Browsing by Subject "DNA Methylation"

Now showing 1 - 10 of 13
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    Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation
    (American Society for Clinical Investigation, 2016-05-19) Maeshima, Keisuke; Stanford, Stephanie M.; Hammaker, Deepa; Sacchetti, Cristiano; Zeng, Li-Fan; Ai, Rizi; Zhang, Vida; Boyle, David L.; Aleman Muench, German R.; Feng, Gen-Sheng; Whitaker, John W.; Zhang, Zhong-Yin; Wang, Wei; Bottini, Nunzio; Firestein, Gary S.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.
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    Analysis of differentiation capacity of Cfp1 null embyronic stem cells
    (2014) Bowen, Tamara R.; Skalnik, David Gordon; Marrs, James; Chang, Hua-Chen
    Epigenetics is defined as “the study of stable, often heritable, changes that influence gene expression that are not mediated by DNA sequence” (Fingerman et al., 2013). Epigenetic marks such as covalent histone modifications and DNA methylation are important for maintaining chromatin structure and epigenetic inheritance. Several proteins have been found to bind and/ or regulate epigenetic marks. One such protein, CXXC finger protein 1 (Cfp1) is an important chromatin regulator that binds to unmethylated CpG islands. It has been found to be essential for mammalian development. Mice lacking Cfp1 exhibit an embryonic- lethal phenotype. However, the function of Cfp1 can be studied using Cfp1 Null mouse ES cells, which are viable. Thus far, Cfp1 has been shown to be important for cell growth, cytosine methylation, histone modifications, subnuclear localization of Set1A histone H3K4 methyltransferase, and cellular differentiation. When Cfp1 Null ES cells are induced to differentiate by removal of Leukemia Inhibitory Factor (LIF), the cells are not able to turn off pluripotency markers such as Oct4 and alkaline phosphatase and fail to express differentiation markers such as Gata4 and Brachyury. In this study, we used established protocols to further examine the differentiation capacity of Cfp1 Null cells. Specifically, we tested the ability of Cfp1 Null ES cells to retain stem cell properties in the absence of LIF, differentiate into cardiomyocytes in the presence of TGF-β2 and differentiate into neuron precursors in the presence of retinoic acid (RA). While the differentiation effects of RA were inconclusive, Null cells were able to start differentiating in the absence of LIF, either as individual cells or EBs, and the presence of TGF-β2 when seeded on gelatin coated tissue culture dishes. However, no difference was seen between cells treated without LIF and those treated with TGF-β2. In both conditions, only a small portion of cells were able to differentiate, while the majority of the cell population retained stem cell characteristics. Cell growth and the differentiation capacity of Cfp1 Null cells were also compromised in comparison to WT cells. Thus, further supporting the need for the correct epigenetic patterns maintained by Cfp1 during cellular differentiation.
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    Comparing Nanopore to MethylationEPIC Array and EM-Seq in DNA Methylation Detection
    (2024-12) Brooks, Steven; Liu, Yunlong; Peng, Gang; Zhang, Pengyue
    DNA Methylation is an important biological process in epigenetics, and many methods have been developed to profile DNA methylation. Recently a growing number of studies use Nanopore long-read sequencing technology in DNA methylation detection, in contrast to widely used Infinium arrays and short-read whole genome sequencing (WGS) methods. In this study, we evaluate the performance of Nanopore sequencing in DNA methylation detection by comparing it to the Illumina MethylationEPIC microarray (EPIC) and Enzymatic Methyl-Sequencing. We first compare Oxford Nanopore Technologies’ Nanopore with MethylationEPIC array. Among the ~850,000 CpG sites covered by both methods, we observed high concordance (R ≥ 0.94 across all four samples). After downsampling Nanopore data from an average coverage of 26.6 reads per site to 10 reads per site, the correlation in CpG methylation remained high (R≥ 0.935). Next, we compare Nanopore with EM-Seq in the context of low coverage. The lower CpG methylation correlation (R ≥ 0.8), can be attributed to reduced coverage of hypomethylated CpG sites by EM-Seq. Furthermore, we highlight Nanopore’s unique capabilities, including native DNA sequencing that can differentiate modification types and the use of long reads for haplotype phasing. Overall, Nanopore demonstrated high concordance with the EPIC array and more uniform coverage across the genome than EM-Seq. This study provides insights for researchers in selecting appropriate DNA methylation detection methods, considering factors such as cost, DNA input, and the complexity of downstream analysis.
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    Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes
    (American Diabetes Association, 2015-11) Fisher, Marisa M.; Watkins, Renecia A.; Blum, Janice; Evans-Molina, Carmella; Chalasani, Naga; DiMeglio, Linda A.; Mather, Kieren J.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Department of Pediatrics, School of Medicine
    Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease.
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    Methylation array data can simultaneously identify individuals and convey protected health information: an unrecognized ethical concern
    (Springer (Biomed Central Ltd.), 2014) Philibert, Robert A.; Terry, Nicolas P.; Erwin, Cheryl; Philibert, Winter J.; Beach, Steven Rh; Brody, Gene H.; School of Law
    BACKGROUND: Genome-wide methylation arrays are increasingly used tools in studies of complex medical disorders. Because of their expense and potential utility to the scientific community, current federal policy dictates that data from these arrays, like those from genome-wide genotyping arrays, be deposited in publicly available databases. Unlike the genotyping information, access to the expression data is not restricted. An underlying supposition in the current nonrestricted access to methylation data is the belief that protected health and personal identifying information cannot be simultaneously extracted from these arrays. RESULTS: In this communication, we analyze methylation data from the Illumina HumanMethylation450 array and show that genotype at 1,069 highly informative loci, and both alcohol and smoking consumption information, can be derived from the array data. CONCLUSIONS: We conclude that both potentially personally identifying information and substance-use histories can be simultaneously derived from methylation array data. Because access to genetic information about a database subject or one of their relatives is critical to the de-identification process, this risk of de-identification is limited at the current time. We propose that access to genome-wide methylation data be restricted to institutionally approved investigators who accede to data use agreements prohibiting re-identification.
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    Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis
    (American Association for Cancer Research, 2017-07-01) Maiuri, Ashley R.; Peng, Michael; Sriramkumar, Shruthi; Kamplain, Caitlin M.; DeStefano Shields, Christina E.; Sears, Cynthia L.; O’Hagan, Heather M.; Medicine, School of Medicine
    Aberrant silencing of genes by DNA methylation contributes to cancer, yet how this process is initiated remains unclear. Using a murine model of inflammation-induced tumorigenesis, we tested the hypothesis that inflammation promotes recruitment of epigenetic proteins to chromatin, initiating methylation and gene silencing in tumors. Compared with normal epithelium and noninflammation-induced tumors, inflammation-induced tumors gained DNA methylation at CpG islands, some of which are associated with putative tumor suppressor genes. Hypermethylated genes exhibited enrichment of repressive chromatin marks and reduced expression prior to tumorigenesis, at a time point coinciding with peak levels of inflammation-associated DNA damage. Loss of MutS homolog 2 (MSH2), a mismatch repair (MMR) protein, abrogated early inflammation-induced epigenetic alterations and DNA hypermethylation alterations observed in inflammation-induced tumors. These results indicate that early epigenetic alterations initiated by inflammation and MMR proteins lead to gene silencing during tumorigenesis, revealing a novel mechanism of epigenetic alterations in inflammation-driven cancer. Understanding such mechanisms will inform development of pharmacotherapies to reduce carcinogenesis.
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    The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer
    (American Association for Cancer Research, 2014-12-15) Fang, Fang; Munck, Joanne; Tang, Jessica; Taverna, Pietro; Wang, Yinu; Miller, David F. B.; Pilrose, Jay; Choy, Gavin; Azab, Mohammad; Pawelczak, Katherine S.; VanderVere-Carozza, Pamela; Wagner, Michael; Lyons, John; Matei, Daniela; Turchi, John J.; Nephew, Kenneth P.; Department of Medicine, IU School of Medicine
    PURPOSE: To investigate SGI-110 as a "chemosensitizer" in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. EXPERIMENTAL DESIGN: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. RESULTS: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. CONCLUSIONS: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting.
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    Persistent Changes in Stress-Regulatory Genes in Pregnant Women or Children Exposed Prenatally to Alcohol
    (Wiley, 2019-07-22) Sarkar, Dipak K.; Gangisetty, Omkaram; Wozniak, Jeffrey R.; Eckerle, Judith K.; Georgieff, Michael K.; Foroud, Tatiana M.; Wetherill, Leah; Wertelecki, Wladimir; Chambers, Christina D.; Riley, Edward; Zymak-Zakutnya, Natalya; Yevtushok, Lyubov; Medical and Molecular Genetics, School of Medicine
    Background: We have recently shown that binge or heavy levels of alcohol drinking increases DNA methylation and reduces gene expression of POMC and PER2 in adult human subjects (Gangisetty et al., 2019). One hypothesis would be that methylation of these two genes is consistently associated with alcohol exposure and could be used as biomarkers to predict risk of PAE. Results of the present study provided some support for this hypothesis. Methods: We conducted a series of studies to determine DNA methylation changes in stress regulatory genes proopiomelanocortin (POMC) and period 2 (PER2) using biological samples from three separate cohorts of patients i) pregnant women who consumed moderate to high levels of alcohol or low/unexposed controls, ii) children with PAE and non-alcohol exposed controls, and iii) children with PAE treated with or without choline. Results: We found pregnant women who consumed moderate to high levels of alcohol and gave birth to PAE children had higher DNA methylation of POMC and PER2. PAE children also had increased methylation of POMC and PER2. The differences in the gene methylation of PER2 and POMC between PAE and controls did not differ by maternal smoking status. PAE children had increased levels of stress hormone cortisol and adrenocorticotropic hormone (ACTH). Choline supplementation reduced DNA hypermethylation and increased expression of POMC and PER2 in children with PAE. Conclusions: These data suggest that PAE significantly elevates DNA methylation of POMC and PER2 and increases levels of stress hormones. Furthermore, these results suggest the possibility that measuring DNA methylation levels of PER2 and POMC in biological samples from pregnant women or from children may be useful for identification of a woman or a child with PAE.
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    The role of DNA methylation in regulating LHX3 gene expression
    (2013-07) Malik, Raleigh Elizabeth; Rhodes, Simon J.; Harrington, Maureen A.; Mirmira, Raghavendra G.; Skalnik, David Gordon; Day, Richard N.
    LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.
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    The Role of DNA Methylation in Regulation of the Murine Lhx3 Gene
    (Elsevier, 2014-01-25) Malik, Raleigh E.; Rhodes, Simon J.; Department of Biology, School of Science
    LHX3 is a LIM-homeodomain transcription factor with critical roles in pituitary and nervous system development. Mutations in the LHX3 gene are associated with pediatric diseases featuring severe hormone deficiencies, hearing loss, developmental delay, and other symptoms. The mechanisms that govern LHX3/Lhx3 transcription are poorly understood. In this study, we examined the role of DNA methylation in the expression status of the mouse Lhx3 gene. Pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with 5-aza-2’-deoxycytidine, a demethylating reagent. This treatment lead to activation of Lhx3 gene expression suggesting that methylation contributes to Lhx3 regulation. Treatment of Pit-1/0 pituitary cells with a combination of a demethylating reagent and a histone deacetylase inhibitor led to rapid activation of Lhx3 expression, suggesting possible crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA was subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation at specific sites in the Lhx3 locus compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the MeCp2 methyl binding protein and Lhx3 promoter regions in the Pit-1/0 cell line. Overall, this study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.