential lies in the characterization of genome-wide changes of DNA methylation patterns in tumors. The authors propose that tumor type- and tumor subtype-specific DNA methylation patterns exist and can be exploited for the classification of cancers, their response to therapy and their metastatic potential, and thus may have predictive value. Various methods for genome-wide analysis of DNA methylation have been developed. These methods are described briefly and the methylated-CpG island recovery assay will be reviewed. This assay has been used in combination with microarray analysis to map CpG island methylation across cancer genomes.

Cartilage matrix protein (CMP) is synthesized by chondrocytes in a developmentally regulated manner. Here we have dissected promoter upstream elements involved in its transcriptional regulation. We show that although the 79-base pair CMP minimal promoter is promiscuous, 1137 base pairs of 5'-flanking region are capable of directing tissue- and developmental stage-specific transcription when fused to a reporter gene. This results from two positive control regions which, in proliferating chondrocytes, relieve the repression mediated by two non-tissue-specific negative control regions. Characterization of the promoter proximal silencer by DNase I footprinting and gel shifts revealed the presence of two elements, SI and SII, which bound mesenchymal cell proteins. Methylation interference analysis indicated a gapped palindromic binding site similar to nuclear factor I (NF-I) family proteins within SI, but only a half-site within SII. Gel shift assays with specific NF-I and mutated SI competitors, binding of recombinant NF-I, as well as supershift analysis with NF-I-specific antiserum verified the binding of NF-I family proteins to the SI element. Double-stranded SI and SII oligonucleotides inserted in single copy in either orientation were found to repress both homologous and heterologous promoters upon transfection into mesenchymal cells. Transcriptional repression also occurred when a consensus NF-I site itself was fused to the CMP minimal promoter. We conclude that NF-I-related protein(s) can mediate transcriptional repression in cells of mesenchymal origin.

Summary T cell receptor transgenic (TCR-Tg) mice specific for the arthritogenic 5/4E8 epitope in the G1 domain of cartilage proteoglycan were generated and back-crossed into arthritis-prone BALB/c background. Although more than 90% of CD4+ T cells of all TCR-Tg lines were 5/4E8-specific, one (TCR-TgA) was highly sensitive to G1-induced or spontaneous arthritis, while another (TCR-TgB) was less susceptible. Here we studied whether fine differences in TCR signalling controlled the onset and severity of arthritis. Mice from the two TCR-Tg lines were immunized side by side with purified recombinant human G1 (rhG1) domain for G1 domain of cartilage proteoglycan (PG)-induced arthritis (GIA). TCR-TgA mice developed severe and early-onset arthritis, whereas TCR-TgB mice developed weaker arthritis with delayed onset, although TCR-TgB CD4+ T cells expressed approximately twice more TCR-Vβ4 chain protein. The more severe arthritis in TCR-TgA mice was associated with higher amounts of anti-G1 domain-specific antibodies, larger numbers of B cells and activated T helper cells. Importantly, TCR-TgB CD4+ T cells were more sensitive to in vitro activation-induced apoptosis, correlating with their higher TCR and CD3 expression and with the increased TCR signal strength. These findings indicate that TCR signal strength determines the clinical outcome of arthritis induction: ‘optimal’ TCR signal strength leads to strong T cell activation and severe arthritis in TCR-TgA mice, whereas ‘supra-optimal’ TCR signal leads to enhanced elimination of self-reactive T cells, resulting in attenuated disease.

Lung carcinomas have been the leading causes of cancer-associated death in both sexes worldwide. Epidemiological studies demonstrated that cigarette smoking and other environmental factors such as radon or asbestos can also contribute to lung cancer development. Environmental chemicals can promote cancer formation by causing lesions in genomic DNA and/or by inducing various alterations in the epigenomes of cells lining the airways. Epithelial cells whose epigenome has significantly become altered or damaged can become transformed (i.e., reprogrammed) and start to exhibit malignant properties. With the advent of the postgenomic era and its advanced methodological arsenal, genome-wide analysis tools have become available that have contributed to the identification of the involved epigenetic factors and map disease-associated epigenome profile changes. These studies proved that DNA methylation, posttranslational modification of histones, and noncoding RNAs are also involved in lung cancer development. Coordinated epigenetic and genetic studies can provide new insights into the etiology of lung carcinomas, and perhaps extend the currently used diagnostic and therapeutic arsenal for cancer patients.

The base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, we investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC were depleted substantially with up to 5-fold reduction compared with normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to more than 30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1. Furthermore, immunohistochemical analysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. The data therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis. Cancer Res; 71(24); 7360–5. ©2011 AACR.

Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n=8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.

To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.

Publisher Summary This chapter discusses the more commonly used genome-scale methods for DNA methylation analysis. DNA methylation was the first epigenetic modification. To explore DNA methylation profiles at genome scale, a wide range of approaches have been developed. Most of the methods were originally used for detecting methylation changes at the single gene level but by coupling them with extensive cloning and sequencing work or combining them with microarray platforms, genome-wide analysis tools have been developed. Most methylation analysis methods can be categorized into several well-characterized groups on the basis of their principles. Several methods are based on restriction endonucleases that possess altered sensitivity towards methylated cytosine residues present in the cleavage site. In this way, the restriction endonuclease digestion pattern depends on the methylation status of the cleavage sites and ultimately reflects methylation profiles of the given chromosomal region. Techniques are based on antibodies or proteins that bind to methylated DNA. Resolution at the single nucleotide level often requires bisulfite-sequencing approaches for which high throughput techniques are currently being developed.

We have found a B2 repeat insertion in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6) in a mouse that developed a skin disorder with clinical and histopathological features resembling those seen in human neutrophilic dermatoses. Neutrophilic dermatoses are a group of complex heterogeneous autoinflammatory diseases that all demonstrate excessive neutrophil infiltration of the skin. Therefore, we tested the cDNA and genomic DNA sequences of PTPN6 from patients with Sweet's syndrome (SW) and pyoderma gangrenosum and found numerous novel splice variants in different combinations. Isoforms resulting from deletions of exons 2, 5, 11, and 15 and retention of intron 1 or 5 were the most common in a patients with a familial case of SW, who had a neonatal onset of an inflammatory disorder with skin lesions and a biopsy specimen consistent with SW. These isoforms were associated with a heterozygous E441G mutation and a heterozygous 1.7-kbp deletion in the promoter region of the PTPN6 gene. Although full-length PTPN6 was detected in all other patients with either pyoderma gangrenosum or SW, it was always associated with splice variants: a partial deletion of exon 4 with the complete deletion of exon 5, alterations that were not detected in healthy controls. The defect in transcriptional regulation of the hematopoietic PTPN6 appears to be involved in the pathogenesis of certain subsets of the heterogeneous group of neutrophilic dermatoses.

Astrocytomas are common and lethal human brain tumors. We have analyzed the methylation status of over 28,000 CpG islands and 18,000 promoters in normal human brain and in astrocytomas of various grades using the methylated CpG island recovery assay. We identified 6,000 to 7,000 methylated CpG islands in normal human brain. Approximately 5% of the promoter-associated CpG islands in the normal brain are methylated. Promoter CpG island methylation is inversely correlated whereas intragenic methylation is directly correlated with gene expression levels in brain tissue. In astrocytomas, several hundred CpG islands undergo specific hypermethylation relative to normal brain with 428 methylation peaks common to more than 25% of the tumors. Genes involved in brain development and neuronal differentiation, such as BMP4, POU4F3, GDNF, OTX2, NEFM, CNTN4, OTP, SIM1, FYN, EN1, CHAT, GSX2, NKX6-1, PAX6, RAX, and DLX2, were strongly enriched among genes frequently methylated in tumors. There was an overrepresentation of homeobox genes and 31% of the most commonly methylated genes represent targets of the Polycomb complex. We identified several chromosomal loci in which many (sometimes more than 20) consecutive CpG islands were hypermethylated in tumors. Seven such loci were near homeobox genes, including the HOXC and HOXD clusters, and the BARHL2, DLX1, and PITX2 genes. Two other clusters of hypermethylated islands were at sequences of recent gene duplication events. Our analysis offers mechanistic insights into brain neoplasia suggesting that methylation of the genes involved in neuronal differentiation, in cooperation with other oncogenic events, may shift the balance from regulated differentiation towards gliomagenesis. Cancer Res; 70(7); 2718–27

The methylated-CpG island recovery assay (MIRA) exploits the intrinsic specificity and the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to methylated CpG dinucleotides in genomic DNA. The MIRA approach works on double-stranded DNA and does not depend on the application of methylation-sensitive restriction enzymes. It can be performed on a few hundred nanograms of genomic DNA. Recently, the MIRA technique has been used to profile DNA methylation patterns at a resolution of 100 base pairs along the entire genome of normal human B-lymphocytes. The MIRA method is compatible with microarray and next generation DNA sequencing approaches. We describe the principles and details of this method applied for methylation profiling of genomes containing methylated CpG sequences.

The following information was missing from the Funding section: Additional funding was provided by NIH grant 5R01DK081587-01.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Using a methylated-DNA enrichment technique (methylated CpG island recovery assay, MIRA) in combination with whole-genome tiling arrays, we have characterized by MIRA-chip the entire B cell “methylome” of an individual human at 100-bp resolution. We find that at the chromosome level high CpG methylation density is correlated with subtelomeric regions and Giemsa-light bands (R bands). The majority of the most highly methylated regions that could be identified on the tiling arrays were associated with genes. Approximately 10% of all promoters in B cells were found to be methylated, and this methylation correlates with low gene expression. Notably, apparent exceptions to this correlation were the result of transcription from previously unidentified, unmethylated transcription start sites, suggesting that methylation may control alternate promoter usage. Methylation of intragenic (gene body) sequences was found to correlate with increased, not decreased, transcription, and a methylated region near the 3′ end was found in approximately 12% of all genes. The majority of broad regions (10–44 kb) of high methylation were at segmental duplications. Our data provide a valuable resource for the analysis of CpG methylation patterns in a differentiated human cell type and provide new clues regarding the function of mammalian DNA methylation.

DNA methylation patterns are often altered in human cancer and aberrant methylation is considered a hallmark of malignant transformation. Several methods have been developed for the characterization of gene-specific and genome-wide DNA methylation patterns. In this chapter, we describe the methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2b/MBD3L1 complex for double-stranded CpG-methylated DNA. MIRA has been used in combination with microarray platforms to map DNA methylation patterns across the human genome.

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80–100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription.

The complete nucleotide sequences of two parvoviruses isolated from goose and muscovy duck were determined. The two virus genomes share 81.9% nucleotide sequence identity, indicating that they are closely related. The coding regions are bracketed by inverted terminal repeats containing palindromes. This is similar to the genome organization of human pervoviruses, adeno-associated virus 2, and B19. Amino acid sequence comparison shows that the closest relative of the goose and muscovy duck pervoviruses is adeno-associated virus 2. This is surprising, because the goose and muscovy duck parvoviruses do not require any helper virus for productive replication, suggesting that adeno-associated virus 2 has been derived from a helper-independent ancestor.

Genomic imprints—parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes—are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP–SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF–dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia., Author Summary Allele-specific DNA methylation is considered the primary mark that distinguishes the parental alleles of imprinted genes. Whereas allele-specific chromatin also exists at imprinted genes in the soma, this has not been assessed in the germ line. It will be important to understand what extent the chromatin composition provides clues in the germ line to the erasure and establishment of methylation imprints. Our novel methods provide the sensitivity required to answer these questions. Our results argue that the erasure of the DNA methylation imprint is complete before, and therefore does not depend on, the erasure of allele-specific chromatin marks at the H19/Igf2 imprint control region. Additionally, we show that incomplete erasure of the allele-specific chromatin is responsible for the delayed DNA methylation imprint establishment of the maternal ICR allele in prospermatogonia. The chromatin bias—the transient epigenetic memory of the mother—in fetal germ cells depends on functional CTCF insulator binding sites in this imprint control region.