Reversal of methylation-mediated repression with short-chain fatty acids: evidence for an additional mechanism to histone deacetylation.

We have constructed a stable cell line, human embryonal kidney 293M+, containing a lacZ reporter gene controlled by an in vitro methylated hormone-responsive enhancer. Methylation of the enhancer-promoter abolishes lacZ expression controlled by ponasterone A (an analogue of ecdysone). Ponasterone A-induced expression is restored by the short-chain fatty acids valeric > butyric > propionic > acetic acid, but not by the histone deacetylase inhibitors trichostatin A and suberoylanilide hydroxamic acid (SAHA). lacZ expression is restored to levels approaching that from an unmethylated counterpart. Incubation with short-chain fatty acids alone does not promote demethylation of the lacZ promoter, however, some demethylation (30%) is observed when transcription is triggered by addition of ponasterone A. Similar levels of hyperacetylated histones H3 and H4 were observed in cells treated with short-chain fatty acids, trichostatin A or SAHA. In vivo DNase I footprinting indicates a more open chromatin structure at the promoter region for butyric acid-treated cells. A synergistic effect in reversing the methylation-mediated repression of the lacZ gene is obtained by combined treatments with the normally ineffective compounds trichostatin A and the short-chain fatty acid caproic acid. Our results suggest the existence of an alternative silencing mechanism to histone deacetylation in executing methylation-directed gene silencing.

5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation.

Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or L-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence.

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.

A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens.

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluor. escent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin. The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules. The fluorescent stain for vitellogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parentchymal cells. The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.

Cloning in a cosmid vector of complete 37 kb and 25 kb ribosomal DNA repeat units from the chicken.

DNA fragments of up to 40 kb containing rRNA-coding sequences have been isolated from a chicken liver DNA library prepared in the cosmid pHC79. Characterization of the cloned DNA by R-loop and restriction mapping has shown that there are two size classes of repeat unit, one of 37 kb and one of 25 kb, the larger of which is a family of units which vary slightly in size. These two classes were shown to be present in the DNA of a single chicken. The size of the internal transcribed spacer in the chicken was measured to be 4.4 kb from analysis of R-loops and heteroduplexes between chicken and Xenopus laevis rDNAs. No introns were observed in either the 18 S or the 28 S coding sequences. The number of copies of the chicken rDNA unit was measured by titration against the cloned sequences to be 202 +/- 51 per haploid genome.

Recombinant plasmids containing avian vitellogenin structural gene sequences derived from complementary DNA.

Purified mRNA coding for chicken vitellogenin, a precursor of egg yolk proteins, was transcribed to complementary DNA (cDNAvit) with avian myeloblastosis virus (AMV) reverse transcriptase. Double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I (fragment A) using the self priming ability of the cDNA. Following S1 nuclease digestion the double-stranded cDNA was inserted into the Hind III site of plasmid pBR322 using the poly(dA) . poly(dT) tailing method, and the hybrid molecules were used to transform Escherichia coli chi 1776. Ampicillin-resistant colonies were screened by colony hybridization with 125I-labeled vitellogenen mRNA. Further screening of positive clones was done by agarose gel electrophoresis and in situ hybridization with 125I-labeled vitellogenin mRNA. In addition, plasmid DNA covalently bound to diazotized paper was used to select complementary mRNA sequences. The cloned vitellogenin sequences were shown to hybridize to a mRNA which directs the synthesis of immunoprecipitable vitellogenin when translated in a reticulocyte lysate cell-free system. The length of the inserted cDNA was determined by agarose gel electrophoresis and heteroduplex mapping. The largest insertion was about 2500 base pairs. Restriction mapping indicates that at least three plasmids out of four have different sequences.

A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells.

In the developing chicken embryo, active DNA demethylation requires both RNA and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550, 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in the 5- and 12-day-old embryos the highest specific activity of 5-methylcytosine DNA glycosylase is found in the brain, the eyes and the skin. In situ hybridization with antisense CpG-rich RNA tightly associated to the DNA demethylation complex shows a restricted expression pattern only in proliferating tissues such as the neuroepithelia of the brain in 5-day-old embryos. The RNA is absent in differentiated tissues like the skeletal and heart muscle, liver and the crystallin-producing cells in the lens. The CpG-rich RNA is transcribed in a developmental stage-specific rather than in a cell-specific manner. In contrast transcripts of DNA methyltransferase are found in dividing and quiescent cells. In situ hybridization with a probe of a RNA helicase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA. The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. The results indicate that proliferating cells have on the average 15% more methylated cytosines than non-dividing cells. This represents roughly 3x10(6) more methylation sites per haploid genome.

Optimized genomic sequencing as a tool for the study of cytosine methylation in the regulatory region of the chicken vitellogenin II gene.

Some critical parameters of genomic sequencing are described. As an example we show a 200-nucleotide (nt) sequence of the estradiol-regulated avian vitellogenin gene II upstream region containing four CpG nt pairs. The two CpG's at positions -612 and -618 are in the sequence binding estradiol-receptor complex. While all four CpG's are methylated in erythrocytes, they are hypomethylated in the DNA of estradiol-responsive organs of egg-laying hens. A simple electroblot DNA transfer system which gives no distortion of DNA bands and quantitative transfer of denatured DNA from the gel to the filter membrane is described. Using Gene Screen membranes, a maximal hybridization signal was obtained when 30-50% of the input DNA was stably bound to the filters. Hybridization background signal and exposure time could be largely reduced by using highly purified fractionated DNA. Using a 90-120 nt long homogeneous single-stranded DNA probe of high specific activity it was possible to read a genomic sequence of up to 200 nt. The resolution was further improved by reducing the extent of chemical modifications of the DNA during the Maxam-Gilbert sequencing reactions.

Mechanism of active DNA demethylation during embryonic development and cellular differentiation in vertebrates.

Incubation of hemimethylated and labelled oligodeoxynucleotides with nuclear extracts from differentiating chicken embryos and mouse myoblasts resulted in the replacement of m5C by C. One of the enzymes involved is m5CpG endonuclease. It cleaves only m5CpG and not, m5CpT, m5CpA, m5CpC or m6ApT. The enzyme is not sequence specific and catalyses the reaction in the presence of high concentrations of EDTA or EGTA.

Approaches to characterize protein-DNA interactions in vivo.

With the availability of direct genomic footprinting techniques the study of native genomes has been greatly facilitated. This review provides an overview of the techniques involved and gives also a description of the mode of action of different DNA modifying agents which can be used for such methods. These include exonuclease III, deoxyribonuclease, DNase I, micrococcal nuclease, dimethyl sulfate, diethyl sulfate, ethyl methanesulphonate, ethylnitrosourea, diethylpyrocarbonate, bromoacetaldehyde, potassium permanganate, osmium tetroxide, methidiumpropyl-EDTA-iron(II), formaldehyde, psoralen, 1,10 phenanthroline-copper and UV light. We also describe the limitations of the currently existing techniques and give some potential developments.

Isolation and fine structure organisation of an avian vitellogenin gene coding for the major estrogen-inducible mRNA.

Two phage lambda recombinant DNA clones covering the entire sequence of an avian vitellogenin gene, plus flanking regions, have been isolated from an erythrocyte DNA gene library and characterized by R-loop and restriction mapping. The total length of this avian vitellogenin gene is 23 kb. The cloned sequences flanking the gene at the 5' and 3' end are 7 and 3 kb, respectively. The total length of exons in the two clones is 6.7 kb (vitellogenin mRNA is 6.6 kb). The gene is interrupted by at least 25 introns with a mean intron length of 940 bp. Some 6--10 additional very small introns may also be present but they were not observed reproducibly. The mean exon length is 250 bp. Restriction endonuclease digests of total liver genomic DNA and lambda recombinant DNA were also analyzed by electrophoresis. Southern blotting and hybridization with cloned vitellogenin cDNA. The results show an identity of organisation of this vitellogenin in the DNA from the two sources, thus ruling out a possible cloning artifact. In contrast to Xenopus vitellogenin we have found no evidence to suggest that avian vitellogenin is encoded by a small family of related genes.

A novel 52 kDa protein induces apoptosis and concurrently activates c-Jun N-terminal kinase 1 (JNK1) in mouse C3H10T1/2 fibroblasts.

A 52 kDa protein (p52) was purified from chicken embryos and its corresponding cDNA was cloned. The p52 cDNA is 1768 bp long and has an open reading frame of 465 amino acids. The sequence of the p52 cDNA shows significant homology with mouse and human cDNAs from the EST database, so do the deduced amino acid sequences, indicating the existence of human and mouse homologues of p52. Northern blot hybridization showed that the p52 mRNA was expressed in a wide range of embryonic and adult tissues. There was more p52 mRNA in embryonic heart and liver than in the brain or muscle. The adult testis had the highest level of p52 mRNA, whereas adult liver had the lowest. Expression of p52 in mouse C3H10T1/2 fibroblasts caused apoptotic cell death, upregulation of transcription factor c-Jun and activation of c-Jun N-terminal kinase 1 (JNK1). In addition, expression of Bcl-2, but not of the dominant negative mutant JNK1, can block the p52-mediated apoptosis. These results indicate that p52 may represent a new cell-death protein inducing apoptosis and activating JNK1 through different pathways.

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.

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

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.

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.

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.

Comparison of dietary patterns between population samples in the three French MONICA nutritional surveys.

A nutritional survey was carried out from 1985 to 1987 in three French areas covered by the ischaemic heart disease registers: Bas-Rhin (BR), Haute-Garonne (HG) and the Urban Community of Lille (UCL). 1,128 men aged 45-64 were included in a survey, using the 3-day record method. The results, after adjustment for age, socio-economic status, residence and prescribed diet, showed differences, sometimes significant, between the three samples. Fat represented 37% of the total energy intake in BR, 36% in UCL, and 34% in HG. The P:S ratio ranged from 0.50 in HG, to 0.47 in BR, and 0.40 in UCL. These differences were due to a higher intake of vegetable fat and a lower intake of animal fat in HG. In spite of a very important alcohol intake, with no big differences between the centres (33 to 36 g/day), we noticed that HG drank mainly wine, whereas BR and UCL drank wine and beer in about the same proportions.