Promotor Demethylation as a Mechanism of HOX11 Activation in Adult T-ALL?.

Abstract Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

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Diagnostic and Prognostic Value of B4GALT1 Hypermethylation and Its Clinical Significance as a Novel Circulating Cell-Free DNA Biomarker in Colorectal Cancer

Cancers ◽

10.3390/cancers11101598 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1598 ◽

Cited By ~ 3

Author(s):

Francesco Picardo ◽

Antonella Romanelli ◽

Laura Muinelo-Romay ◽

Tommaso Mazza ◽

Caterina Fusilli ◽

...

Keyword(s):

Colorectal Cancer ◽

Lung Metastases ◽

Methylation Status ◽

Therapy Response ◽

Progression Free Survival ◽

Prognostic Impact ◽

Aberrant Expression ◽

Predictive Values ◽

Methylation Specific Pcr ◽

Specific Pcr

Epigenetic modifications of glyco-genes have been documented in different types of cancer and are tightly linked to proliferation, invasiveness, metastasis, and drug resistance. This study aims to investigate the diagnostic, prognostic, and therapy-response predictive value of the glyco-gene B4GALT1 in colorectal cancer (CRC) patients. A Kaplan–Meier analysis was conducted in 1418 CRC patients (GEO and TCGA datasets) to assess the prognostic and therapy-response predictive values of the aberrant expression and methylation status of B4GALT1. Quantitative methylation-specific PCR (QMSP) and droplet digital quantitative methylation-specific PCR (dd-QMSP) were respectively used to detect hypermethylated B4GALT1 in metastasis and plasma in four cohorts of metastatic CRC cases (mCRC). Both the downregulated expression and promoter hypermethylation of B4GALT1 have a negative prognostic impact on CRC. Interestingly a low expression level of B4GALT1 was significantly associated with poor cetuximab response (progression-free survival (PFS) p = 0.01) particularly in wild-type (WT)-KRAS patients (p = 0.03). B4GALT1 promoter was aberrantly methylated in liver and lung metastases. The detection of hypermethylated B4GALT1 in plasma of mCRC patients showed a highly discriminative receiver operating characteristic (ROC) curve profile (area under curve (AUC) value 0.750; 95% CI: 0.592–0.908, p = 0.008), clearly distinguishing mCRC patients from healthy controls. Based on an optimal cut-off value defined by the ROC analysis, B4GALT1 yield a 100% specificity and a 50% sensitivity. These data support the potential value of B4GALT1 as an additional novel biomarker for the prediction of cetuximab response, and as a specific and sensitive diagnostic circulating biomarker that can be detected in CRC.

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Active Demethylation of Non-CpG Moieties in Animals: A Neglected Research Area

International Journal of Molecular Sciences ◽

10.3390/ijms20246272 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6272 ◽

Cited By ~ 1

Author(s):

Marco Lucarelli ◽

Giampiero Ferraguti ◽

Andrea Fuso

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Functional Role ◽

Cytosine Methylation ◽

Research Area ◽

Cpg Methylation ◽

Biological Functions ◽

Active Demethylation ◽

Modulation Of Gene Expression

The functional role of cytosine methylation in the CpG moieties of DNA, is well established in several biological functions. The interplay between CpG methylation and hypomethylation is a well-known mechanism of modulation of gene expression. However, the role of non-CpG methylation and active dynamics of demethylation is not clearly recognized. Although some evidence exists of a role of active non-CpG demethylation in the fast dynamics of transcriptional activation in animals, few studies deal with this topic. At present, active demethylation of non-CpG moieties is a neglected research area, in spite of the promise of significant novelties.

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Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level

10.1101/2021.04.24.441265 ◽

2021 ◽

Author(s):

Andreas Nerlich ◽

Nina Janze ◽

Ralph Goethe

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Leucine Zipper ◽

Methylation Status ◽

Gene Activation ◽

Cell Types ◽

Cpg Methylation ◽

Basic Leucine Zipper ◽

Murine Macrophages ◽

Copy Numbers

Interleukin-36α (Il-36α) is a member of the novel Il-1-like proinflammatory cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types. We have recently shown that CCAAT enhancer binding proteinβ (C/EBPβ) binds specifically to an essential half cAMP response element (half-CRE)•C/EBP motif in the Il36A promoter to induce Il36A expression upon LPS stimulation. C/EBPs are transcription factors belonging to the basic leucine zipper (bZIP) family of transcriptional regulators. C/EBP proteins can form homo- and heterodimers and regulate gene expression by binding to C/EBP specific recognition sequences and composite sites that can contain 5’-cytosine-phosphate-guanine-3’ dinucleotides (CpG). CpG methylation of such elements has been shown to influence transcription factor binding and gene expression. Here we show that the half-CRE•C/EBP element in the Il36A promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. By using electrophoretic mobility gel shift and fluorescence polarization assays we demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36A promoter following LPS stimulation is insensitive to CpG methylation. Transfection assays also show that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36A promoter activity. A direct comparison of Il36A mRNA copy numbers as well as the pro-Il-36α protein level in RAW264.7 and primary macrophages revealed similar amounts in both cell types. Taken together, our data suggest that C/EBPβ binding to the half-CRE•C/EBP element and C/EBPβ mediated gene activation occurs independently of the CpG methylation status of the target DNA sequence and underline the potential of C/EBPβ to recognize methylated as well as unmethylated binding sites.

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SUV39H1-Mediated DNMT1 is Participated in the Epigenetic Regulation of Smad3 in Cervical Cancer

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721110016 ◽

2020 ◽

Vol 20 ◽

Author(s):

Li Zhang ◽

Sijuan Tian ◽

Minyi Zhao ◽

Ting Yang ◽

Shimin Quan ◽

...

Keyword(s):

Cervical Cancer ◽

Cell Lines ◽

Epigenetic Regulation ◽

Promoter Region ◽

Methylation Status ◽

Regulation Mechanism ◽

Methylation Specific Pcr ◽

Specific Pcr ◽

Immune Factor ◽

Cancer Tissues

Background: Smad3 is a pivotal intracellular mediator for participating in the activation of multiple immune signal pathway. Objective: The epigenetic regulation mechanism of the positive immune factor Smad3 in cervical cancer remains unknown. Therefore, the epigenetic regulation on Smad3 is investigated in this study. Methods: The methylation status of SMAD3 was detected by Methylation-specific PCR (MS-PCR) and Quantitative Methylation-specific PCR (MS-qPCR) in cervical cancer tissues and cell lines. The underlying molecular mechanisms of SUV39H1-DNMT1-Smad3 regulation was elucidated using cervical cancer cell lines containing siRNA or/and overexpression system. Confirmation of the regulation of DNMT1 by SUV39H1 used Chromatin immunoprecipitation-qPCR (ChIP-qPCR). The statistical methods used for comparing samples between groups were paired t tests and one-way ANOVAs. Results: H3K9me3 protein which regulated by SUV39H1 directly interacts with the DNMT1 promoter region to regulate its expression in cervical cancer cells, resulting in the reduce expression of the downstream target gene DNMT1. In addition, DNMT1 mediates the epigenetic modulation of the SMAD3 gene by directly binding to its promoter region. The depletion of DNMT1 effectively restores the expression of Smad3 in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1-DNMT1 was found to correlate with Smad3 expression in accordance with the expression at the cellular level. Notably, the promoter region of SMAD3 was hypermethylated in cervical cancer tissues, and this hypermethylation inhibits the subsequent gene expression. Conclusion: These results indicate that SUV39H1-DNMT1 is a crucial Smad3 regulatory axis in cervical cancer. SUV39H1-DNMT1 axis may provide a potential therapeutic target for the treatment of cervical cancer.

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Promoter CpG methylation status affects ADRP gene expression level and intramuscular fat content in pigs

Italian Journal of Animal Science ◽

10.1080/1828051x.2020.1729261 ◽

2020 ◽

Vol 19 (1) ◽

pp. 783-791

Author(s):

Yongyan Zhao ◽

Xiangsheng Lin ◽

Kaiqing Liu ◽

Ye Tian ◽

Lifan Zhang ◽

...

Keyword(s):

Gene Expression ◽

Methylation Status ◽

Intramuscular Fat ◽

Fat Content ◽

Cpg Methylation ◽

Gene Expression Level ◽

Expression Level ◽

Intramuscular Fat Content

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Hypermethylation of the p15/INK4B Gene in Fanconi Anemia.

Blood ◽

10.1182/blood.v104.11.4296.4296 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4296-4296

Author(s):

Satoshi Hamanoue ◽

Miharu Yabe ◽

Hiromasa Yabe ◽

Takayuki Yamashita

Keyword(s):

Bone Marrow ◽

Fanconi Anemia ◽

Restriction Analysis ◽

Bone Marrow Failure ◽

Methylation Status ◽

Age At Onset ◽

Refractory Anemia ◽

Molecular Abnormalities ◽

Methylation Specific Pcr ◽

Specific Pcr

Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome with multiple complementation groups, characterized by genomic instability and predisposition to MDS and AML. Recent evidence indicates that multiple FA proteins are involved in DNA repair. Thus, increased genetic damage and secondary dysregulation of cell proliferation, differentiation and apoptosis are thought to play important roles in the development of bone marrow failure and subsequent progression to MDS/AML. However, little is known about molecular abnormalities responsible for these hematological disorders. Numerous studies indicated that epigenetic silencing of p15/INK4B, an inhibitor of cyclin-dependent kinases, plays an important role in the pathogenesis of MDS and AML. In the present study, we examined methylation status of 5′ CpG islands of the p15 gene in bone marrow mononuclear cells of FA patients, using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Bone marrow samples were analyzed in 10 patients and serially studied in 4 of them. Hypermethylation of the p15 promoter region was detected in 5 patients (50%). This group included 3 patients with MDS: FA28-1 with refractory anemia (RA), FA87 with RAEB (RA with excess of blasts), and FA88 with later development of RA and progression to RAEB; whereas myelodysplasia was not observed in 2 patients (FA89, FA90). In two cases (FA88, FA90), p15 hypermethylation became negative during their courses, perhaps because of decreased myeloid cells. On the other hand, none of 5 patients without p15 hypermethylation had MDS. These results suggest that p15 hypermethylation is associated with development of MDS and occurs in the early phase of clonal evolution in the disease. Methylation status of p15 may be a useful prognostic factor of FA. Patient Age at onset (year old) Time from onset (month) Cytopenia MDS Cytogenetic abnormalities p15 methylation MSP b p15 methylation COBRA c a siblings, b MSP: methylation specific PCR, c COBRA: combined bisulfite restriction analysis, d ND: not determined FA28-1a 5 128 severe RA − − + 133 severe RA − + ++ FA87 8 252 severe RAEB + + +++ FA88 5 31 moderate − − + +++ 45 severe RA + − − 58 severe RAEB + + + FA89 5 49 mild − − + + 56 severe − − + + FA90 2 2 mild − − + ++ 31 severe − − − − FA28-2a 5 51 mild − − − NDd FA28-3a 3 12 mild − − − NDd FA47 3 15 mild − − − NDd FA68 5 46 moderate − − − NDd FA91 5 129 mild − − − NDd

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Intragenic hypomethylation of DNMT3A in patients with myelodysplastic syndrome

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0142 ◽

2018 ◽

Vol 56 (3) ◽

pp. 485-491 ◽

Cited By ~ 2

Author(s):

Ying-Ying Zhang ◽

Jing-Dong Zhou ◽

Dong-Qin Yang ◽

Pin-Fang He ◽

Dong-Ming Yao ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Dna Methyltransferase ◽

De Novo ◽

Methylation Status ◽

World Health ◽

Differentially Methylated Region ◽

International Prognostic Scoring System ◽

Aberrant Expression ◽

Specific Pcr ◽

Cell Counts

AbstractBackground:DNMT3Ais a DNA methyltransferase that acts inde novomethylation. Aberrant expression ofDNMT3Ahas been reported in several human diseases, including myelodysplastic syndrome (MDS). However, the pattern ofDNMT3Amethylation remains unknown in MDS.Methods:The present study was aimed to investigate the methylation status ofDNMT3Aintragenic differentially methylated region 2 (DMR2) using real-time quantitative methylation-specific PCR and analyze its clinical significance in MDS.Results:Aberrant hypomethylation ofDNMT3Awas found in 57% (51/90) MDS cases. There were no significant differences in age, sex, white blood cell counts, platelet counts, hemoglobin counts and World Health Organization, International Prognostic Scoring System and karyotype classifications betweenDNMT3Ahypomethylated andDNMT3Ahypermethylated groups. However, the patients withDNMT3Ahypomethylation had shorter overall survival time than those withoutDNMT3Ahypomethylation (11 months vs. 36 months, p=0.033). Multivariate analysis confirmed the independent adverse impact ofDNMT3Ahypomethylation in MDS.Conclusions:Our data suggest thatDNMT3ADMR2 hypomethylation may be a negative prognostic hallmark in MDS.

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Ethnic Differences in MicroRNA-375 Expression Level and DNA Methylation Status in Type 2 Diabetes of Han and Kazak Populations

Journal of Diabetes Research ◽

10.1155/2014/761938 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Xiangyun Chang ◽

Siyuan Li ◽

Jun Li ◽

Liang Yin ◽

Ting Zhou ◽

...

Keyword(s):

Type 2 Diabetes ◽

Methylation Status ◽

Cpg Methylation ◽

Expression Level ◽

Related Factor ◽

Expression Levels ◽

Relative Expression ◽

Cpg Sites ◽

Significant Difference

Han population is six times as likely as Kazak population to present with type 2 diabetes mellitus (T2DM) in China. We hypothesize that differential expression and CpG methylation of miR-375 may be an ethnic-related factor that influences the incidence of T2DM. The expression level of miR-375 was examined using real-time PCR on Kazak and Han T2DM plasma samples. Furthermore, the methylation levels of CpG sites of miR-375 promoter were determined by MassARRAY Spectrometry in these samples. The relative expression levels of plasma miR-375 in Kazak T2DM samples are 1, and the relative expression levels of plasma miR-375 in Han T2DM samples are 3. The mean level of miR-375 methylation, calculated from the methylation levels of the CpG sites, was 8.47% for the Kazak T2DM group and 10.38% for the Han T2DM group. Further, five CpG units showed a statistically significant difference between Kazak and Han T2DM samples, and, among them, four were hypomethylated and only one CpG unit showed hypermethylation in Kazak T2DM samples. These findings indicate that the expression levels of plasma miR-375 and its CpG methylation in the promoter region are ethnically different, which may contribute to the different incidence of diabetes observed in Kazak and Han populations.

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Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

Molecular Endocrinology ◽

10.1210/me.2003-0461 ◽

2004 ◽

Vol 18 (7) ◽

pp. 1740-1755 ◽

Cited By ~ 37

Author(s):

Leo Tsz-On Lee ◽

Kian-Cheng Tan-Un ◽

Ronald Ting-Kai Pang ◽

David Tai-Wai Lam ◽

Billy Kwok-Chong Chow

Keyword(s):

Gene Expression ◽

Hepg2 Cells ◽

Cpg Island ◽

Methylation Status ◽

Gene Promoter ◽

Mobility Shift ◽

Specific Pcr ◽

E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

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