Different Degrees Of Methylation Research Articles

Gut Epithelial Barrier Function is Impacted by Hyperglycemia and Secondary Bile Acids in Vitro: Possible Rescuing Effects of Specific Pectins.

Gut epithelial barrier disruption is commonly observed in Western diseases like diabetes and inflammatory bowel disease (IBD). Enhanced epithelial permeability triggers inflammatory responses and gut microbiota dysbiosis. Reduced bacterial diversity in IBD affects gut microbiota metabolism, altering microbial products such as secondary bile acids (BAs), which potentially play a role in gut barrier regulation and immunity. Dietary fibers such as pectin may substitute effects of these BAs. The study examines transepithelial electrical resistance of gut epithelial T84 cells and the gene expression of tight junctions after exposure to (un)sulfated secondary BAs. This is compared to the impact of the dietary fiber pectin with different degrees of methylation (DM) and blockiness (DB), with disruption induced by calcium ionophore A23187 under both normal and hyperglycemic conditions. Unsulfated lithocholic acid (LCA) and deoxycholic acid (DCA) show a stronger rescuing effect, particularly evident under 20mM glucose levels. DM19 with high DB (HB) and DM43HB pectin exhibit rescuing effects under both glucose conditions. Notably, DM19HB and DM43HB display higher rescue effects under 20mM glucose compared to 5mM glucose. The study demonstrates that specific pectins such as DM19HB and DM43HB may serve as alternatives for preventing barrier disruption in the case of disturbed DCA metabolism.

Tuning Properties of Layered Materials Based on Hexagonal Boron Nitride by Methylation: A Density Functional Theory Study.

In this work, the rational design of optoelectronic properties of two-dimensional materials based on hexagonal boron nitride (h-BN) by functionalization by methyl (CH3) groups is proposed. Using density functional theory, we examine the functionalization of single- or double-layer systems with either CH3 radicals alone or with both CH3 (cations) and chlorine (anions), i. e., under conditions of homolytic or heterolytic splitting of CH3Cl precursor molecules, respectively. Different degrees of methylation (coverages) are considered. The methylation of pure h-BN leads to a reduction of the band gap, while in h-BN/G heterostructures (with methylated graphene layer), methylation increases the band gap. As a consequence, h-BN/G heterostructures offer a high tunability of their optoelectronic properties. To guide possible experiments, vibrational properties and spectra of methylated h-BN and methylated h-BN/G are determined.

CONTROL OF THE SELECTIVITY OF THE SEPARATION OF GRAPE ANTHOCYANIDIN 3-GLUCOSIDES AND 3,5-DIGLUCOSIDES: DETERMINATION OF ANTHOCYANINS IN GRAPE FRUIT GROWN IN THE BELGOROD REGION

For the first time, a new version of the separation of 3-glucosides and 3,5-diglucosides of the five main anthocyanidins for grape fruits - delphinidin, cyanidin, petunidin, peonidin and malvidin under reverse-phase HPLC conditions was proposed. It has been shown that the replacement of the traditionally used acetonitrile with environmentally more acceptable ethanol upon acidification with ortho-phosphoric rather than formic acid makes it possible to significantly change the selectivity of the separation of two types of glucosides at their joint presence. The proposed separation option allows differentiating between Vitis vinifera grapes, in the fruit peel of which only 3-glucosides of the above anthocyanidins are synthesized, and grapes of other species or hybrid grape varieties. For a complete review of the anthocyanin composition, it is necessary to use a gradient mode, since the acylation of anthocyanins with acetic and p-coumaric acids significantly changes the lipophilicity of anthocyanins. The type of anthocyanins was analyzed according to the previously proposed system, which takes into account the activity of three types of enzymes: 1) 5-О-glucosyltransferase that leads to 3,5-diglucosides synthesis; 2) 3',5'-hydroxylase responsible for ring B hydroxylation; 3) anthocyanin O-methyltransferase for converting cyanidin into peonidin derivatives as well as delphinidine into petunidin and malvidin glycosides. The method was used to determine anthocyanins in 43 grape varieties grown in Belgorod on farms and private farms. Among the studied grapes, varieties were found with grape fruits with the accumulation of only 3-glucosides, as well as 3-glucosides and 3,5-diglucosides simultaneously, with delphinidin and cyanidin types of anthocyanins, and with different degrees of methylation. The calculated parameters for all grape varieties are presented in two tables and a brief discussion of some varieties is given.

Probing strong steric hindrance effects in aqueous alkanolamines for CO2 capture from first principles

Carbon dioxide (CO2) absorption into aqueous amine solvents is known to be governed by two competing reactions leading to carbamate or bicarbonate formation. Sterically hindered alkanolamines with different degrees of methylation are experimentally known to cause different favorability in forming carbamate or bicarbonate. However, the fundamental mechanisms responsible for the preferential formation of carbamate or bicarbonate still remain unclear. Herein, we present CO2 absorption behavior with several different sterically hindered amines, especially the effects of methylation and temperature. Our ab initio metadynamics simulations clearly demonstrate that free-energy barriers for the carbamate and bicarbonate formation reactions strongly depend on the degree of steric hindrance of hydrophobic methyl groups. This in turn directly impacts on the kinetic favorability of the two competing reaction pathways. Our analysis shows that various degrees of steric effects cause considerably different temperature-dependent degrees of solvation of the nitrogen atom of hindered amines, which may directly affect CO2 accessibility to form carbamate. Moreover, the enhanced solvation as a result of increased steric effects particularly at low absorption temperatures lead to kinetically facile protonation of the nitrogen atom of amines, initiating and promoting bicarbonate formation. Our work sheds light on the effects of various degrees of steric hindrance, with different temperature-dependent solvation degrees governing reaction mechanisms and rates during CO2 capture.

Abstract 3376: Novel reference materials for the analysis of methylation in liquid biopsies

Abstract Here we describe new reference materials (RMs) for liquid biopsies that are designed to mimic the methylation found in circulating cell-free DNA (ccfDNA). Methylation is an important epigenetic modification that influences cellular differentiation and gene expression. Recently, liquid biopsies have started to incorporate analyses of epigenetic modifications for screening purposes to detect cancer-derived DNA in the blood. Additionally, epigenetic modifications are being used to also assign a tissue of origin to the cancer to direct confirmatory diagnostic procedures. Obtaining sufficient amounts of ccfDNA for assay development, validation, and proficiency testing is difficult. Furthermore, methods of analyzing the epigenetic modifications, such as bisulfite conversion, can damage a significant fraction of the input material; but, failing to carry them out to completion can result in an over-estimation of methylation. To address the challenges associated with assessing the methylation of ccfDNA, we created and characterized three types of RMs. The first type of RM is a fragmented genome of the GM24385 reference cell line that is used to create a set of RMs with different degrees of methylation (0%, ~25%, ~50%, ~75% and close to 100%). The exact degree of methylation was assigned by digital PCR using assays for heterozygous SNPs, where one allele is both a site for CpG methylation and a recognition site for a methylation-sensitive restriction enzyme while the other allele is not. The second type of RM is an amplified ccfDNA sample that has been methylated similarly but also reproduces the fragment lengths and genomic representation biases of ccfDNA. The degree of methylation was assigned by digital PCR, but only a subset of assays was useful because not all SNPs were heterozygous. The third type of RM is an amplified ccfDNA sample that has been processed to mimic the methylation found in the original ccfDNA. Analyses of the RMs by whole genome sequencing (WGS) showed that, while bisulfite conversion and/or the amplification of the resulting dU-containing DNA appeared to lead to significant biases in genomic representation, the resulting data was generally in agreement with the amount of methylation assigned by digital PCR. Additionally, it was possible to prepare an RM that generally reproduces the methylation found in ccfDNA; although, this was limited to individual molecules having either no or close to full methylation, which is unlikely to be the case for all molecules of ccfDNA in vivo. Citation Format: Jayanthi Ramprakash, Matthew G. Butler, Russell K. Garlick, Yves Konigshofer. Novel reference materials for the analysis of methylation in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3376.

Methylation of Cyanidin-3-O-Glucoside with Dimethyl Carbonate.

The approach presented in this study is the first for the hemisynthesis of methylated anthocyanins. It was possible to obtain cyanidin-3-O-glucoside derivatives with different degrees of methylation. Cautious identification of 4′-, 5-, and 7-OH monomethylated derivatives was also accomplished. The methylation agent used was the “green chemical” dimethyl carbonate (DMC), which is characterized by low human and ecological toxicity. The influence of the temperature, reaction time, and amount of the required diazabicyclo[5.4.0]undec-7-en (DBU) catalyst on the formation of the products was examined. Compared to conventional synthesis methods for methylated flavonoids using DMC and DBU, the conditions identified in this study result in a reduction of reaction time, and an important side reaction, so-called carboxymethylation, was minimized by using higher amounts of catalyst.

Mobility Tuning of Polyrotaxane Surfaces to Stimulate Myocyte Differentiation

Polyrotaxanes, consisting of poly(ethylene glycol) and α-cyclodextrins, are mechanically interlocked supermolecules. The structure allows α-cyclodextrins to move along the polymer, referred to as molecular mobility. Here, polyrotaxane-based triblock copolymers, composed of polyrotaxanes with different degrees of methylation and poly(benzyl methacrylate) at both terminals, are coated on culture surfaces to fabricate dynamic biointerfaces for myocyte differentiation. The molecular mobility increases with the degree of methylation and the contact angle hysteresis of water droplets and air bubbles. When the mouse myoblast cell line C2C12 is cultured on methylated polyrotaxane surfaces, the expression levels of myogenesis-related genes, myogenin (Myog) and myosin heavy chain (Myhc) are altered by the degree of methylation. Polyrotaxane surfaces with intermediate degrees of methylation promote the highest expression levels among all the surfaces. The polyrotaxane surface provides an appropriate environment for myocyte differentiation by accurately adjusting the degrees of methylation.

Study on the exposure of polychlorinated biphenyl contamination and DNA methylation in male employees in an e-waste dismantling area in Tianjin

Objective: To study on the exposure of polychlorinated biphenyl (PCB) contamination and DNA methylation in male employees in an e-waste dismantling area in Tianjin. Methods: In 2016, an e-waste dismantling area in Tianjin and an area 50 km away from the e-waste dismantling area (no e-waste or other chemical, industrial and agricultural pollution nearby) were selected as the study area and the reference area. Male residents of the study area and male farmers who planted vegetables, fruits, and crops in the reference area were selected as the exposed and reference group by using the convenient sampling method. A total of 60 subjects (30 in each of the exposed group and the reference group) were included. The peripheral blood (5 ml) of the study subject was collected, and the PCB concentration was detected. Eight independent subjects in the exposed group and the reference group were randomly selected by random number table method to detect the methylation level of the promoter region of all gene loci, and the mRNA transcript levels. Results: The PCB concentration in peripheral blood of the exposed group was higher than that of the reference group, and the difference was statistically significant (allP values <0.001). The methylation levels of the promoter region of the exposed group and the reference group showed obvious clustering, and 994 gene loci had different degrees of methylation. Compared with the reference group, there were 391 hypomethylation sites and 553 hypermethylation sites in the exposed group. The proportion of methylation sites in the high CpG-rich region was 59.2% and 48.1%, respectively. The mRNA level of the hypomethylated gene in the exposed group was higher (FAM131A, HBM), and the transcription level of the hypermethylated gene was lower (CAPN15, NFIC, SHISA5, FGF13, GRAMD1A, CLEC3B, LILRB2, DCAF7). The mRNA transcription levels of 10 genes above in the exposed group and the reference group were statistically significant (all P values <0.001). Conclusion: The PCB concentration of peripheral blood in the exposed population of e-waste is high. PCB exposure changes the methylation level of specific genes and affects the mRNA transcription level of some genes.

Methylation of Epstein-Barr Virus-Associated Gastric Cancer Suppressor Genes

Objective: To identify the methylation silenced tumor suppressor genes in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC). Methods: EBV-positive (GT38, PT and SNU719) and negative (SGC7901) gastric cancer cell lines were selected and treated with 5-Aza-CdR. Then real-time fluorescence quantitative PCR was used to validate the results of microarray, and methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) were adopted to detect the CpG island methylation levels of gene promoters. Results: The expression levels of 6 differentially expressed genes (H19, LOXL1, ARMCX2, LXN, CDH3 and MMP7) before and after 5-Aza-CdR treatment were confirmed by real-time qPCR, which are consistent with the results of microarray analysis. There were different degrees of methylation in LOXL1 gene promoter in EBVaGC. GT38 and PT were fully methylated, and SGC7901 and HGC-27 was unmethylated, suggesting that this gene is a candidate methylation silenced tumor suppressor gene. The methylation rate of LOXL1 in EBVaGC was significantly higher than that in EBV-negative gastric cancer (EBVnGC). Conclusion: The promoter region of candidate tumor suppressor gene LOXL1 shows high methylation status, indicating that EBV critically accounts for the methylation of LOXL1 gene regulatory region. EBV is involved in the pathogensis of EBVaGC that aberrant methylation occurred in promoter CpG island, which inactivates tumor suppressor genes.

Mesoporous Methyl-Functionalized Sn-Silicates Generated by the Aerosol Process for the Sustainable Production of Ethyl Lactate

The aerosol-assisted sol–gel process was used to synthesize in a straightforward one-pot procedure a series of methyl-functionalized tin silicates with different degrees of methylation. The successful incorporation of isolated Sn as single site within the silica framework was confirmed via 119Sn solid-state NMR measurements, while 29Si and 13C solid-state magic-angle-spinning NMR experiments revealed a degree of methylation close to the theoretical value, hence proving the efficacy of the adopted cosynthetic approach. These materials were tested as catalysts in the synthesis of ethyl lactate from dihydroxyacetone and ethanol. The methylated solids display enhanced performances in terms of both activity and selectivity compared to the nonmethylated analogues, highlighting a strong beneficial role of surface hydrophobicity. Under proper conditions a total conversion and a selectivity higher than 95% were achieved. Moreover, the efficient separation and reuse of the heterogeneous catalyst as well as the poss...

Effects of cytosine methylation on DNA morphology: An atomic force microscopy study

Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.

Aminoarenethiolate Aluminum Complexes: Synthesis, Characterization, and Use in l-Lactide Polymerization

Reaction of AlMe3 with S(SiMe3)(C6H3-2-CH2NRR′-5-tBu) (RR′ = C5H10 (1a), C4H8 (1b), Me2 (1c)), at ambient temperature, affords the amino adducts [AlMe3{S(SiMe3)(C6H3-2-CH2NRR′-5-tBu)}-κN] (RR′ = C5H10 (2a), C4H8 (2b), Me2 (2c)), which undergo TMS elimination upon heating to give the monomeric aminoarenethiolate aluminum complexes [AlMe2{S(C6H3-2-CH2NRR′-5-tBu)-κ2S,N}] (RR′ = C5H10 (3a), C4H8 (3b), Me2 (3c)). Following the same procedure, treatment of AlCl2Me and AlCl3 with 1 yields analogous aminoarenethiolate aluminum complexes with different degrees of methylation, the chloro methyl and dichloro complexes [AlClMe{S(C6H3-2-CH2NRR′-5-tBu)-κ2S,N}] (RR′ = C5H10 (4a), C4H8 (4b), Me2 (4c)) and [AlCl2{S(C6H3-2-CH2NRR′-5-tBu)}-κ2S,N] (RR′ = C5H10 (5a), C4H8 (5b) Me2 (5c)), respectively. These complexes have been characterized by multinuclear NMR spectroscopy and elemental analysis. Moreover, the molecular structures of 3a,b have been determined by X-ray diffraction methods. Aluminum complexes 3 have been investigated for the ring-opening polymerization (ROP) of l-lactide, achieving high conversions in relatively short periods of time. The PLAs obtained feature an aminoarenethiolate end functionality, as inferred from MALDI-TOF mass analysis.

Methylation of ethanolamine groups in phosphoethanolamines is relevant for L-arginine insertion in lipid membranes

The interaction of L-arginine with membranes composed by phospholipids with different degrees of methylation of the ethanolamine group was studied by means of surface and dipole potentials and surface pressure variations. The subsequent methylation of the amine head group appears to hinder the synergic response of the adsorption observed in phosphatidylethanolamine membranes. The kinetics of the binding process denotes that the methyl groups are relevant in regulating the specific interaction of the amino acid with the interface by hydrogen bonds. This response can be put in correlation with the function of signal transduction assigned previously to methyl lipids [F. Hirata and J. Axelrod, 1980] and appears to be relevant to understand the mechanism of insertion of arginine residues in peptides of biological interest.

Urinary arsenic speciation profiles in mice subchronically exposed to low concentrations of sodium arsenate in drinking water

Arsenic is a proven human carcinogen. Although the mechanism of its carcinogenicity is still largely unknown, methylation is thought to have an important role to play in arsenic toxicity. In this study, urinary methylation profiles were investigated in female C57BL/6J black mice given drinking water containing 500 μg arsenate (As V)/L, 250 μg As V/L, or 100 μg As V/L as sodium arsenate for 2 months. The concentrations of arsenic chosen reflected those in the drinking water often encountered in arsenic-endemic areas. Urine samples were collected from the mice at the end of the exposure period, and the arsenic species were analyzed by high performance liquid chromatography-inductively coupled plasma-mass spectrometry. All detectable arsenic species showed strong linear correlation with the administered dosage. The methylation patterns were similar in all three groups with a slight decrease of dimethylarsinic acid/As V ratio in the 500-μg/L group, which corresponded to the significantly higher arsenic retention in the tissue. The results indicate that urinary arsenic could be used as a good biomarker for internal dose and potential biological effects. Different doses of arsenic exposure could result in different degrees of methylation, excretion, and tissue retention, and this may contribute to the understanding of arsenic carcinogenicity.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

Molecular Mechanisms of Lysine Methylation

SET domain lysine methyltransferases (KMTs) are S‐adenosylmethionine (AdoMet)‐dependent enzymes that catalyze the site‐specific methylation of lysine residues in histones, transcription factors, and other protein substrates. SET domain KMTs also display product specificity, which is defined as their ability to catalyze different degrees of methylation of the lysine‐epsilon amine group, thus imparting an additional hierarchy in methyllysine signaling. To understand the mechanism underlying product specificity, we have characterized two active site mutants of the monomethylase SET7/9 that alter its specificity to a dimethylase and a trimethylase, respectively. Structures of the SET7/9 mutants in complex with peptides bearing unmodified, mono‐, di‐, and trimethylated lysines reveal that water molecules within the active site function as place holders that linearly align the lysine epsilon‐amine group with the methyl group of AdoMet to promote methyltransfer. As the methylation state of the lysine substrate increases during successive reactions, the water molecules dissociate from the active site, thereby enlarging the lysine binding channel to accommodate the increasing bulk of the methylated epsilon‐amine group. Collectively, our findings illuminate the catalytic roles of active site water molecules in facilitating lysine multiple methylation by SET domain KMTs.

Molecular Mechanisms of Lysine Methylation

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysine residues in histones, transcription factors, chromatin modifying enzymes, and other protein substrates. These modifications mediate protein:protein interactions with signaling factors that possess effector modules which can recognize methyllysines in a sequence-dependent manner. In addition to their site specificity, SET domain KMTs exhibit product specificity, which is defined as their ability to catalyze different degrees of methylation of the lysine epsilon amine group, thus imparting an additional hierarchy in methyllysine signaling. To understand the mechanism underlying the product specificity of SET domain KMTs, we have structurally and functionally characterized two active site mutants of the human monomethylase SET7/9 that alter its specificity to a dimethylase and a trimethylase, respectively. Structures of the SET7/9 mutants in complex with peptides bearing unmodified, mono, di, and trimethylated lysines reveal that water molecules within the active site function as molecular place holders that align the lysine epsilon amine group in a linear geometry with the sulfonium methyl group of AdoMet to promote methyltransfer. As the methylation state of the lysine substrate increases during successive methyltransfer reactions, the water molecules dissociate from the active site, thereby enlarging the lysine binding channel to accommodate the increasing bulk of the multiply methylated epsilon amine group. Taken together, our findings illuminate the catalytic roles of active site water molecules in facilitating lysine multiple methylation by SET domain KMTs.

Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

Insulin-like Growth Factor-II Methylation Status in Lymphocyte DNA and Colon Cancer Risk in the Northern Sweden Health and Disease Cohort

Loss of imprinting (LOI) of the insulin-like growth factor II (IGFII) gene is a frequent phenomenon in colorectal tumor tissues. Previous reports indicated that subjects with colorectal neoplasias show LOI of IGFII in circulating lymphocytes. Furthermore, LOI of IGFII is strongly related to the methylation of a differentially methylated region (DMR) in intron 2 of IGFII, suggesting that the methylation status could serve as a biomarker for early detection. Thus, hypermethylation of this DMR, even at a systemic level, e.g., in lymphocyte DNA, could be used for screening for colon cancer. To validate this, we performed a case-control study of 97 colon cancer cases and 190 age-matched and gender-matched controls, nested within the prospective Northern Sweden Health and Disease Study cohort. Methylation levels of the IGFII-DMR in lymphocyte DNA were measured at two specific CpG sites of the IGFII-DMR using a mass-spectrometric method called short oligonucleotide mass analysis, the measurements of which showed high reproducibility between replicate measurements for the two CpG sites combined and showed almost perfect validity when performed on variable mixtures of methylated and unmethylated standards. Mean fractions of CpG methylation, for the two CpG sites combined, were identical for cases and controls (0.47 and 0.46, respectively; P(difference) = 0.75), and logistic regression analyses showed no relationship between colon cancer risk and quartile levels of CpG methylation. The results from this study population do not support the hypothesis that colon cancer can be predicted from the different degrees of methylation of DMR in the IGFII gene from lymphocyte DNA.

Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.