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PubMed1995

DNA recognition by the EcoP15I and EcoPI modification methyltransferases.

The DNA-binding properties of the EcoP15I DNA methyltransferase (M.EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate cross-linking that M.EcoP15I is a dimer in solution. While M.EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.

Adenosine Triphosphate↗
PubMed2005

Simple modifications of the standard DNA sequencing protocol allow for sequencing through siRNA hairpins and other repeats.

RNAi is a relatively new but powerful technology used for monitoring gene silencing in many species. The RNA corresponding to the gene of interest can be delivered into cells using various protocols, but the approach where both sense and antisense strands are located in the same transcript seems to be the most promising. However, the DNA sequencing step is a challenge due to the formation of a strong hairpin structure. In this work we present a very simple modification to the standard DNA sequencing protocol that allows sequencing through such difficult regions. The modification does not require any additional enzymatic or chemical manipulations. It simply adds a 5-min heat-denaturation step at 98 degrees C, prior to addition of a Taq mix. The inclusion of this step is very effective in sequencing through many different categories of difficult templates. Other examples of the implementation of this step have been described previously and more detailed information on heat-denaturation of plasmid DNAs has been presented.

RNA, Small Interfering↗
PubMed1993

Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein-DNA interactions at basal and enhancer promoter elements.

In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [TATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transcribe the OC gene. Furthermore, the protein-DNA complexes that reflect hormonal control are also developmentally regulated, mediating both the transcriptionally active and repressed states of the OC gene. For example, in proliferating osteoblasts, a vitamin D receptor-antibody-sensitive complex is formed that is different from the DNA binding complex induced by vitamin D postproliferatively when the OC gene is transcribed. Mutational analysis of the steroid hormone binding domain and the overlapping AP-1 site at the VDRE supports mutually exclusive occupancy by Fos-Jun heterodimers and vitamin D receptor. Such protein-DNA interactions at the VDRE are consistent with repression of competency for vitamin D-mediated transcriptional enhancement in proliferating osteoblasts expressing high levels of Fos and Jun.

Animals↗
PubMed2002

SNPs in the CpG island of NAP1L2: a possible link between DNA methylation and neural tube defects?

Deletion of the murine X-linked Nap1l2 gene causes lethality from midgestation onwards. The affected embryos exhibit neural tube defects (NTDs) closely resembling spina bifida and anencephaly in humans. X-linked familial and spontaneous cases of NTD were analyzed for sequence alterations in the human NAP1L2. No differences were found in the familial cases. However, a number of single nucleotide polymorphisms (SNPs) within the 5' region of NAP1L2 were identified both in cases of spontaneous NTD and in normal controls. Most of these SNPs lead to the replacement of guanidines or cytosines within a CpG island that is conserved between the human and the mouse promoter regions. Demethylation in vitro activates Nap1l2 transcriptional activity, suggesting the importance of the CpG island in regulating the activity of the Nap1l2/NAP1L2 genes, and the potential importance of the polymorphisms in modifying their transcriptional activity. NAP1L2/Nap1l2 expression may therefore depend on the genetic-environmental factors that are frequently associated with NTDs.

Animals↗
PubMed2006

MGMT methylation: a marker of response to temozolomide in low-grade gliomas.

The methylation status of the O6-methylguanine-methyltransferase promoter (MGMTP) was evaluated in 68 low-grade gliomas treated by neoadjuvant temozolomide. Methylated MGMTP was detected in 63 of 68 (92.6 %) patients and was a favorable predictor of progression-free survival as compared with unmethylated MGMTP tumors (p < 0.0001). Assessment of MGMTP status could help identifying low-grade gliomas patients more likely to respond to chemotherapy or to benefit from MGMT depletion strategies.

Adult↗
PubMed1995

Analysis of the joining sequences of the t(15;17) translocation in human acute promyelocytic leukemia: sequence non-specific recombination between the PML and RARA genes within identical short stretches.

Molecular analysis of the t(15;17) translocation in 70 patients with acute promyelocytic leukemia (APL) confirmed that the breakpoints of chromosome 15 were located in two regions of the promyelocytic leukemia (PML) gene, mainly introns 3 and 6, whereas the breakpoints of chromosome 17 were consistently in intron 2 of the retinoic acid receptor alpha (RARA) gene. To study the reason for the clustering of the breakpoints and the underlying mechanism of the chromosomal translocation, we characterized the joining sequences of der(15) and der (17) by polymerase chain reaction in samples from eight patients with APL. There was no cluster of the breakpoints within the introns, and no consensus sequence-motif was found around them. One or nine extra nucleotides were inserted into two joining sites. There were identical stretches of one to seven nucleotides between the PML and RARA genes in the majority of the joining sequences. These data provide a potential model of the t(15;17) translocation: random DNA double strand cleavage, modification of DNA ends by enzymes including terminal deoxynucleotidyl transferase, and single strand base-pairing within identical short stretches. Furthermore, APL develops only when the PML and RARA genes are rearranged, within restricted genomic regions and a functional PML-RARA chimeric product is produced, and this might lead to a clustering of the breakpoints.

Base Sequence↗
PubMed2003

Expression profiling reveals that methylation of TIMP3 is involved in uveal melanoma development.

Uveal melanoma is associated with a high tumor-related mortality due to the propensity to develop metastases. The mechanisms that are responsible for malignant dissemination are largely unknown and need to be explored to facilitate diagnosis and treatment of metastases. To identify molecules involved in dissemination, we used cell lines derived from a primary uveal melanoma and 2 liver metastases from the same patient as a model for tumor progression. Using a microarray representing 1,176 genes, we identified 63 differentially expressed genes. Forty genes showed a higher expression and 23 showed a lower expression in the primary cell line compared to the metastasis cell lines. These genes are involved in processes like angiogenesis, apoptosis, macrophage stimulation, and extracellular matrix regulation. In contrast, the 2 liver metastasis cell lines produced nearly identical expression profiles. Demethylation of the primary melanoma cell line by 5-aza-2'deoxycytidine treatment revealed that 19 genes were suppressed by hypermethylation. An important finding was the 5-fold decreased expression of TIMP3 in the metastatic cell lines, a molecule involved in extracellular matrix degradation. We demonstrate in the cell lines that TIMP3 expression is regulated by methylation. These observations were confirmed in primary uveal melanoma and suggests a role for TIMP3 in uveal melanoma development.

Azacitidine↗
PubMed2002

Characterization of XAGE-1b, a short major transcript of cancer/testis-associated gene XAGE-1, induced in melanoma metastasis.

Suppression subtractive hybridization, comparing mRNA expression profiles of common nevocellular nevi and melanoma metastases, was used to identify potential markers of melanoma progression. From the metastases we isolated XAGE-1b, a 470 bp transcript of the XAGE-1 gene. In general, expression of XAGE-1b was much more prominent than expression of the longer XAGE-1 transcript, isolated from Ewing's sarcoma. The XAGE-1b open-reading frame codes for a putative protein of 81 amino acids, harboring a functional bipartite nuclear localization signal and a C-terminal acidic transcription-activation-like domain. On the nucleotide level, XAGE-1b has a 50% homology with members of the GAGE family. However, homology between the corresponding proteins is weak. Expression of XAGE-1b in normal tissues was mainly restricted to testis, while placenta and brain were sporadically positive. In human tumor cell lines as well as in human tumor lesions, expression was most frequently found in melanocytic tumors and Ewing's sarcoma. In the different stages of melanocytic tumor progression, expression was exclusively seen in melanoma metastases (38%; n = 61), while all tested common and atypical nevi (n = 10) as well as primary melanomas (n = 8) were negative. Upregulation of expression after treatment with demethylating agent 5-aza-2'-deoxycytidine was detected in 1 of 4 human melanoma cell lines tested. The XAGE-1 gene consists of 4 exons and is located on chromosome Xp11.21-Xp11.22. After transfection into COS cells, the corresponding protein can direct the coupled fluorescent protein to the nucleus, showing a distinct speckled staining aspect. Our data imply the nuclear cancer/testis-associated XAGE-1b to be a marker for late melanocytic tumor progression.

Amino Acid Sequence↗
PubMed2005

Downregulation of connexin 26 in human lung cancer is related to promoter methylation.

Cell-Cell communication via gap junctions plays a key role in carcinogenesis and in growth control. One of the gap junction proteins, Connexin 26 (Cx26) was considered as tumor suppressor in various cancers. In our study, the expression of Cx26 was analyzed in human lung cancer. The reduced mRNA expression was observed in 17 lung cancer cell lines examined by Northern blot analysis and RT-PCR. In 138 primary carcinomas comprising all subtypes analyzed by immunohistochemistry, 85 cases (62%) exhibited no expression of Cx26, whereas in other 53 cases the Cx26 staining was positive (38%). Additionally, an association between Cx26 protein expression and higher grading of tumors was found in whole tumor samples (p =0.028) but no statistically significant correlations could be observed with tumor stage, tumor size and node status. In squamous cell carcinoma, tumors with higher stage and grading were linked to higher expression of Cx26 (p = 0.015 and 0.017, respectively). To explore the mechanism responsible for the downregulation of Cx26, we treated 2 lung cancer cell lines H2170 and H226 with the demethylation agent 5-aza-2'-deoxycytidine and found the reexpression of Cx26 mRNA. Methylation status of these 2 cell lines was further analyzed by PCR amplification of bisulfite modified DNA and sequencing. A heterogeneous methylation pattern turned out. Our results suggest the inactivation of Cx26 in lung cancer may be explained by promoter methylation.

Actins↗
PubMed1992

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact.

Base Sequence↗
PubMed2002

Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts.

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI-38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI-38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI-38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI-38 cells. These studies indicate that reduced genome-wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene-localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase.

Base Sequence↗
PubMed2005

Deregulation of DNA methyltransferases and loss of parental methylation at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development.

To ascertain whether p53 deficiency in vivo leads to the deregulation of DNA methylation machinery prior to tumor development, we investigated the expression profile of DNA methyltransferases in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice at 7 weeks of age before tumor development. The expression of DNA methyltransferases was examined in the thymus at 7 weeks of age, since the malignant T-cell lymphoma develops most frequently in p53(-/-) mice around 20 weeks of age. Both mRNA and protein levels of Dnmt1 and Dnmt3b were increased in the thymus and the liver of p53-deficient mice. The expression of Dnmt3a was also increased in the liver but not in the thymus of p53-deficient mice. Dnmt3L expression was reduced in the thymus of p53(+/-) and p53(-/-) mice. The total 5-methylcytosine (5-MeC) in the genomic DNA of p53(+/+), p53(+/-), and p53(-/-) mice was quantitated by dot-blot using antibody against 5-MeC. Global methylation was increased in the thymus and the liver of p53-deficient mice. To correlate the deregulated expression of DNA methyltransferases with the disturbance of the epigenetic integrity, we examined the DNA methylation of the imprinting control region (ICR) at the insulin-like growth factor II (Igf2)/H19 loci in the thymus and the liver of p53(+/+), p53(+/-), and p53(-/-) mice. The region containing two CCCTC binding factor (CTCF) binding sites in the 5'-ICR tended to be hypomethylated in the thymus of p53(-/-) mice, but not in the liver. The expression profile of Igf2 and H19 indicated that the thymus-specific changes of Igf2 and H19 expression were coherent to the hypomethylation of the ICR in the thymus. Our results suggest that p53 is required for the maintenance of DNA methylation patterns in vivo.

Animals↗
PubMed2004

Protection and selection for gene therapy in the hematopoietic system.

Hematopoietic stem cell gene therapy is potentially curative for a number of inherited and acquired disorders. However, poor gene transfer and expression in repopulating hematopoietic stem cells attenuate this potential. Here we review potential means of conferring a selective advantage to hematopoietic stem cells and their progeny, and discuss the issues that surround the use of selective advantages in vivo.

ATP-Binding Cassette Transporters↗
PubMed2003

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma.

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.

Adult↗
PubMed2006

Relationship between point gene mutation, chromosomal abnormality, and tumour suppressor gene methylation status in colorectal adenomas.

Epigenetic mechanisms in carcinogenesis may have a significant role in the development of colorectal cancer. To investigate this phenomenon in early-stage disease, promoter methylation status in the tumour suppressor genes APC, MGMT, hMLH1, P14/P14ARF, and CDKN2A/P16 was investigated in 78 colorectal adenomas. These had previously been characterized for mutations of APC, KRAS, and TP53 genes and for chromosomal abnormality by comparative genomic hybridization (CGH). APC hypermethylation was seen in 52 tumours (66.7%). APC showed either methylation or mutation in 66 lesions (84.6%), but these events were not statistically associated. MGMT methylation was detected in 39 cases (50%). Adenomas with this abnormality showed a significantly lower number of chromosomal changes by CGH (p < 0.02), confirming that DNA repair defect of this type is associated with a lower level of chromosomal instability. An hMLH1 methylation defect was seen in only one adenoma (1.3%), from a patient who had a synchronous cancer showing the same defect. Methylation of P14 (P14ARF) was seen in 31 adenomas (39.7%) and CDKN2A (P16) abnormality in 25 (32.1%). DNA methylation at two or more loci was seen in 46 tumours (59%), while 11 lesions (14.1%) showed no evidence of hypermethylation at any of the loci studied. Methylation at any or all of MGMT, P14 or P16 was significantly associated with APC methylation (p = 0.01). Those neoplasms with more than two methylated genes showed significantly fewer chromosomal abnormalities than adenomas with one or no methylated loci (p < 0.001). There was no association between specific individual chromosomal abnormalities, APC, KRAS or TP53 mutations and any pattern of methylation abnormality. We conclude that methylation abnormality is very common in pre-invasive colorectal neoplasia, and that high level methylation is associated with low level chromosomal instability.

Adaptor Proteins, Signal Transducing↗
PubMed1995

A bisulfite method of 5-methylcytosine mapping that minimizes template degradation.

The bisulfite method is a highly sensitive approach to 5-methylcytosine mapping that utilizes the capability of the polymerase chain reaction to exponentially amplify DNA. We have observed that the bisulfite reaction results in a significant level of template degradation due to DNA depurination. Furthermore, our data suggest that the DNA fragmentation which occurs limits the sensitivity of the method. We describe a simple solution to limit degradation of the DNA template.

5-Methylcytosine↗
PubMed2000

Cloning and characterization of mRNA capping enzyme and mRNA (Guanine-7-)-methyltransferase cDNAs from Xenopus laevis.

The mRNA cap structure, which is synthesized by a series of reactions catalyzed by capping enzyme, mRNA (guanine-7-)-methyltransferase, and mRNA (ribose-2'-O-)-methyltransferase, has crucial roles for RNA processing and translation. Methylation of the cap structure is also implicated in polyadenylation-mediated translational activation during Xenopus oocyte maturation. Here we isolated two Xenopus laevis cDNAs, xCAP1a and xCAP1b, for mRNA capping enzyme and one cDNA for mRNA (guanine-7-)-methyltransferase, xCMT1, which encode 598, 511, and 402 amino acids, respectively. The deduced amino acid sequence of xCAP1a was highly homologous to that of human capping enzyme hCAP1a, having all the characteristic regions including N-terminal RNA 5'-triphosphatase as well as C-terminal mRNA guanylyltransferase domains which are conserved among animal mRNA guanylyltransferases, whereas in xCAP1b the most C-terminal motif was missing. The amino acid sequence of xCMT1 was also similar to human (guanine-7-)-methyltransferase, hCMT1a, with all the conserved motifs among cellular (guanine-7-)-methyltransferases, except for its N-terminal portion. The recombinant xCAP1a and xCMT1 exhibited cap formation and mRNA (guanine-7-)-methyltransferase activities, respectively. RT-PCR analysis showed that mRNA for xCAP1a and xCMT1 exist abundantly in fertilized eggs as maternal mRNAs, but xCMT1 mRNA gradually decreased in its amount in later stages of early development.

Amino Acid Sequence↗
PubMed1995

DNA palindromes adopt a methylation-resistant conformation that is consistent with DNA cruciform or hairpin formation in vivo.

Long DNA palindromes present a threat to genomic stability and are not tolerated in Escherichia coli. It has been suggested that this is a consequence of cruciform or hairpin formation by palindromic sequences. This work describes a methylation inhibition assay for unusual DNA secondary structure in vivo that is both internally controlled and non-invasive. If a palindrome with a central GATC target site for Dam methylase assumes a cruciform or hairpin conformation in vivo, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of a long perfect palindrome located in bacteriophage lambda is shown to be methylation-resistant in vivo. Changes to the central sequence and insertions of 10 base-pairs of asymmetric sequence do not alter the degree of under-methylation, but insertions of 20 base-pairs or more of asymmetric sequence reduce the under-methylation of the palindrome centre. We also show that the centres of long palindromes are more under-methylated than equivalent sequences in a non-palindromic context. These results are consistent with an unusual secondary structure, such as DNA cruciform or hairpin, and indicate that the formation pathway of the structure detected is independent of the composition and symmetry of the central 10 base-pairs of the palindrome.

Bacteriophage lambda↗