In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.     

Vector methylation inhibits transcription from the SV40 early promoter.

Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.     

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.     

Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.     

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.