Srimoyee Ghosh

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Me thylated D NA I mmunoP recipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify T issue and D evelopmental S tage specific D ifferentially M ethylated DNA R egions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues

Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of twelve human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.

Identification of Novel Methylation Markers in Cervical Cancer Using Restriction Landmark Genomic Scanning

Aberrant methylation of CpG islands in gene promoters often represents an early clonal event in carcinogenesis. Accordingly, defining methylation profiles may be useful for developing marker panels for early detection or predicting the risk of cancer precursors. To identify specific genes frequently methylated in cervical cancer, we conducted methylation profiling of 20 primary human cervical cancers using NotI-based restriction landmark genomic scanning (RLGS). Of 2,172 RLGS fragments analyzed (average, 1,753 CpG islands per patient), 186 RLGS fragments were lost in at least one tumor and 40 were lost in three or more. Methylation was identified in 19 (95%) of 20 tumor samples compared with normal DNA. Bisulfite sequencing was conducted to confirm RLGS results. Of the confirmed markers frequently methylated, we developed Methylight assays for two corresponding genes, nucleolar protein 4 (NOL4), and lipoma HMGIC fusion partner-like protein 4 (LHFPL4), which were methylated in 85% and 55% of cancers, respectively. Using these assays, we further confirmed frequent CpG island methylation in the original cancers and in another independent series of 15 cervical cancers. We also showed methylation at a reduced frequency in a set of carefully reviewed cytology specimens demonstrating cells exfoliated from cancer precursor lesions. In summary, we identified, for the first time, NOL4 and LHFPL4 as novel methylation targets specific for cervical cancer. Inclusion of NOL4 and LHFPL4 in evaluating methylation panels for early detection, risk prediction, and etiologic research on cervical cancer is warranted.

Epigenetics: differential DNA methylation in mammalian somatic tissues

Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally conserved mechanism to regulate gene expression in mammals. Because DNA methyation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi‐stable changes in gene expression in somatic tissues. Although it remains controversial whether germ‐line DNA methylation in mammalian genomes is stably heritable, frequent tissue‐specific and disease‐specific de novo methylation events are observed during somatic cell development/differentiation. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylated regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL/6J mouse tissues. Comparative analysis between mice and humans suggests that some, but not all, tissue‐specific differentially methylated regions are conserved. A deeper understanding of cell‐type‐specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue‐specific differentiation in mammals.

Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.

Identification and analysis of an early diagnostic marker for malignant melanoma: ZAR1 intra-genic differential methylation

BACKGROUND Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma. OBJECTIVE To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma. METHODS The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed. RESULTS The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines. CONCLUSION Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.

Genome‐wide screening of aberrant DNA methylation which associated with gene expression in mouse skin cancers

Epigenetic alteration of genomic DNA is a common and key process in carcinogenesis. There is considerable evidence indicating that some of the somatic alterations occurring during carcinogenesis in humans also involve the same processes as those observed in mice. Therefore, we analyzed mouse skin cancer tissues induced by the 2‐stage carcinogenesis model to identify skin tumor‐specific differentially methylated regions (ST‐DMRs) during the multistep carcinogenesis process. We have previously identified ST‐DMRs using the restriction landmark genomic scanning (RLGS) technique and reported that some of the mouse ST‐DMRs were also epigenetically modified in human cancers, such as melanoma, neuroblastoma, and brain tumor. These results encouraged us to pursue global methylation screening in mouse skin carcinogenesis. Using the methylated DNA immunoprecipitation (MeDIP) method combined with the NimbleGen promoter plus CpG island (CpGi) array, we identified 615 ST‐DMRs. In combination with global gene expression analysis, 91 of these ST‐DMRs were shown to be located on or around the genes differentially expressed between normal skin and tumor tissues, including a candidate human tumor suppressor gene Tfap2e. As observed in human colorectal cancers, Tfap2e was methylated at a CpGi located in intron 3 and downregulated in skin tumors. Our results identified aberrant methylated regions that were associated with gene expression regulation during carcinogenesis, which may indicate critical genetic regions also involved in human carcinogenesis.

Nr4a3, a possibile oncogenic factor for neuroblastoma associated with CpGi methylation within the third exon

Aberrant methylation of Nr4a3 exon 3 CpG island (CpGi) was initially identified during multistep mouse skin carcinogenesis. Nr4a3 is also known as a critical gene for neuronal development. Thus, we examined the Nr4a3 exon 3 CpGi methylation in mouse brain tissues from 15-day embryos, newborns and 12-week-old adults and found significant increase of its methylation and Nr4a3 expression during mouse brain development after birth. In addition, homologous region in human genome was frequently and aberrantly methylated in neuroblastoma specimens. A quantitative analysis of DNA methylation revealed that hypomethylation of CpG islands on NR4A3 exon 3, but not on exon 1 was identified in three neuroblastomas compared with matched adrenal glands. Additional analysis for 20 neuroblastoma patients was performed and 8 of 20 showed hypomethylation of the CpGi on NR4A3 exon 3. The survival rate of those 8 patients was significantly lower compared with those in patients with hypermethylation. Immunohistochemical NR4A3 expression was generally faint in neuroblastoma tissues compared with normal tissues. Moreover, the MYCN amplified NB9 cell line showed hypomethylation and low expression of NR4A3, while the non-MYCN amplified NB69 cell line showed hypermethylation and high expression. These results indicate that DNA hypomethylation of the CpGi at NR4A3 exon 3 is associated with low NR4A3 expression, and correlates with poor prognosis of neuroblastoma. Since NR4A3 upregulation associated with the hypermethylation and neuronal differentiation in mice, poor prognosis of neuroblastoma associated with NR4A3 low expression may be partly explained by dysregulation of its differentiation.

Identification of aberrant methylation regions in neuroblastoma by screening of tissue-specific differentially methylated regions†‡§

The identification of tissue‐specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process.

Whole genome DNA methylation profiling of oral cancer in ethnic population of Meghalaya, North East India reveals novel genes

Oral Squamous Cell Carcinoma (OSCC) is a serious and one of the most common and highly aggressive malignancies. Epigenetic factors such as DNA methylation have been known to be implicated in a number of cancer etiologies. The main objective of this study was to investigate physiognomies of Promoter DNA methylation patterns associated with oral cancer epigenome with special reference to the ethnic population of Meghalaya, North East India. The present study identifies 27,205 CpG sites and 3811 regions that are differentially methylated in oral cancer when compared to matched normal. 45 genes were found to be differentially methylated within the promoter region, of which 38 were hypermethylated and 7 hypomethylated. 14 of the hypermethylated genes were found to be similar to that of the TCGA-HNSCC study some of which are TSGs and few novel genes which may serve as candidate methylation biomarkers for OSCC in this poorly characterized ethnic group.