Hypomethylation Of CpG Sites Research Articles

SPC25 Functions as a Prognostic-Related Biomarker, and Its High Expression Correlates with Tumor Immune Infiltration and UCEC Progression.

Most tumor tissues expressed spindle pole body component 25 (SPC25), one of the four subunits of the NDC80 complex, at greater levels compared to surrounding normal tissues. According to earlier researches, this subunit strongly encouraged tumor cell proliferation and tumor growth, which resulted in worse prognoses in patients with hepatocellular, breast, lung, and prostate cancer. Precisely because SPC25's role in uterine corpus endometrial carcinoma (UCEC) is understudied, we chose to concentrate on UCEC for gaining a more scientific and thorough understanding of SPC25. Along with examining SPC25's differential expression, prognostic significance, and biological function in UCEC, our research sought to clarify the underlying mechanism by which SPC25 influences the course of UCEC and patient prognosis from the viewpoints of methylation and immune infiltration. We observed differential expression of SPC25 gene in different clinicopathological features of UCEC and identified SPC25 as a hazard factor for poorer overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI) in UCEC, particularly in its multiple clinical subtypes. In addition, we also discovered that SPC25 and its co-expressed genes mostly engaged in biological processes and signal transduction routes linked to cell cycle and cell division in UCEC. After investigating SPC25's methylation status, we discovered that patients with UCEC had elevated SPC25 expression and a poor prognosis due to hypomethylation of CpG sites in the SPC25 gene sequence. Finally, we investigated SPC25's potential role in immunotherapy and discovered that SPC25 might alter the major immune cell infiltration levels in the tumor microenvironment (TME) by regulating the expression of immunoregulatory molecules and chemokines, which would be beneficial for SPC25 to control the progression of UCEC. In conclusion, SPC25 was a useful predictive biomarker as well as a possible therapeutic target for UCEC.

CNSC-25. NEURAL SIGNATURE OF GBM EXHIBITS SYNAPTOGENIC AND OPC-LIKE FEATURES AND INDEPENDENTLY PREDICTS PATIENT SURVIVAL

Abstract Interactions of neural cells with glioma cells drive tumor growth. In this study, we identified an epigenetically-defined, malignant neural signature of IDH-wildtype glioblastoma (GBM) that significantly influenced patients’ outcome. We used DNA methylation-based reference signatures of neural cells to deconvolve 1.058 primary patient GBM samples. Samples were classified based on the neural reference signature and annotated as low or high neural GBM. A high neural GBM was linked to hypomethylation of CpG sites associated with invasiveness, neuron-to-glioma-interactions, and transsynaptic signaling. Through spatially resolved, single cell transcriptomic and proteomic profiling, we discovered a correlation between the high neural signature and the upregulation of early development programs in OPC and NPC-like cells. These traits were accompanied by diminished immune infiltration and immunological response, further highlighting the distinctive neural-like nature of GBM. High neural GBM demonstrated increased connectivity as evidenced by magnetoencephalography and resting state magnet resonance imaging. Spatiotemporal tumor sampling showed a homogeneous and stable neural signature in newly diagnosed and recurrent GBM. In newly diagnosed GBM, a high neural signature correlated with decreased survival in 306 patients (median OS 15.0 versus 20.0 months, p< 0.001). Patient-derived xenograft models corroborated the aggressive nature of high neural GBM, showing a significantly reduced median OS of 58 days compared to 73 days (p< 0.01). These high neural GBM xenografts also exhibited an elevated tumor burden and an increase in neuron-glioma synapse formation. Consideration of the extent of resection showed a dependency on the neural subgroup as high neural GBM had an increased benefit from a complete contrast enhancing tumor removal when compared to low neural GBM. As a preoperative biomarker, the concentration of BDNF was increased in serum of high neural GBM patients (p < 0.01). Overall, we present an epigenetically-defined malignant neural signature in GBM that is associated with an unfavorable patient survival.

Long-term environmental metal exposure is associated with hypomethylation of CpG sites in NFKB1 and other genes related to oncogenesis

BackgroundLong-term environmental exposure to metals leads to epigenetic changes and may increase risks to human health. The relationship between the type and level of metal exposure and epigenetic changes in subjects exposed to high concentrations of metals in the environment is not yet clear. The aim of our study is to find the possible association of environmental long-term exposure to metals with DNA methylation changes of genes related to immune response and carcinogenesis. We investigated the association of plasma levels of 21 essential and non-essential metals detected by ICP-MS and the methylation level of 654 CpG sites located on NFKB1, CDKN2A, ESR1, APOA5, IGF2 and H19 genes assessed by targeted bisulfite sequencing in a cohort of 40 subjects living near metal mining area and 40 unexposed subjects. Linear regression was conducted to find differentially methylated positions with adjustment for gender, age, BMI class, smoking and metal concentration.ResultsIn the metal-exposed group, five CpGs in the NFKB1 promoter region were hypomethylated compared to unexposed group. Four differentially methylated positions (DMPs) were associated with multiple metals, two of them are located on NFKB1 gene, and one each on CDKN2A gene and ESR1 gene. Two DMPs located on NFKB1 (chr4:102500951, associated with Be) and IGF2 (chr11:2134198, associated with U) are associated with specific metal levels. The methylation status of the seven CpGs located on NFKB1 (3), ESR1 (2) and CDKN2A (2) positively correlated with plasma levels of seven metals (As, Sb, Zn, Ni, U, I and Mn).ConclusionsOur study revealed methylation changes in NFKB1, CDKN2A, IGF2 and ESR1 genes in individuals with long-term human exposure to metals. Further studies are needed to clarify the effect of environmental metal exposure on epigenetic mechanisms and pathways involved.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

SNCA Gene Methylation in Parkinson's Disease and Multiple System Atrophy.

In recent years, epigenetic mechanisms have been implicated in the development of multifactorial diseases including neurodegenerative disorders. In Parkinson's disease (PD), as a synucleinopathy, most studies focused on DNA methylation of SNCA gene coding alpha-synuclein but obtained results were rather contradictory. In another neurodegenerative synucleinopathy, multiple system atrophy (MSA), very few studies investigated the epigenetic regulation. This study included patients with PD (n = 82), patients with MSA (n = 24), and a control group (n = 50). In three groups, methylation levels of CpG and non-CpG sites in regulatory regions of the SNCA gene were analyzed. We revealed hypomethylation of CpG sites in the SNCA intron 1 in PD and hypermethylation of predominantly non-CpG sites in the SNCA promoter region in MSA. In PD patients, hypomethylation in the intron 1 was associated with earlier age at the disease onset. In MSA patients, hypermethylation in the promotor was associated with shorter disease duration (before examination). These results showed different patterns of the epigenetic regulation in two synucleinopathies-PD and MSA.

DNA Hypomethylation of the MPO Gene in Peripheral Blood Leukocytes Is Associated with Cerebral Stroke in the Acute Phase

Dysregulation of the oxidant-antioxidant system contributes to the pathogenesis of cerebral stroke (CS). Epigenetic changes of redox homeostasis genes, such as glutamate-cysteine ligase (GCLM), glutathione-S-transferase-P1 (GSTP1), thioredoxin reductase 1 (TXNRD1), and myeloperoxidase (MPO), may be biomarkers of CS. In this study, we assessed the association of DNA methylation levels of these genes with CS and clinical features of CS. We quantitatively analyzed DNA methylation patterns in the promoter or regulatory regions of 4 genes (GCLM, GSTP1, TXNRD1, and MPO) in peripheral blood leukocytes of 59 patients with CS in the acute phase and in 83 relatively healthy individuals (controls) without cardiovascular and cerebrovascular diseases. Wefound that in both groups, the methylation level of CpG sites in genes TXNRD1 and GSTP1 was ≤5%. Lower methylation levels were registered at a CpG site (chr1:94,374,293, GRCh37 [hg19]) in GCLM in patients with ischemic stroke compared with the control group (9% [7%; 11.6%] (median and interquartile range) versus 14.7% [10.4%; 23%], respectively, p<0.05). In the leukocytes of patients with CS, the methylation level of CpG sites in the analyzed region of MPO (chr17:56,356,470, GRCh3 [hg19]) on average was significantly lower (23.5% [19.3%; 26.7%]) than that in the control group (35.6% [30.4%; 42.6%], p<0.05). We also found increased methylation of MPO in smokers with CS (27.2% [23.5%; 31.1%]) compared with nonsmokers with CS (21.7% [18.1%; 24.8%]). Thus, hypomethylation of CpG sites in GCLM and MPO in blood leukocytes is associated with CS in the acute phase.

Association of Promoter Methylation and Expression of Inflammatory Genes IL-6 and TNF-α with the Risk of Coronary Artery Disease in Diabetic and Obese Subjects among Asian Indians.

The inflammatory cytokines such as interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) are considered as the most important contributors to the endothelial dysfunction in subjects with type 2 diabetes mellitus (T2DM) and obesity. The hypomethylation of CpG sites in the promoter region of the IL-6 and TNF-α have shown to be associated with the increased expression of IL-6 and TNF-α. However, there are no studies on the methylation and expression of IL-6 and TNF-α with the risk of coronary artery disease (CAD) in subjects with T2DM and obesity in Asian Indians. Hence, the present study was aimed to investigate whether the IL-6, TNF-α promoter methylation and expression in blood leukocyte DNA is associated with the risk of CAD in diabetic and obese subjects in Asian Indians. For this study, we recruited 574 subjects which includes, 207 angiographically confirmed CAD patients, 100 T2DM patients, 82 obese subjects and 185 healthy controls. The methylation status of IL-6 and TNF-α gene loci was determined by methylation specific PCR (MPCR) and gene expression was determined by qPCR. We found significant hypomethylation of IL-6 in CAD and T2DM subjects (OR 1.98 95% CI: 1.32-2.97, p = 0.001, OR: 2.23 95% CI:1.34-3.76, p = 0.001, respectively). Further, a significant increase in the expression of IL-6 in CAD and T2DM subjects (fold change: 26.39 & 14.7, p = 0.0001) compared to the control subjects was observed. A significant increase in the hypomethylation of TNF-α in CAD, T2DM and obese subjects was observed as compared to the control (OR: 2.04 95% CI: 1.36-3.05, p = 0.0005, OR: 1.81 95% CI 1.10-2.96, p = 0.01, and OR: 2.1 95% CI 1.24-3.57, p = 0.007, respectively).We also found an increased expression of TNF-α in CAD, T2DM and obese subjects as compared to controls. In addition, presence of low folate, and hyperhomocysteinemia was observed in the present study, may be the contributing factors for the hypomethylation of IL-6 and TNF-α and oxidative stress. In conclusion, increased expression of IL-6 and TNF-α due to hypomethylation in T2DM and obese individuals may contribute to CAD risk in these subjects. The presence of hyperhomocysteinemia and increased oxidative risk may enhance the CAD risk further.

Epigenetic Regulation of the Clinical Signs of Friedreich’s Disease

Objectives. To study the methylation profile of the FXN gene and its influence on the formation of the clinical presentation of Friedreich’s disease (FD). Materials and methods. The promoter area and intron 1 of the FXN gene up to the GAA expansion (UP-GAA) and after the GAA expansion (DOWN-GAA) regions were studied in 17 patients with FD, with analysis of a total of 45 CpG sites. Results. Studies of genetic-epigenetic interactions identified correlations between the extent of methylation of a series of CpG sites in the UP-GAA and DOWN-GAA and the number of GAA repeats in both expanded alleles of the FXN gene in patients with FD. We also found a link between methylation and the presence of the extraneural signs of FD: cardiomyopathy was more likely to be present when the CpG site of the promoter region was hypermethylated, while impairments to carbohydrate metabolism were more common in hypomethylation of CpG sites in the DOWN-GAA area. Conclusions. The data obtained here provide evidence that epigenetic modifications of the FXN gene make a significant contribution to forming the clinical picture of FD.

Abstract A54: Epigenetic activated IL1B promotes tumor progression of tongue cancer

Abstract Alterations in host immunity, inflammation, angiogenesis, and metabolism have been noted to be prominent clinical features in patients with head and neck squamous cell carcinoma (HNSCC). IL-1β, a prototypical inflammatory cytokine, is induced and activated following infection and tissue damage. High IL-1 levels in the tumor microenvironment are associated with a more aggressive tumor phenotype and generally poor prognoses. It was reported that single-nucleotide polymorphisms (SNPs) identified in IL1B gene influence the risk of many kinds of cancer, but the role of IL1B in the development and progress of HNSCC and the related molecular mechanism were not well understood. In the present study, we found IL1B was upregulated in tongue cancer tissues according to our RNA-Seq data and was validated in other 60 paired clinical samples. Using data from The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSC), we found that the expression of IL1B in HNSCC tumor samples (n=501) is significantly higher than the adjacent noncancer tissues (n=44) and hypomethylation of CpG sites was found around the promoter region of IL1B in the cancer samples. Regression analysis identified a moderately negative correlation between IL1B expression and the DNA methylation of these CpG sites, even though there is not CpG island around its promoter. Knockdown of IL1B in Cal-27 and TCA-8113 tongue cancer cells inhibited cell proliferation, migration and invasion ability, and overexpression of IL1B or recombinant IL1B protein can promote proliferation, migration and invasion of tongue cancer cells. The results indicated that aberrant IL1B expression might be modulated by its DNA hypomethylation and IL1B produced by cancer cells can promote tumor progression and metastasis, and the inhibition of IL1B pathway may be a novel therapeutic approach in the treatment of HNSCC. Citation Format: Dali Zheng, Weitao Chen, Youguang Lu. Epigenetic activated IL1B promotes tumor progression of tongue cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A54.

In Utero Exposure to Gestational Diabetes Alters DNA Methylation and Gene Expression of CDKN2A/B in Langerhans Islets of Rat Offspring.

Objective DNA methylation, a major epigenetic reprogramming mechanism, contributes to the increased prevalence of type 2 diabetes mellitus (T2DM). Based on genome-wide association studies, polymorphisms in CDKN2A/B are associated with T2DM. Our previous studies showed that gestational diabetes mellitus (GDM) causes apoptosis in β-cells, leading to a reduction in their number in pancreatic tissue of GDM-exposed adult rat offspring. The aim of this study was to examine the impact of intrauterine exposure to GDM on DNA methylation, mRNA transcription, as well as protein expression of these factors in the pancreatic islets of Wistar rat offspring. Our hypothesis was that the morphological changes seen in our previous study might have been caused by aberrant methylation and expression of CDKN2A/B. Materials and Methods In this experimental study, we delineated DNA methylation patterns, mRNA transcription and protein expression level of CDKN2A/B in the pancreatic islets of 15-week-old rat offspring of streptozotocin-induced GDM dams. We performed bisulfite sequencing to determine the DNA methylation patterns of CpGs in candidate promoter regions of CDKN2A/B. Furthermore, we compared the levels of mRNA transcripts as well as the cell cycle inhibitory proteins P15 and P16 in two groups by qPCR and western blotting, respectively. Results Our results demonstrated that hypomethylation of CpG sites in the vicinity of CDKN2A and CDKN2B genes is positively related to increased levels of CDKN2A/B mRNA and protein in islets of Langerhans in the GDM offspring. The average percentage of CDKN2A promoter methylation was significantly lower in GDM group compared to the controls (P<0.01). ConclusionWe postulate that GDM is likely to exert its adverse effects on pancreatic β-cells of offspring through hypomethylation of the CDKN2A/B promoter. Abnormal methylation of these genes may have a link with β-cell dysfunction and diabetes. These data potentially lead to a novel approach to the treatment of T2DM.

Cross-Generational Effects of Alcohol Dependence in Humans on HRAS and TP53 Methylation in Offspring

We hypothesized that cross-generational effects of alcohol exposure could alter DNA methylation and expression of the HRAS oncogene and TP53 tumor suppressor gene that drive cancer development. DNA methylation of the HRAS and TP53 genes was tested in samples from young participants (Mean age of 13.4years). Controlling for both personal use and maternal use of substances during pregnancy, familial alcohol dependence was associated with hypomethylation of CpG sites in the HRAS promoter region and hypermethylation of the TP53 gene. The results suggest that ancestral exposure to alcohol can have enduring effects that impact epigenetic processes such as DNA methylation that controls expression of genes that drive cancer development such as HRAS and TP53.

Methylation pattern of preferentially expressed antigen of melanoma in acute myeloid leukemia and myelodysplastic syndromes.

Preferentially expressed antigen of melanoma (PRAME), a tumor-associated antigen, is overexpressed in a variety of hematologic malignancies with a great variation in expression. The majority of patients with acute myeloid leukemia (AML) 1-eight-twenty one (ETO)+ AML and a certain number of myelodysplastic syndromes (MDS) have an abnormally high increase in PRAME expression level. The landscape of PRAME methylation requires evaluation in order to determine the most relevant sites and the exact association of its methylation with expression level and type of disease. In the present study, bone marrow samples collected from 8 AML1-ETO+ AML, 4 MDS, 3 AML1-ETO- AML and 2 normal volunteers underwent bisulfate sequencing to analyze the methylation status of all four 5'-C-phosphate-G-3' (CpG) regions within the entire PRAME gene. The median PRAME transcript level of 15 patients was 204.5% (range, 0.02-710.3%). PRAME transcript levels were inversely associated with the degree of methylation of the -389 to -146 CpG sites (r=-0.69; P=0.002) in the 3' part of the promoter region and the +132 to +363 CpG sites (r=-0.69; P=0.006) in the exon 1b region. However, not every sample strictly followed this correlation: Certain samples with high degrees of methylation demonstrated abnormally high expression levels, and vice versa. The methylation ratios of CpG sites in exon 1a were low for all samples (range, 0.0-13.8%), and those in exon 2 were similar in 16 samples (range, 72.4-93.4%), with the exception of one patient with high expression (425.2%) and significantly low degree of methylation in the PRAME gene (22.2%). MDS patients revealed similar methylation ratios in the 3' section of the promoter region, but tended to have lower methylation ratios in the exon 1b region (P=0.62 and P=0.09, respectively) compared with those observed in AML1-ETO+ patients with AML and similar degree of PRAME overexpression. Therefore, the hypomethylation of CpG sites in the 3' part of the promoter region and in exon 1b was typically found with PRAME overexpression in AML and MDS. Methylation of other CpG islands, epigenetic and genetic mechanisms, and type of disease may also be involved.

Rapid and specific hypomethylation of enhancers in endothelial cells during adaptation to cell culturing

ABSTRACTEpigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene HDAC9 increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. HDAC9 displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.

Epigenetic Regulation of the Homeobox GeneMSX1Associates with Platinum-Resistant Disease in High-Grade Serous Epithelial Ovarian Cancer

Although high-grade serous ovarian cancer (HGSOC) is frequently chemoresponsive, a proportion of patients do not respond to platinum-based chemotherapy at presentation or have progression-free survival (PFS) of less than 6 months. Validated predictive biomarkers of lack of response would enable alternative treatment stratification for these patients and identify novel mechanisms of intrinsic resistance. Our aim was to identify DNA methylation biomarkers of poor response to chemotherapy and demonstrate involvement of the associated gene in platinum drug cell sensitivity. DNA methylation was investigated in independent tumor cohorts using Illumina HumanMethylation arrays and gene expression by Affymetrix arrays and qRT-PCR. The role of Msh homeobox 1 (MSX1) in drug sensitivity was investigated by gene reintroduction and siRNA knockdown of ovarian cancer cell lines. CpG sites at contiguous genomic locations within the MSX1 gene have significantly lower levels of methylation in independent cohorts of HGSOC patients, which recur by 6 months compared with after 12 months (P < 0.05, q < 0.05, n = 78), have poor RECIST response (P < 0.05, q < 0.05, n = 61), and are associated with PFS in an independent cohort (n = 146). A decrease in methylation at these CpG sites correlates with decreased MSX1 gene expression. MSX1 expression is associated with PFS (HR, 0.92; 95% CI, 0.85-0.99; P = 0.029; n = 309). Cisplatin-resistant ovarian cancer cell lines have reduced MSX1 expression, and MSX1 overexpression leads to cisplatin sensitization, increased apoptosis, and increased cisplatin-induced p21 expression. Hypomethylation of CpG sites within the MSX1 gene is associated with resistant HGSOC disease at presentation and identifies expression of MSX1 as conferring platinum drug sensitivity. Clin Cancer Res; 22(12); 3097-104. ©2016 AACR.

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%–5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%–16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.

Genome-Wide Assessment of Differential DNA Methylation Associated with Autoantibody Production in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.

Abstract 32: Improper Histone Modification Of Gene Expression In Kawasaki Disease May Influence Both Its Development And Outcome

Kawasaki disease (KD) is characterized by development of an autoimmune vasculitis and potential coronary artery lesions (CAL). Patients present with significantly marked expression of various immune cells and the inflammatory cells that they produce, recruit, or secrete, particularly cytokines. Concurrently, regulatory immune cells that suppress immune and inflammatory cells are significantly reduced in KD, particularly regulatory T cells (Tregs) such as CD4+CD25+FOXP3+ Tregs. Therefore, the clinical picture results from an unchecked, marked increase of immune and inflammatory cells infiltrating passed activated endothelial cells and into the vascular wall. This appears to largely be a consequence of amplified cell signaling via the Ca2+/NFAT pathway in KD, which is involved in targeted gene expression. As a result, we hypothesized that epigenetic control of gene expression through histone modifications may play a leading role in development of KD, its complications, and recovery. A previous study found significant DNA hypomethylation of CpG sites for the SOCS1 gene in KD patients (n=32), which was higher in patients without CAL. We conducted a preliminary study to widely examine epigenetic changes in DNA methylation of CpG sites before and after IVIG treatment in KD patients (n=9) using age-matched febrile controls (n=8). Our results demonstrated ≥10% differences in DNA methylation rates for 67 genes in KD patients, in addition to 10 genes with ≥15% methylation difference. Larger studies are required to accurately determine the role of epigenomic changes in KD development, in addition to investigations of the methylation reactions which regulate them (see Figure 1).

Integrated Analysis of Genome-Wide Methylation and Gene Expression Shows Epigenetic Regulation of CYP11B2 in Aldosteronomas

Differential methylation of CpG regions is the best-defined mechanism of epigenetic regulation of gene expression. Our objective was to determine whether any changes in methylation are associated with aldosteronomas. We performed integrated genome-wide methylation and gene expression profiling in aldosteronomas (n = 25) as compared with normal adrenal cortical tissue (n = 10) and nonfunctioning adrenocortical tumors (n = 13). To determine the effect of demethylation on gene expression of CYP11B2, the H295R cell line was used. The methylome of aldosteronomas, normal adrenal cortex, and nonfunctioning adrenocortical tumors was distinct, with hypomethylation of aldosteronomas. Integrated analysis of gene expression and methylation status showed that 53 of 60 genes were hypermethylated and downregulated, or hypomethylated and upregulated, in aldosteronomas. Of these, 3 genes that regulate steroidogenic signals and synthesis in adrenocortical cells were differentially methylated: AVPR1α and PRKCA were downregulated and hypermethylated, and CYP11B2 was upregulated and hypomethylated. Demethylation treatment resulted in upregulation of these genes, with direct hypomethylation of CpG sites associated with the genes. The CpG island in the promoter region of CYP11B2 was hypomethylated in aldosteronomas but not in blood DNA from the same patients (P = .0004). Altered methylation in aldosteronomas is associated with dysregulated expression of genes involved in steroid biosynthesis. Aldosteronomas are hypomethylated, and CYP11B2 is overexpressed and hypomethylated in these tumors.

PPARγ forms a bridge between DNA methylation and histone acetylation at the C/EBPα gene promoter to regulate the balance between osteogenesis and adipogenesis of bone marrow stromal cells

The balance between osteogenesis and adipogenesis of bone marrow stromal cells is impaired in many human diseases. Knowledge of how to fine-tune this balance is of medical importance. CCAAT/enhancer binding protein α (C/EBPα) has been shown to regulate the balance between osteogenesis and adipogenesis of C3H10T1/2 cells, with epigenetic modifications of the C/EBPα promoter playing an important role. The present study aimed to elucidate the underlying molecular mechanisms. The results showed that peroxisome proliferator-activated receptor γ (PPARγ) binds the -1286 bp/-1065 bp region of the C/EBPα promoter to activate C/EBPα expression during osteogenesis and adipogenesis of C3H10T1/2 cells. DNA hypermethylation in the -1286 bp/-1065 bp region, observed at the terminal stage of osteogenesis, prevented PPARγ binding, and then histone deacetylase 1 (HDAC1) occupied this region to reduce the level of histone acetylation. We regulated the balance between osteogenesis and adipogenesis of mouse bone marrow stromal cells through modulation of DNA methylation and histone acetylation status. In addition, in bone marrow stromal cells from the glucocorticoid-induced osteoporosis (GIO) mouse, hypomethylation of CpG sites, higher binding of PPARγ, acetylated histones 3 and 4, and reduced binding of HDAC1 in the -1286 bp/-1065 bp region of C/EBPα promoter were observed, compared with normal mice. This study provides a deeper insight into the molecular mechanisms underlying the balance between osteogenesis and adipogenesis regulated by C/EBPα in synergy with PPARγ, and suggests a molecular model for how DNA methylation and histone acetylation are linked by PPARγ to regulate differentiation of bone marrow stromal cells.

Hypomethylation of ETS Transcription Factor Binding Sites and Upregulation of PARP1 Expression in Endometrial Cancer

Although PARP1 promoter methylation is involved in the regulation of PARP1 expression in human keratinocyte lines and lymphoblastoid cell lines, its roles in human endometrial cancer are unknown. DNA from forty normal endometrium (NE) and fifty endometrial adenocarcinoma (EAC) tissues were analyzed by bisulfite sequencing using primers focusing on the core promoter region of PARP1. Expression levels of PARP1 were assessed by immunohistochemistry and real-time PCR. Associations between patient clinicopathological characteristics and PARP1 protein levels were assessed by Fisher's exact test. Here, PARP1 mRNA and protein were overexpressed in EAC tissues (P < 0.05). CpG sites within the ETS motif in the PARP1 promoter exhibited significant hypomethylation in EAC tissues, and there was a significant negative correlation between PARP1 mRNA levels and the number of methylated sites in both NE and EAC tissues (R 2 = 0.262, P < 0.001). Notably, PARP1 protein expression was associated with FIGO stage (P = 0.026), histological grade (P = 0.002) , and body mass index (P = 0.04). Our findings imply that PARP1 overexpression may participate in endometrial cancer progression, and abnormal hypomethylation of CpG sites within the ETS motif in the core promoter region may be responsible for PARP1 overexpression in EAC tissues.