Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction.

The study of changes in genome-wide levels of DNA methylation has become a key focus for understanding the epigenetic regulation of gene expression. Many procedures exist to study DNA methylation, falling into two categories: gene-specific and genome-wide. Genome-wide methylation analysis is best performed by DNA hydrolysis followed by HPLC; however, it requires access to an HPLC machine, which is not always available. Alternative procedures, such as the radioactive labeling of CpG sites using SssI DNA methyltransferase, have been developed to address this problem, but it can only monitor CpG methylation changes, and CpNpG methylation is not detected. Here, we present a method for the analysis of DNA methylation in any sequence context by fluorescent labeling. We present control analyses using synthetic oligonucleotides of known methylation levels and a comparison of genomic DNA from two transgenic tobacco lines known to differ in their methylation levels. The results indicate that hygromycin-induced hypermethylation acts equally on all classes of methylatable cytosine, perhaps indicating a common mechanism.

Molecular analysis of animal and plant cells is facilitated by their attachment to collodion (cellulose nitrate) films.

A procedure for the efficient transfer of cell monolayers, cultured on glass coverslips, to microscopy slides has been developed. This technique involves the coating of the upper surface of an ethanol-fixed cultured cell layer with a film of collodion dissolved in n-amyl acetate. The dry collodion-coated cell layer can then be detached by rehydrating it for 1 h under water or phosphate-buffered saline and then carefully peeling it away from the coverslip using a pair of tweezers. Once a cell layer has been so mounted, it can be subjected to rough treatment such as proteolytic degradation (which greatly improves the signal-to-noise ratio in procedures like in situ hybridization [ISH] or primed in situ synthesis [PRINS]) without running the risk of cell detachment because of the partial or total degradation of the extracellular matrix. As an example of its application, we show a PRINS of telomeres from mouse fibroblasts. The high mechanical strength of collodion ensures that the structural integrity and morphology of the cell layer is maintained under experimental conditions where the collodion itself is insoluble. In addition to its use on cell layers, collodion can be used for the production of support films for (i) attaching suspension cell cultures, (ii) immobilizing cells normally cultured in suspension (such as tobacco BY2 cells or germinating tobacco pollen grains) or (iii) planting cryostat sections to microscopy slides. The value of this technique lies in its ease of use and the large number of different applications, in both the plant and animal fields of research, to which it may be applied.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation.

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of (1)/(5) of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.

Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants.

Plant genomic DNA methylation was analyzed by an improved SssI methyltransferase assay and by genomic sequencing with sodium bisulfite. Kanamycin, hygromycin, and cefotaxime (also called Claforan) are commonly used as selective agents for the production of transgenic plants. These antibiotics caused DNA hypermethylation in tobacco plants grown in vitro, which was both time- and dose-dependent. An exposure of the plantlets to 500 mg/liter cefotaxime for 1 month caused the de novo methylation of 3 x 10(7) CpG sites/haploid genome of 3.5 x 10(9) base pairs. It occurred in high, moderate, and low repetitive DNA and was not reversible upon the removal of the antibiotics. Reversion was only observed in progeny grown in the absence of drugs. Analysis of the promoter regions of two single-copy genes, an auxin-binding protein gene and the class I chitinase gene, showed the hypermethylation to be heterogeneous but biased toward CpGs. The hypermethylation of the class I chitinase and the auxin-binding protein promoters was not a consequence of a drug-induced gene amplification.

Non-symmetrical cytosine methylation in tobacco pollen DNA.

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants.

Phosphorylation/dephosphorylation of the repressor MDBP-2-H1 selectively affects the level of transcription from a methylated promoter in vitro.

We have previously shown that in vivo estradiol-dependent dephosphorylation of MDBP-2-H1 (a member of the histone H1 family) correlates with the loss of in vitro preferential binding to methylated DNA. To study the effects of the phosphorylation/dephosphorylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in vitro transcription system using HeLa nuclear extracts. We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, methylation of the vitellogenin II promoter does not affect the transcription. Addition of purified MDBP-2-H1 from rooster to the in vitro transcription system inhibits transcription more efficiently from a methylated than an unmethylated DNA template. Dephosphorylation of rooster MDBP-2-H1 by phosphatase treatment or estradiol treatment of rooster lead to the loss of inhibitory activity of the protein when added to the in vitro transcription assays. These findings indicate that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. Taken together these results establish the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.

Non-histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes.

We have previously purified and characterized a ubiquitous non-histone protein (NHP1) which has a high affinity (Kd 10(-11) M) for different avian vitellogenin gene sequences containing CpGs (Hughes et al. (1989) Biochemistry 28, 9137-9142; Hughes and Jost (1989) Nucleic Acids Res. 17, 8511-8520). Here we show by microsequencing that the peptides derived from the purified p75 and p85 subunits of NHP1 from HeLa cells have between 64 and 100% identity with the human Ku autoantigen. During the differentiation of human HL-60 promyelocytes there is an increase in the amount of p85 subunit protein whereas the level of the p75 subunit is unchanged. In differentiating mouse G8 myoblasts there is, however, an upregulation of both the p75 and p85 subunits and of the p85 mRNA. An inhibition of mouse myoblast differentiation by either cAMP, 3-aminobenzamide or sodium butyrate abolishes the upregulation of the p85 subunit. In G8 myoblasts chemical, or physical stress by UV light or X-rays does not upregulate the level of the p85 subunit. The possible involvement of NHP1 in the active demethylation of bifilarly methylated DNA will be discussed.

DNA binding and methyl transfer catalysed by mouse DNA methyltransferase.

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

AP-1 binds to a putative cAMP response element of the MyoD1 promoter and negatively modulates MyoD1 expression in dividing myoblasts.

We have studied the transcriptional activity of the mouse MyoD1 gene promoter in vivo and in vitro using mouse G8 myoblasts and muscle cell nuclear extracts. 5' deletion analysis of the promoter and transcription-competition analysis using oligonucleotides corresponding to several cis-acting elements revealed that the basal activity of the MyoD1 promoter is conferred by two SP1 boxes, an AP-2 box, and a CAAT box. We have identified a negative regulatory sequence located between nucleotide position -342 to -322 with respect to the cap site. The negative regulatory element shows sequence homology with cAMP-responsive element (CRE) and AP-1 binding site (5'-GAGCACTGAGGTCAGTACAG-3'). As determined by gel mobility shift competition analysis, oligonucleotides containing AP-1 binding sites inhibit protein interactions with the MyoD1 CRE-like element. We also show that binding to this element is down-regulated during myogenic differentiation and can be reinduced by the addition of serum. Furthermore, mutation of the CRE-like element induces MyoD promoter activity in diving myoblasts. By using anti-c-Fos antibodies we show that AP-1 is binding to the MyoD1 CRE-like element. Our results indicate that AP-1 negatively modulates MyoD1 expression in growing myoblasts and strongly suggest that c-Fos and c-Jun inhibit myogenesis and MyoD1 expression by direct binding to a negative cis-acting element in the MyoD1 promoter.

Characterisation of a genomic clone covering the structural mouse MyoD1 gene and its promoter region.

We have isolated the mouse MyoD1 gene flanked by its promoter region by screening a genomic library with synthetic oligonucleotides. The structural gene is interrupted by two G + C rich introns. Transfection of the cloned gene inserted into an expression vector converts fibroblasts to myoblasts. Sequence analysis of about 650 bp of the 5' upstream region revealed the presence of several potential regulatory elements such as a TATA-box, an AP2-box, two SP1-boxes and a CAAT-box. In addition, there are three half palindromic estrogen response elements, a potential cAMP response element and various muscle specific elements such as a muscle-specific CAAT-box (MCAT) and four potential binding sites for MyoD1. Using S1 protection analysis the major start site of transcription in muscle and myoblast cells was mapped 3 bp upstream of the published cDNA 5' end. Promoter activity of the 650 bp upstream fragment was tested by in vitro transcription and by transfection analysis of myoblasts and fibroblasts. In all promoter test systems used, MyoD1 promoter activity was detected in myoblasts as well as in fibroblasts. Furthermore, DNA methylation was found to turn off MyoD1 promoter activity both in myoblasts and in fibroblasts.

Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site.

A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATGAGGGGGATCATACTGG3' of the avian vitellogenin II gene (Nucleic Acids Res. 19, 1029-1034, 1991). Treatment of adult and immature roosters with estradiol results in a 90% decrease in the binding activity of MDBP-2 within three days. This corresponds to the level found in egg laying hens. The decrease in the binding activity of MDBP-2 precedes the onset of vitellogenin gene transcription. At the same time, there is a two-fold increase in the binding activity of NHP-1 (tested with the same oligonucleotide as for MDBP-2), a protein thought to be involved in the active demethylation of DNA. The methylated oligonucleotide binds either MDBP-2 or NHP-1 and there is no complex formation between the two proteins and DNA. Estradiol treatment does not change the equilibrium binding constant of MDBP-2 which is about 10(-9)M for the methylated oligonucleotide. The early kinetics of demethylation of the mCpG pair in the binding site of MDBP-2 was studied by means of genomic sequencing. A low level of demethylation of mCpG starts gradually on both DNA strands already 4 hours after estradiol treatment during the lag phase of vitellogenin mRNA synthesis. It is concluded that the lowering of the binding activity of MDBP-2 may have a stronger effect on the derepression of the gene than the slow demethylation of MDBP-2 DNA binding site. The role of the methylated CpG is to assure a high binding affinity of the repressor to DNA.

An estrogen-dependent polysomal protein binds to the 5' untranslated region of the chicken vitellogenin mRNA.

An estrogen-dependent protein present in chicken liver polysomes binds to the 5' untranslated region of the chicken vitellogenin II mRNA. Competition binding assays with different RNAs indicate that the binding of the polysomal protein to this region is sequence specific. Of the tissues tested, this RNA binding activity is liver specific. In vivo kinetics of appearance of the binding activity following a single injection of estrogen to immature chicks are similar to the rate of accumulation of vitellogenin mRNA. The molecular weight of the polysomal protein has been estimated to be 66,000 on the basis of UV crosslinking and subsequent SDS polyacrylamide gel electrophoresis. In vitro RNA decay assays carried out with a minivitellogenin mRNA suggest that the estrogen-dependent polysomal protein may be involved in the estrogen-mediated stabilization of the chicken vitellogenin II mRNA.

An avian 40 KDa nucleoprotein binds preferentially to a promoter sequence containing one single pair of methylated CpG.

In vitro transcription competition with oligonucleotides has shown that a down regulating factor can be displaced by a methylated oligonucleotide covering a specific region of the avian vitellogenin II gene promoter (Proc. Natl. Acad. Sci USA, (1990) 87, 3047-3051). Gel mobility shift and competition assays show that a protein binding preferentially to methylated DNA (MDBP-2) is present in fractionated hen and rooster nuclear extracts. The protein(s) bind to the methylated sequence 5' TTCACCTTmCGCTATG-AGGGGGATCATACTGG' 3' (nucleotide positions +2 to +32) of the vitellogenin II promoter and not to other methylated DNA sequences. Contact points of the MDBP-2 with DNA were studied by DNA binding interference experiments with partially depurinated and depyrimidinated oligonucleotides. The protein has an approximate molecular weight of 40 KDa and is mainly found in the liver and oviduct. Proteolytic clipping bandshift assays of the MDBP-2 from rooster and hen liver nuclear extracts indicate that the protein from the two sources are different. In vitro transcription experiments show that the addition of a purified nuclear fraction containing the addition of a purified nuclear dependent manner the transcription of vitellogenin II gene.

In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: evidence for the same protein binding to this sequence in hen and rooster liver.

The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERED) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5-0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERED and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays with hen and rooster liver nuclear extracts identified the ERE binding protein to be the same or very closely related in both tissues. Furthermore, the protein from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides, u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with Mr of about 75,000-80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene.

Positive and negative regulatory elements of chicken vitellogenin II gene characterized by in vitro transcription competition assays in a homologous system.

A homologous in vitro transcription system was developed in which the cloned chicken vitellogenin II gene is faithfully transcribed by extracts prepared from chicken liver nuclei. The use of template deleted of its upstream region resulted in poor transcriptional efficiency, as did the use of extracts prepared from rooster liver, in which the gene is silent. The influence of individual cis elements was determined by transcription competition analysis. Oligonucleotides covering greater than 500 base pairs of the promoter region were used as competitor DNA in the in vitro reactions. Competition with an oligonucleotide covering part of the expression-specific DNase I hypersensitivity site B2, which contains a demethylation site, mCpG, at nucleotide position + 10, increased transcription of the gene, suggesting the binding of a repressor to this region. The enhancement of transcription was even more pronounced when the same oligonucleotide was methylated at the corresponding + 10 cytosine. Competition with oligonucleotides covering the TATA box, or the estrogen response element half-palindromic motif (GGTCA) at nucleotide positions -198 to -194, resulted in a large decrease in vitellogenin gene transcription, indicating that strongly activating factors bind to these regions. Competing oligonucleotides covering other GGTCA-containing motifs situated further upstream at nucleotide positions -292 to -288, -367 to -351, and -626 to -614 were increasingly less effective in inhibiting transcription. The results indicate that factors other than the estrogen receptor are involved in transcriptional activation of the vitellogenin II gene.

Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions.

The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor.

The ubiquitous nuclear protein, NHP1, binds with high affinity to different sequences of the chicken vitellogenin II gene.

In gel shift assays, affinity chromatography-purified NHP1 forms a stable complex with different sequences of the chicken vitellogenin II gene. The apparent KD of NHP1 with the estrogen response element (ERE) containing 5-methylcytosine is 1 X 10(-11) M. NHP1 does not form a complex with the Xenopus vitellogenin ERE where the GCG bases are replaced by CAG. NHP1 is closely related if not identical to the other ubiquitous proteins NHP2, NHP3 and NHP4 that bind specifically to different sequences. All four proteins behave identically on chromatography and give identical patterns in proteolytic bandshift assays. NHP1, NHP2 and NHP3 have a native molecular weight of 170,000 and are composed of two polypeptides of 85 and 75 kDa. The possible function of NHP1 is discussed.

Purification and characterization of a protein from HeLa cells that binds with high affinity to the estrogen response element, GGTCAGCGTGACC.

A non-histone protein, NHP1, that binds with high affinity to the estrogen response element (ERE), GGTCAGCGTGACC, has been purified approximately 45,000-fold from HeLa cells by a combination of chromatography on Sephacryl S-300, heparin-Sepharose, Mono Q (FPLC), and sequence-specific oligonucleotide-Sepharose. The native protein has a molecular weight of 170,000 and is composed of two polypeptides of 85 and 75 kDa. The two polypeptides are different as judged by peptide mapping, and only the 85-kDa polypeptide can be cross-linked to the bromodeoxyuridine-substituted synthetic ERE by UV irradiation. The native protein binds to the ERE with an apparent KD of 1 x 10(-11) M and has a pI of 5. The contact points of the protein with individual bases of the ERE have been determined by using partially depurinated and depyrimidinated synthetic oligonucleotides. The strongest contact points of NHP1 with the ERE are 5'AGCG3' in the center of the palindrome and differ from those of the estrogen receptor. NHP1 appears to produce specific nicks around the central CpGs of the ERE, thereby suggesting that it may play a role in active demethylation of mCpGs.