Dot1 and histone H3K79 methylation in natural telomeric and HM silencing

The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a URA3 reporter located at TEL-VII-L of Saccharomyces cerevisiae, it was proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation. URA3 reporter assays also indicated that H3K79 methylation is required for HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as COS12 at TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural HML silencing. Therefore, commonly used URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.     

Prokaryotic cytochromes P450 (Review)

The research on the structure and role of bacterial cytochromes P450 are summarized in this review. We consider the organizational features of these enzymes, cytochrome-catalyzed reactions, the distribution of cytochromes among prokaryotes, and their functions in bacterial cells. We cite the data on cytochrome genes and the regulation of their expression in prokaryotes and classify cytochromes by components involved in the electronic transition. We consider the role of bacterial cytochromes in the biodegradation of carbohydrates and xenobiotics by microorganisms and the possible involvement of reactive oxygen species, which are generated in the catalytic cycle of these enzymes, at the initial stages of carbohydrate biodegradation.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

Purification of cytochromes P-450(scc) and P-450(17 alpha) by steroid-binding affinity column chromatography.

The preparation, testing and use of a variety of cholesterol-, deoxycorticosterone (DOC)- and pregnenolone-binding 1,6-diaminohexyl (EAH)-Sepharose 4B supports for affinity column chromatography of cytochromes P-450(scc) and P-450(17 alpha) from bovine adrenal and pig testis are described. EAH-Sepharose 4B has free amino groups at the end of a 10-atom spacer arm. Hydroxyl groups of cholesterol (3 beta), deoxycorticosterone (21 beta) and pregnenolone (3 beta) are linked to succinic anhydride in pyridine through an ester linkage. These coupling ligands of hemisuccinate were synthesized by a general procedure. Free amino groups of EAH-Sepharose 4B were used to couple ligands, containing carboxyl groups, by the carbodiimide coupling method. Both the purified cytochromes P-450(scc) and P-450(17 alpha) were found to be homogeneous and estimated to have a molecular weight of 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectra with peaks at 450 and 448 nm exhibit the absorption spectra of typical cytochromes P-450(scc) and P-450(17 alpha), respectively. Cytochromes P-450(scc) and P-450(17 alpha) were determined to have isoelectric points of 8.0 and 6.5 in isoelectric focusing on a pH gradient gel. Cytochrome P-450s can be purified between 425- and 1000-fold from the crude extracts.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

Development of bacterial cytochrome P-450(cam) (cytochrome m) production

Cytochrome P-450(cam) monooxygenase is an important bacterial redox enzyme system with potential commercial value for detoxifying trace hydrocarbon contaminants, catalyzing regiospecific hydroxylations, and amperometric biosensing. The present study was undertaken to increase productivity of this enzyme, which is induced in its host, pseudomonas putida PpG 786, by D(+)-camphor. Culture processes were studied in batch, fed-batch, and continuous modes to evaluate metabolic behavior and develop constitutive equations for specific rate of growth (mu), camphor utilization (q(p)). Fed-batch culture was characterized by an extended linear growth phase which is often encountered in hydrocarbon fermentations. Inhibition by the camphor solvent, dimethylformamide, was assessed. Production of the terminal protein of the p-450(cam) enzyme system, cytochrome m, was shown to depend on growth medium iron content in fed-batch culture and was increased by 130% over previously protocols by eliminating iron deficiency. A continuous process that enables greater production rates was developed by using oxygen enrichment while simultaneously reducing gas throughput. Camphor and oxygen requirements were determined for fedbatch and continuous growth. (c) 1993 John Wiley & Sons, Inc.     

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.     

Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips

Illumina’s Infinium HumanMethylation450 BeadChip arrays were used to examine genome-wide DNA methylation profiles in 22 sample pairs from colorectal cancer (CRC) and adjacent tissues and 19 colon tissue samples from cancer-free donors. We show that the methylation profiles of tumors and healthy tissue samples can be clearly distinguished from one another and that the main source of methylation variability is associated with disease status. We used different statistical approaches to evaluate the methylation data. In general, at the CpG-site level, we found that common CRC-specific methylation patterns consist of at least 15,667 CpG sites that were significantly different from either adjacent healthy tissue or tissue from cancer-free subjects. Of these sites, 10,342 were hypermethylated in CRC, and 5,325 were hypomethylated. Hypermethylated sites were common in the maximum number of sample pairs and were mostly located in CpG islands, where they were significantly enriched for differentially methylated regions known to be cancer-specific. In contrast, hypomethylated sites were mostly located in CpG shores and were generally sample-specific. Despite the considerable variability in methylation data, we selected a panel of 14 highly robust candidates showing methylation marks in genes SND1, ADHFE1, OPLAH, TLX2, C1orf70, ZFP64, NR5A2, and COL4A. This set was successfully cross-validated using methylation data from 209 CRC samples and 38 healthy tissue samples from The Cancer Genome Atlas consortium (AUC = 0.981 [95% CI: 0.9677–0.9939], sensitivity = 100% and specificity = 82%). In summary, this study reports a large number of loci with novel differential methylation statuses, some of which may serve as candidate markers for diagnostic purposes.     

A framework for analyzing DNA methylation data from Illumina Infinium HumanMethylation450 BeadChip

Background

DNA methylation has been identified to be widely associated to complex diseases. Among biological platforms to profile DNA methylation in human, the Illumina Infinium HumanMethylation450 BeadChip (450K) has been accepted as one of the most efficient technologies. However, challenges exist in analysis of DNA methylation data generated by this technology due to widespread biases.

Results

Here we proposed a generalized framework for evaluating data analysis methods for Illumina 450K array. This framework considers the following steps towards a successful analysis: importing data, quality control, within-array normalization, correcting type bias, detecting differentially methylated probes or regions and biological interpretation.

Conclusions

We evaluated five methods using three real datasets, and proposed outperform methods for the Illumina 450K array data analysis. Minfi and methylumi are optimal choice when analyzing small dataset. BMIQ and RCP are proper to correcting type bias and the normalized result of them can be used to discover DMPs. R package missMethyl is suitable for GO term enrichment analysis and biological interpretation.

     

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.     

Quantification of DNA methylation in electrofluidics chips (Bio-COBRA)

Alterations of normal gene expression patterns are a hallmark of human cancers. It is now clear that the dysregulation of epigenetic modifications of the DNA and surrounding histones contributes to aberrant gene silencing, thus being major participants not only in the progression but also the initiation of the disease phenotype. The best-studied epigenetic modification is DNA methylation, which converts cytosine to 5-methylcytosine. Aberrant hypermethylation of the promoter is frequently observed in cancer and is generally associated with gene silencing. Currently, accurate and reproducible quantification of DNA methylation remains challenging. Here, we describe Bio-COBRA, a modified protocol for Combined Bisulfite Restriction Analysis (COBRA), that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. In the life sciences, microfluidics usually entails the scaling down of at least one application, such as electrophoresis, to chip format, which often results in increased efficiency and reliability. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also makes it a tool for the analysis of DNA methylation in clinical samples. The Bio-COBRA assay can be performed on 12 samples in less than 1 h. If the protocol is started at the DNA isolation step, however, approximately 48 h would be required to complete the entire procedure.     

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (V_max) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.     

Effective cytochrome P450 (CYP) inhibitors isolated from tarragon (Artemisia dracunculus)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 μM for compound 1 and 3.3 ± 0.2 μM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.     

Effective Cytochrome P450 (CYP) Inhibitors Isolated from Tarragon (Artemisia dracunculus)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC₅₀ values of 10.0 ± 1.3 µM for compound 1 and 3.3 ± 0.2 µM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips

Hepatocellular carcinoma (HCC) incidence has increased in the US and also has one of the fastest growing death rates of any cancer. The purpose of the current study was to discover novel genome-wide aberrant DNA methylation patterns in HCC tumors that are predominantly HCV-related. Infinium HumanMethylation 450K BeadChip arrays were used to examine genome-wide DNA methylation profiles in 66 pairs of HCC tumor and adjacent non-tumor tissues. After Bonferroni adjustment, a total of 130,512 CpG sites significantly differed in methylation level in tumor compared with non-tumor tissues, with 28,017 CpG sites hypermethylated and 102,495 hypomethylated in tumor tissues. Absolute tumor/non-tumor methylation differences ≥ 20% were found in 24.9% of the hypermethylated and 43.1% of the hypomethylated CpG sites; almost 10,000 CpG sites have ≥ 30% DNA methylation differences. Most (60.1%) significantly hypermethylated CpG sites are located in CpG islands, with 21.6% in CpG shores and 3.6% in shelves. In contrast, only a small proportion (8.2%) of significantly hypomethylated CpG sites are situated in islands, while most are found in open sea (60.2%), shore (17.3%) or shelf (14.3%) regions. A total of 2,568 significant CpG sites (2,441 hypermethylated and 127 hypomethylated) covering 589 genes are located within 684 differentially methylated regions defined as regions with at least two significant CpG sites displaying > 20% methylation differences in the same direction within 250-bp. The top 500 significant CpG sites can significantly distinguish HCC tumor from adjacent tissues with one misclassification. Within adjacent non-tumor tissues, we also identified 75 CpG sites significantly associated with gender, 228 with HCV infection, 17,207 with cirrhosis, and 56 with both HCV infection and cirrhosis after multiple comparisons adjustment. Aberrant DNA methylation profiles across the genome were identified in tumor tissues from US HCC cases that are predominantly related to HCV infection. These results demonstrate the significance of aberrant DNA methylation in HCC tumorigenesis.