scholarly journals Methylation Analysis of CpG Islands in Pineapple SERK1 Promoter

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 425
Author(s):  
Aiping Luan ◽  
Chengjie Chen ◽  
Tao Xie ◽  
Junhu He ◽  
Yehua He
Keyword(s):  
Methylation Status ◽  
Cpg Islands ◽  
Embryonic Cell ◽  
Regulatory Mechanisms ◽  
Ananas Comosus ◽  
Regulatory Sequences ◽  
Methylation Analysis ◽  
Embryonic Callus ◽  
Methylation Inhibitor ◽  
Traditional Breeding

Somatic embryogenesis (SE) is a more rapid and controllable method for plant propagation than traditional breeding methods. However, it often suffers from limited efficiency. SERK1 promotes SE in several plants, including pineapple (Ananas comosus L.). We investigate the embryonic cell-specific transcriptional regulation of AcSERK1 by methylation analysis of CpG islands in AcSERK1 regulatory sequences. This revealed differences in the methylation status of CpG islands between embryonic callus and non-embryonic callus; the methylation inhibitor 5-azaC increased AcSERK1 expression and also accelerated SE. These findings indicate that the expression of AcSERK1 is regulated epigenetically. This study lays the foundation for further analysis of epigenetic regulatory mechanisms that may enhance the efficiency of SE in pineapple and other plants.

Epigenetic Dysregulation of Candidate Cis-Regulatory Sequences in Hematological Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2229-2229
Author(s):  
Reid F. Thompson ◽  
Maria E. Figueroa ◽  
Ari M. Melnick ◽  
John M. Greally
Keyword(s):  
Cytosine Methylation ◽  
Hematological Malignancies ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Regulatory Sequences ◽  
Cpg Island Methylator Phenotype ◽  
Global Methylation ◽  
Methylator Phenotype ◽  
Tumor Types

Abstract Epigenetic changes (in particular, altered cytosine methylation) have been described in a variety of tumors. The CpG Island Methylator Phenotype (CIMP) is a well-known instance of this phenomenon wherein cytosine methylation is markedly dysregulated (normally hypomethylated loci shift to a methylated state). CIMP has been demonstrated in a number of different cancer types including hematological malignancies like AML. While methylation status has been studied predominantly at CpG islands, we used a novel assay (HELP; Khulan et al., Genome Res. 2006) to look for changes in cytosine methylation in large contiguous regions of the genome. We assessed global patterns of cytosine methylation by HELP analysis in a variety of tumor samples including leukemias and lymphomas. We found significant changes in the global methylation patterns of malignant cells, confirming prior observations of epigenetic dysregulation in these tumor types. We also discovered that the majority of the changes in cytosine methylation are occurring not at CpG islands but at other loci in the genome, including constitutively hypomethylated loci that we are finding to be candidate cis-regulatory sequences. We conclude that cytosine methylation changes in cancer occur much more extensively than analysis of CpG islands alone would indicate, and that the epigenetic dysregulation in cancer may be predominantly targeted to cis-regulatory sequences rather than to promoters.

scholarly journals Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 883 ◽  
Author(s):  
Luan ◽  
He ◽  
Xie ◽  
Chen ◽  
Mao ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Specific Activity ◽  
Marker Gene ◽  
Immature Embryo ◽  
Embryonic Cell ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Embryonic Callus

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS（Beta-glucuronidase） reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

scholarly journals Studying Methylation Status of Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) in Chronic Myeloid Leukemia Patients in the State of Qatar

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5124-5124
Author(s):  
Mohamed Ismail ◽  
Marzia Vezzalini ◽  
Ali Al Sayab ◽  
Maria Monne ◽  
Vinod Gupta ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Phosphatase ◽  
Myeloid Leukemia ◽  
Tyrosine Phosphatase ◽  
Methylation Status ◽  
Cpg Islands ◽  
P Value ◽  
Methylation Analysis ◽  
Specific Pcr ◽  
The Mean

Abstract Background: Chronic myeloid leukemia (CML) is a clonal myeloid stem cell disorder associated with increased myeloid cells spectrum in peripheral blood. The disease is due to the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL1 fusion oncogene which is the hallmark of CML. The resultant chimeric protein P210 kD of BCR-ABL1 causes a constitutive activation of tyrosine kinase domain that leads to continuous activation of several cell signaling pathways. The CML is currently treated with several generations of tyrosine kinase inhibitors (TKIs). However, treatment failure, resistance and disease progression are still reported and the molecular mechanisms still remain poorly understood. On the other hand, protein tyrosine phosphatase receptor gamma (PTPRG) is the natural regulatory mechanism to tyrosine kinase family, acts as tumor suppressor gene, well documented to be downregulated in CML patients while its re-expression led to reduced tyrosine phosphorylation and induced apoptosis in CML cells. PTPRG is also an important protein in the JAK/STAT pathway, has a vital role in the downstream of BCR-ABL protein kinase. Several studies suggested that the aberrant DNA methylation is involved in TKIs resistance in CML leading to leukemic clone escape and disease propagation. In different types of tumors, several mechanisms have been suggested for the loss of function of PTPRG activities such as mutations, deletions, or loss of heterozygosity LOH and gene silencing by methylation. Aim: In this study, we aimed to evaluate and characterize the methylation status of PTPRG gene function in 15 CML Patients 1- Group A: CML patients at diagnosis and 2- Group B) CML patients failed treatments according to European Leukemia Net (ELN) 2013 criteria and 10 healthy individuals. (Labeled as Normal in the figure 1) Materials and Methods: Thirty subsequent peripheral blood samples have been collected from the 15 CML patients at diagnosis and at time of failure. Genomic DNA was isolated from 400 ml of blood on EDTA tubes using Maxwell DNA Purification Kits, the quality and quantity of DNA were measured on Nanodrop Spectrophotometer and gel electrophoresis. The methylation-specific PCR and bisulfite-sequencing were adapted, EZ DNA Methylation Kit was utilized for bisulfite-conversion. Methylation Primers were designed to target the promotor and intronic CpG islands of the PTPRG gene. Gradient PCR was performed to detect the two bands of 321bp and 218bp for CpG islands of promoter and intron of PTPRG respectively. Bisulfite-specific PCR technique was carried out to amplify a fragment of bisulfite-converted DNA that has been pre-defined by the designed bisulfite-specific primers. The Methylation PCR amplified product was sequenced via Sanger Sequencing (Applied Biosystems 3130 Genetic Analyzer). The methylation analysis was performed to study the methylation status of the CpG islands using the ESME (Epigenetic Sequencing Methylation) Analysis Software. Results: Results of the methylation in 25 CpG sites of Promoter region of PTPRG: There are significant differences in the mean of methylation status amongst CML patients at diagnosis and healthy individuals of CpG promoter of PTPRG (p value is ˂0.04). However, there are no significant differences in the mean of methylation status between patients failed treatments and other groups (Figure1) Results of the methylation in 26 CpG sites of Intronic region of PTPRG: There are also significant differences in the mean of methylation status amongst all groups (p value is ˂0.003). (Figure1) Discussion and Conclusions: This is the first prospective study to evaluate epigenetic changes of PTPRG gene from CML patients in Qatar. We previously documented that the expression level of PTPRG is inversely correlated with the expression of BCR-ABL1 in CML patients. In this study, the mechanism behind the downregulation of PTPRG in our cohorts of CML patients was explored and the aberrant methylation of PTPRG could be a mechanism associated with CML disease progression and resistance to TKIs. However, further studies are still needed including larger patient's cohort and CML patients responding to the TKIs. Finally, the methylation of PTPRG in the CML patients at diagnosis and failed treatment may allow the identification of new therapeutic targets and using methylation inhibitors could be also suggested to prevent or reverse TKIs resistance and restore TKIs sensitivity. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Alberto Stolfi ◽  
Elijah K Lowe ◽  
Claudia Racioppi ◽  
Filomena Ristoratore ◽  
C Titus Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Embryonic Cell ◽  
Regulatory Mechanisms ◽  
Regulatory Sequences ◽  
Pharyngeal Muscle ◽  
Fate Specification ◽  
Cell Lineages ◽  
Identical Cell ◽  
Cis And Trans

Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or ‘unintelligibility’, of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis.

An Upstream Insulator Element Regulates DLK1 Imprinting in AML

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 202-202
Author(s):  
Haytham Khoury ◽  
Fernando Suarez-Saiz ◽  
Samantha Wu ◽  
Serban San-Marina ◽  
Mark Minden
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Ctcf Binding ◽  
Monoallelic Expression ◽  
Methylation Analysis ◽  
Insulator Element ◽  
Significant Difference

Abstract DLK1 is a transmembrane protein of the epidermal growth factor (EGF) family, encoded by a paternally-imprinted gene located within the chromosomal region 14q32. In addition to DLK1, this region contains other paternally expressed genes, including Dio3 and RTl1 and a set of genes expressed from the maternal chromosome, including MEG3, C/D snoRNA and Mirg. In mice, the expression of these paternally- and maternally-imprinted genes is inversely correlated and controlled by the methylation status intergenic differentially methylated region (IG-DMR). Recently it has been reported that DLK1 is expressed at higher than normal levels in myelodysplasia (MDS) and also in acute myeloblastic leukemia (AML). To determine whether loss of imprinting (LOI) could account for DLK1 overexpression and to study the mechanisms that regulate DLK1 imprinting in AML, we analyzed the expression of 3 informative coding SNPs located in exon 5 (rs#1802710, rs#2295660, rs#1058009) in 11 normal bone marrows (NBMs), the 3 cell lines (OCI/AML-5, NB4 and K562) characterized by DLK1 upregulation and 40 AML patients (pts) with DLK1 overexpression. Furthermore, we undertook quantitative methylation analysis, using the MassARRAY system, of 7 CpG rich-reas: 3 located upstream or within MEG3 and correspond to the CpG islands # 30, 45 and 70, 1 corresponds to the putative IG-DMR, and 3 located upstream or within DLK1 and correspond to the CpG islands # 26, 65 and 79. Informative SNPs were found in 6 NBMs, 28 AML pts and the cell line K562. Of these, biallelic DLK1 expression was found in the cell line K562 as well as 22/28 (72%) AML pts. In contrast, all NBMs and 7 AML pts were found to have monoallelic expression. Pts with biallelic DLK1 expression showed higher DLK1/GAPDH than pts with monoallelic expression (median: 0.0057 vs 0.0024). On the other hand, no significant difference in MEG3 levels was found between the two groups (P = 0.49) and no correlation was found between DLK1 and MEG3 levels (r= −0.1212). The quantitative methylation analysis revealed no difference between the pts with monoallelic and biallelic DLK1 expression in the methylation patterns of the CpG islands # 30, 45, 70, 65 and 26 or the IG-DMR. In contrast, significant difference was found in the methylation the CpG island #79, located 18 kb upstream DLK1 (P <0.0001). In fact, there was a strong association between bi-allelic DLK1 expression and the hypermethylation of the latter CpG island, suggesting that this region contains an insulator element that regulates DLK1 imprinting and transcription through a methylation-sensitive mechanisms. Almost all insulator elements use the zinc finger protein CTCF to achieve their silencing activity. Indeed, a bioinformatic search indicated the presence of at least 4 predicted CTCF-binding sites. The CpG dinucleotides located within or in the vicinity of these binding sites were largely hypermethylated in AML with biallelic DLK1 expression, in sharp contrast with NBMs and pts with monoallelic DLK1 expression. Chromatin immunoprecipitation analysis confirmed that the CTCF protein binds to this region in 1 NBM and 2 pts with monoallelic expression, whereas no immunoprecipitation was seen in the K562 and 2 pts with biallelic DLK1 expression. Taken together, our data suggest DLK1 LOI occurs in 72% of AML and the expression of the paternally-imprinted DLK1 and the maternally-imprinted MEG3 genes are not coordinated in AML. Furthermore, an insulator element located 18 kb upstream DLK1 plays important role in controlling this gene imprinting in AML, through interaction with CTCF.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Aberrant Genes Promoter Methylation in Neural Crest-Derived Tumors

2012 ◽  
Vol 27 (4) ◽  
pp. 389-394 ◽  
Author(s):  
Annamaria La Torre ◽  
Lucia Anna Muscarella ◽  
Paola Parrella ◽  
Teresa Balsamo ◽  
Michele Bisceglia ◽  
...  
Keyword(s):  
Small Bowel ◽  
Neural Crest ◽  
Promoter Methylation ◽  
Methylation Status ◽  
Cpg Islands ◽  
Aberrant Methylation ◽  
Tumor Type ◽  
Epigenetic Landscape ◽  
Molecular Biomarker

Disturbances in the epigenetic landscape by aberrant methylation of CpG islands can lead to inactivation of cancer-related genes in solid tumors. We analyzed the promoter methylation status of 6 genes previously reported as cancer-specific methylated (MCAM, SSBP2, NISCH, B4GALT1, KIF1A and RASSF1A) in 38 neural crest-derived tumors by quantitative methylation-specific real-time PCR (QMSP). The results demonstrated that the determination of the methylation status of RASSF1A is able to distinguish between normal and tumor samples in cutaneous melanomas, lung carcinoids and small bowel carcinoids. MCAM methylation levels were significantly higher in lung carcinoids tumors (p=0.001), suggesting that this alteration may represent a molecular biomarker in this tumor type.

scholarly journals Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value

2021 ◽  
Vol 43 (3) ◽  
pp. 1419-1435
Author(s):  
Walter Pulverer ◽  
Kristi Kruusmaa ◽  
Silvia Schönthaler ◽  
Jasmin Huber ◽  
Marko Bitenc ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Therapy Monitoring ◽  
Methylation Status ◽  
Methylation Analysis ◽  
Microarray Experiments ◽  
Specific Pcr ◽  
Colonic Adenomas ◽  
Methylation Sensitive Restriction Enzyme ◽  
Dna Methylation Analysis

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes’ single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

Reproducibility-optimized detection of differential DNA methylation

Epigenomics ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 747-755
Author(s):  
Veronika Suni ◽  
Fatemeh Seyednasrollah ◽  
Bishwa Ghimire ◽  
Sini Junttila ◽  
Asta Laiho ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Throughput Sequencing ◽  
State Of The Art ◽  
Methylation Status ◽  
Real Data ◽  
Epigenetic Mechanism ◽  
Methylation Analysis ◽  
Test Statistic ◽  
Differentially Methylated Regions ◽  
Dna Methylation Analysis

Aim: DNA methylation is a key epigenetic mechanism regulating gene expression. Identifying differentially methylated regions is integral to DNA methylation analysis and there is a need for robust tools reliably detecting regions with significant differences in their methylation status. Materials & methods: We present here a reproducibility-optimized test statistic (ROTS) for detection of differential DNA methylation from high-throughput sequencing or array-based data. Results: Using both simulated and real data, we demonstrate the ability of ROTS to identify differential methylation between sample groups. Conclusion: Compared with state-of-the-art methods, ROTS shows competitive sensitivity and specificity in detecting consistently differentially methylated regions.

Sign in / Sign up

Export Citation Format

Share Document