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PubMed1998

Chromosomal fragility may be indicative of altered higher-order DNA organization as the underlying genetic diathesis in complex neurobehavioral disorders.

Preliminary observations concerning increased chromosomal fragility in association with behavioural disorders in humans allow an opportunity to suggest a cohesive theory regarding the possible importance of higher-order DNA modifications in the coordination of gene function in brain evolution and during development. Visible or submicroscopic acentric chromosomal fragments are formed as an accompaniment to chromosomal breakage and are associated with sequence amplification. During genomic reintegration of extrachromosomally amplified repeat sequence elements, functional consequences could include unequal crossing over with gain-of-function, and/or deletion with loss-of-function. This process could result in regulatory changes in gene function in association with normal coding regions, since fragile sites appear to be located at or near upstream DNaseI-hypersensitive areas. Earlier research on chromosomal breakage in relation to transposon behaviour in maize has set a precedent by which many elements in a network could be coordinately controlled, a principle which may allow transcriptional control over multiple areas in the genome simultaneously. The hypothesis proposed in this paper implies that a small number of fundamental higher-order changes may be responsible for influencing a wide range of genetic alterations leading to complex phenotypes, sometimes segregating as distinct entities within pedigrees, or alternatively, and perhaps more commonly, presenting with several overlapping phenotypes in some other families. Studying only pure multiplex families in psychiatric genetics may not be sufficient for an understanding of the underlying genetic diathesis in this group of disorders. Validation of the fragile site hypothesis for complex neurobehavioural disorders may offer additional avenues for gene therapy based either on preferential integration of exogenous DNA at fragile sites, or utilizing the acentric fragments to modify sequence amplification extrachromosomally.

Chromosome Breakage↗
PubMed1997

Hypochlorous acid-induced base modifications in isolated calf thymus DNA.

Exposure of calf thymus DNA to hypochlorous acid/hypochlorite leads to extensive DNA base modification. Large concentration-dependent increases in pyrimidine oxidation products [thymine glycol (cis/trans), 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxyhydantoin] but not purine oxidation products (8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine) were observed at pH 7.4. In addition, large increases in 5-chlorouracil (probably formed from 5-chlorocytosine during sample preparation), a novel chlorinated base, were observed. Addition of HOCl to DNA already damaged by .OH generated by a mixture of ascorbate, copper(II) chloride, and hydrogen peroxide showed that hypochlorous acid led to a loss of 8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine, and 5-hydroxycytosine in a concentration- and pH-dependent manner. Nevertheless, time course studies suggested that the formation of purine oxidation products in isolated DNA by hypochlorous acid was not a major oxidation pathway. If this pattern of damage, especially the production of 5-chlorocytosine, is unique to hypochlorous acid, it might act as a "fingerprint" of damage to DNA by HOCl.

Animals↗
PubMed1989

Nucleophilic selectivity of carcinogens as a determinant of enhanced mutational response in excision repair-defective strains in Drosophila: effects of 30 carcinogens.

Consequences on mutation induction of a defective excision repair (exr-) system have been studied in Drosophila for a series of 30 carcinogens, representing 17 mono-, six bi- or trifunctional agents, five cyclic alkylating agents and two polycyclic aromatic compounds. Repair-inactive spermatozoa or (late) spermatids were mutagenized and then transferred to excision-defective (mei-9L1) or appropriate excision proficient (exr+) oocytes. Hypermutability responses in exr- in relation to the exr+ genotype were determined by calculating frep-/frep- (ratio = y) indices at x = 1% X-linked recessive lethal mutations (SLRL); frep- denotes induced SLRL frequency in mei-9L1, frep+ that induced in repair-proficient condition, and x = 1 means 1% SLRL in exr+, A linear positive relationship between frep-/frep+ estimates and nucleophilic selectivity (Swain and Scott's constants s) was established for 18 carcinogens, representing either mono- and cyclic alkylating agents: frep-/frep+ = 12.4s - 1.9; r = 0.79, r2 = 0.62, P less than 0.01 Noticeable exceptions to this linear correlation indicated that, although nucleophilicity is a principle determinator for hypermutability response in exr- mutants, other cellular factors play a significant role as well. These are (i) the faster rate of removal of methyl adducts compared to ethyl derivatives, (ii) the complex metabolism of some of the carcinogens (PC, DTIC, DMPT, Cl3DMPT) and (iii) the length of the time period between DNA modification and onset of replication after fertilization. By contrast, CEO and polyfunctional agents (FA, HMPA, MC, MCT, Thio-TEPA and BCNU) did not follow the linear relationship. They provided lower frep-/frep+ indices than would be anticipated on the basis of their nucleophilic selectivity (MC, BCNU, Thio-TEPA) or were even inactive (FA, MCT, HMPA) in the Drosophila repair assay. Both in terms of consistency (mono- and cyclic alkylating agents) and exceptional behavior (CEO, the six crosslinking agents), there is an intriguing positive correlation between relative efficiency ranking of carcinogens and with respect to their position on the potency scale for hypermutability. Thus, in genetic terms, as most potent carcinogens in rodents appear to be those agents giving no effect or a low activity in the exr- genotype in Drosophila: carcinogens with high potential for direct miscoding [ENU, ENNG, DEN, iPMS, CEO and presumably DMBA (?)], and those capable of forming crosslinks (MC, MCT, HMPA, Thio-TEPA and BCNU).

Alkylating Agents↗
PubMed2005

BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes.

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

Algorithms↗
PubMed1999

Chromosomal fragility may be indicative of altered higher-order DNA organization as the underlying genetic diathesis in complex neurobehavioural disorders.

Preliminary observations concerning increased chromosomal fragility in association with certain behavioural disorders in humans allow an opportunity to suggest a cohesive theory regarding the possible importance of higher-order DNA modification in the coordination of gene function in brain evolution and during development. Visible or submicroscopic acentric chromosomal fragments are formed as an accompaniment to chromosomal breakage and are associated with sequence amplification. During genomic reintegration of such extra chromosomally amplified repeat sequence elements, functional consequences could include unequal crossing over with gain-of-function, and/or deletion with loss-of-function. This process could result in regulatory changes in gene function in association with normal coding regions, since fragile sites appear to be located at or near upstream DNase-I hypersensitive areas. Earlier research on chromosomal breakage in relation to transposon behaviour in maize has set a precedent by which many elements in a network could be coordinately controlled, a principle which may allow transcriptional control over multiple areas in the genome simultaneously. The hypothesis proposed in this paper implies that a small number of fundamental higher order changes may be responsible for influencing a wide range of genetic alterations leading to complex phenotypes, sometimes segregating as distinct entities within pedigrees, or alternatively, and perhaps more commonly, presenting with several overlapping phenotypes in some other families. Current emphasis on the investigation of only pure multiplex families in psychiatric genetics may assist with identification of a number of discrete behaviour-modifying genes, but may not be sufficient for an understanding of the broad underlying genetic diathesis in these, and perhaps other 'multifactorial type' disorders. Validation of a role for altered fragile site expression and the molecular consequences thereof as proposed in this paper may offer additional avenues for gene therapy based either on preferential integration of exogenous DNA at fragile sites, or utilizing the acentric fragments formed during chromosome breakage to modify sequence amplification extrachromosomally.

Animals↗
PubMed2000

Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.

The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1986

The sequence and mom-transactivation function of the C gene of bacteriophage Mu.

The mom gene of bacteriophage Mu encodes a DNA modification function. The gene is regulated on the transcriptional level by Dam-specific methylation and a trans-acting Mu function, and on a post-transcriptional level by the product of gene com. The gene encoding the transactivator has been cloned and mapped. By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C. Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ. Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal. Successful transactivation results in the formation of blue plaques. Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids. The promoter for C (pc) is likely to be located at least 0.5 kb upstream from the gene. A transcription terminator is found directly downstream from the C-coding region.

Amino Acid Sequence↗
PubMed2000

Methylation of conserved CpG sites neighboring the beta retinoic acid response element may mediate retinoic acid receptor beta gene silencing in MCF-7 breast cancer cells.

We investigated the mechanism of retinoic acid receptor (RAR) beta2 gene silencing in breast cancer cells. Transfection experiments indicated that MCF-7 cells transactivate an exogenous beta2 promoter (-1470/+156) to the same extent as MTSV1.7 breast epithelial cells, which express endogenous RARbeta2. This was true even in the context of replicated chromatin, suggesting a cis-acting rather than a trans-acting defect. Cytosine methylation, a cis-acting DNA modification, has been implicated in RARbeta2 silencing in cancer cells. Upon bisulfite genomic sequencing, we found that 3 CpG sites in the beta2 RARE region were variably methylated in MCF-7 cells but were not methylated in MTSV1.7 cells or in 2 MDA-MB-231 subclones that differed in RARbeta2 expression (high in clone A2, low in clone A4). However, the 5'-UTR region was hypermethylated in clone A4 relative to clone A2 cells. Following 5-azacytidine treatment, RA and trichostatin A markedly induced RARbeta2 expression in MCF-7 cells but not in MDA-MB-231 clone A4 cells. A beta2 RARE reporter construct in which the methylation-susceptible cytosines in the sense strand were replaced by thymine displayed marked loss of activity in a replicated chromatin-dependent manner. We conclude that cytosine methylation contributes to RARbeta2 gene silencing in MCF-7 cells and that methylation of the RARE region may be particularly important. Oncogene (2000) 19, 4066 - 4070.

Adenocarcinoma↗
PubMed2006

Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts.

Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.

Animals↗
PubMed1993

Methylation-enhanced binding of Sp1 to the stage selector element of the human gamma-globin gene promoter may regulate development specificity of expression.

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.

Adult↗
PubMed2001

Methylation of O(6)-methylguanine-DNA methyltransferase gene is associated significantly with K-ras mutation, lymph node invasion, tumor staging, and disease free survival in patients with gastric carcinoma.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase (MGMT) can remove O(6)alkylG DNA adducts. If they are not removed, then the adducts mispair with T during DNA replication, resulting in G-to-A mutation. Interrelations between MGMT gene inactivation by promoter methylation, K-ras mutation, and clinicopathologic features in patients with gastric carcinoma were studied. METHODS: Surgically removed tumor tissues from 79 patients were analyzed with MGMT methylation by genomic DNA modification and methylation specific polymerase chain reaction analysis, K-ras mutation by mutant allele specific amplification, TNM classification according to the International Union Against Cancer system, and MGMT protein expression by immunohistochemistry. RESULTS: MGMT-promoter methylation was found in 18 of 79 tumors. Among those 18 tumors, K-ras mutations were found in 33% and 11% of tumors at codons 12 and 13, respectively, corresponding to 20 times and 7 times greater rates of mutation compared with unmethylated tumors. MGMT methylation was associated significantly with lymph node invasion (P < 0.01), tumor stage (P < 0.03) and 5-year disease free survival (P < 0.02). MGMT protein expression was detected in intestinal metaplasia and adenocarcinoma samples, whereas no expression was detected in normal foveolar cells. CONCLUSIONS: MGMT-promoter methylation in patients with gastric carcinoma was associated significantly with point mutations of K-ras at codons 12 and 13, lymph node invasion, tumor stage, and disease free survival. These associations indicate a significant role of MGMT methylation during gastric carcinogenesis.

Adenocarcinoma↗
PubMed2002

The human DNA methyltransferases DNMT3A and DNMT3B have two types of promoters with different CpG contents.

DNA modification that is established by de novo methylation is involved in the epigenetic control of genome functions. The DNMT3A and DNMT3B genes encode putative de novo methyltransferases. In this paper, we investigated the transcriptional regulatory regions of the human DNMT3A and DNMT3B genes. We found that the DNMT3A and DNMT3B genes have multiple transcriptional start points (TSPs) that are separated on the chromosome and the expressions of these genes are controlled by multiple promoters. The DNMT3A gene has at least four TSPs and the expression is controlled by three different promoters. All three promoters lack typical TATA sequences adjacent to the TSPs. Two of them bear CpG-rich promoters and the other a CpG-poor promoter. The DNMT3B gene has at least two TSPs which exist in different exons and the expression is controlled by different promoters. Both promoter regions of the DNMT3B gene lack typical TATA sequences, where one promoter contains a CpG-rich area near the TSP, the other promoter is CpG-poor. Together with the data that the human DNMT1 gene has both CpG-rich and CpG-poor promoters, it is suggested that transcriptional regulation by two types of promoters, CpG-rich and CpG-poor, might be common characteristics in the DNMT gene family.

5' Flanking Region↗
PubMed2001

Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas.

BACKGROUND: Catenin/E-cadherin complex proteins play an important role in cell-cell adhesion with decreased expression correlating with adverse prognostic variables in several human malignancies. METHODS: Archival formalin fixed, paraffin embedded (FFPE) sections from 118 prostatic adenocarcinomas (PACs) were immunostained by an automated method (Ventana Medical Systems, Tuscon, AZ) using monoclonal antibodies to catenins alpha and beta, p120 CTN, and E-cadherin proteins. Immunoreactivity was semiquantitatively graded, and results correlated with traditional prognostic parameters. In a subset of 10 randomly selected cases, E-cadherin gene promoter methylation status was determined on FFPE tissues using sodium bisulfite/hydroquinone DNA modification and polymerase chain reaction (PCR) with methylation specific primers (CpG wiz E-cadherin methylation assay; Intergen Co., Purchase, NY). RESULTS: Decreased expression of alpha-catenin (17%), beta catenin (4%), p120 CTN (45%), and E-cadherin (25%) proteins was noted in PACs with downregulation of each protein correlating with high tumor grade (P = 0.01-0.0001). In addition, p120 CTN and E-cadherin expression levels correlated with pathologic stage (P = 0.05; P = 0.02), aneuploidy (P = 0.001; P = 0.0001), and alpha-catenin with aneuploidy (P = 0.0001). p120 CTN loss also correlated with preoperative serum prostate specific antigen (P = 0.05). Two of 10 cases featured no evidence of E-cadherin gene promoter methylation by PCR and both cases retained expression of E-cadherin protein on immunohistochemistry. Of the 8 cases that showed E-cadherin methylation, 5 (68%) featured loss of expression of the protein on immunohistochemistry (P = 0.11). There was no correlation between E-cadherin methylation and adverse prognostic variables. CONCLUSIONS: Decreased expression of catenin/E-cadherin complex cell adhesion proteins is associated with aggressive phenotype in prostatic adenocarcinoma. E-cadherin gene promote methylation is a common event in prostate carcinoma but does not appear to bear prognostic significance in the subset of cases analyzed.

Adenocarcinoma↗
PubMed2006

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota.

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.

Baculoviridae↗
PubMed1996

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene.

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s, 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0-2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 2B1 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA > or = DMBA trans-5,6-dihydrodiol > or = 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Identification of a novel member of the snail/Gfi-1 repressor family, mlt 1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice.

DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.

Amino Acid Sequence↗
PubMedDate not supplied

How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?

In restriction-modification systems, cleavage of substrate sites in cellular DNA by the restriction endonuclease is prevented by the action of a cognate methyltransferase that acts on the same substrate sites. The PvuII restriction endonuclease (R.PvuII) has been structurally characterized in a complex with substrate DNA (Cheng et al., 1994) and as an apoenzyme (Athanasiadis et al., 1994). We report here a structure, determined to 1.9 A resolution by crystallography, of a complex between R.PvuII and iodinated DNA. The presence of an iodine at the 5-carbon of the methylatable cytosine results in the following changes in the protein: His84 moved away from the modified base; this movement was amplified in His85 and disrupts an intersubunit hydrogen bond; and the base modification disturbs the distribution of water molecules that associate with these histidine residues and the area of the scissile bond. Considering these observations, hypotheses are given as to why a similar oligonucleotide, where a methyl group resides on the 5-carbon of the methylatable cytosine, is slowly cleaved by R.PvuII (Rice et al., 1995).

Crystallography, X-Ray↗
PubMed2007

Multiple binding of methyl-CpG and polycomb proteins in long-term gene silencing events.

Epigenetic regulation is involved in the maintenance of long-term silencing phenomena, such as X-inactivation and genomic imprinting in mammals. Gene repression is mediated by several mechanisms, such as histone modifications, DNA methylation, and recruitment of Polycomb proteins. To understand the mechanistic relationships between these mechanisms for stable gene silencing, we analyzed the mechanisms of X- and Y-inactivation of the PAR2 gene SYBL1, previously showed to be regulated by concerted epigenetic mechanisms. Maintenance of stable repression occurs via the recruitment of both MBDPs and PRC2 complexes to SYBL1 promoter. Their binding is equally sensitive to defective DNA methylation seen in cells derived from ICF syndrome patients. Multiple occupancy is a feature shared within long-term repressed genes, such as the X-inactivated PGK1 and the imprinted IGF2. MBD2, MBD3, and MeCP2 occupy SYBL1 promoter simultaneously, as revealed by sequential ChIP. We did not find this co-occurring binding when looked for members of PRC2 complex together with any of the methyl-binding proteins. Furthermore, in co-transfection assays, MECP2 can silence methylated SYBL1 promoter, whereas the mutated protein fails. However, RNA interference of endogenous MECP2 does not induce the expression of the inactive SYBL1 alleles, suggesting that its silencing activity can be replaced by the other methyl-binding proteins. Our data suggest that maintenance of long-term silencing involves multiple layers of epigenetic control functionally redundant. PRC2 and MBD proteins could collaborate to different phases of this process, the former possibly recruiting DNMTs to the silenced promoters, the latter dictating the lock of the transcription.

Cell Line↗