Identification of highly methylated human DNA sequences using a methyl-CpG binding domain column

A library of highly methylated sequences has been constructed from DNA derived from human blood. Genomic DNA was fractionated using a column which binds preferentially according to methyl-CpG frequency (Cross et al., 1994). The library contains known fragments of methylated CpG Islands (CGIs) and other novel regions with high numbers of m5CpG. Analysis of cloned inserts demonstrated that most were more GC-rich and had a higher CpGobs/Exp than bulk genomic DNA. Cloned inserts were used as probes against southern blots of DNA digested with methylation sensitive or insensitive isoschisomers. The hybridisation patterns demonstrated that majority of the cloned sequences tested were methylated in DNA when derived from blood. One of the most frequently occurring clones in the library matched to a fragment from the rDNA repeat non transcribed spacer (NTS). This entire region has subsequently been shown to be heavily methylated in DNA from somatic cells. The cloned fragments derived from both male and female DNA were used as probes in Fluorescent in situ Hybridisation (FISH) experiments. DNA derived from both male and female gave indistinguishable hybridisation patterns with many fragments apparently hybridising to subtelomeric regions. The other regions of significant hybridisation were to the short arms of the acrocentrics and the centromere of chromosome 9. The rDNA repeats are located on the short arms of the acrocentric chromosomes while the centromere of chromosome 9 has been reported to contain low copy number methylated repeats. These sequences, though methylated in DNA from somatic cells, are often urimethylated in sperm. This apparent lack of germ-line methylation may account for the high frequency of CpG in these regions. Abbreviations AdoMet S-adenosyl-L-methionine Ab antibody bp base pair CCD charge coupled device CGI(s) CpG Island(s) CpGObs /Exp CpG Observed /Expected. cpm counts per minute Dapi 4,6-Diamidino-2-phenylindole ddH20 double distilled water dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine triphosphate dNTPs all four deoxy triphosphates DMSO dimethyl suiphoxide DTT dithiothreitol DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid EtBr ethidium bromide EtOH ethanol FISH fluorescent in situ hybridisation Hepes N(Hydoxyethyl)piperazine N[2 ethanosu1phonic acid] IPTG isopropylthio-j3-D-galactosidase K2HPO4 potassium hydrogen phosphate kb kilo base m5C 5-methylcytosine mSCpG methylated CpG MBD methyl binding domain MeCPs methyl-CpG binding proteins MgSO4 magnesium sulphate min minutes ms milliseconds MTases methyltransferases Mwt molecular weight NaCl sodium chloride (NH4)2SO4 ammonium sulphate NTS non-transcribed spacer PCR polymerase chain reaction PMSF phenylmethylsulfonyl flouride SIDS sodium dodecyl sulphate SDS-PAGE SIDS-polyacrylamide gel electrophoresis TAE tris acetate EDTA buffer TE tris-EDTA buffer TEMED N,N,N',N'-tetra-methylethylenediamine (v/v) volume: volume ratio (w/v) weight: volume ratio X-Gal 5-bromo-4-chloro-3-indolyl-3-galactopyranoside Chapter 1: General Introduction 1.1. The methylation of cytosine in vertebrate genomes (m5CpG) Deoxyribonucleic acid (DNA) consists of four bases; adenine, cytosine, guanine and thymine. The methylation of vertebrate DNA in vivo is a modification to cytosine catalysed by a cellular enzyme or enzymes. The methylation of cytosine involves the enzyme catalysed transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the C-5 position on a cytosine ring (Figure 1.1.A). In vertebrates this occurs when the cytosine is followed by a guanine (5'-CG-3') in a CpG dinucleotide. Cytosine methylation produces a protrusion from the major groove of DNA which may have the affect of altering local protein recognition signals (Eden and Cedar, 1994). However, attachment of a methyl group does not alter interstrand base pairing with guanine as there are still three hydrogen bonds. In vertebrates the modification of newly synthesised CpG dinucleotides takes place during the (S) phase of the cell cycle and may take several hours to complete (Adams and Burdon, 1985). Following replication, the unmethylated CpG in the daughter strand will be paired with the methylated CpG (mSCpG) of the parental strand (Figure 1.1.13). These hemi-methylated DNA sequences are thought to be the preferred substrate for mammalian DNA methyltransferases (MTases). The mammalian MTases are related to bacterial methyltransferases which also modify cytosine bases but have a more complex sequence specificity (Bestor et al., 1988). In mammals, the best studied are the maintenance MTases which act upon hemi-methylated sequences (Gruenbaum et al., 1982). A pattern of DNA methylation once established will then be maintained through many Figure I.I. The modification of cytosine by addition of a methyl-group The addition of a methyl group (CH3) to the C-5 position of the DNA base cytosine to produce 5-methylcytosine. The reaction is enzymatic, with S-adenosyl-L-methionine (Ado Met) as the methyl group donor. Action of mammalian maintenance methyltransferases (MTase) on newly replicated DNA. In the original and daughter strands m5CpG is represented by a black solid circle. The newly synthesised strand has no methyl groups but is paired with a m5CpG from the original strand. This hemi-methylated DNA is thought to be the preferred substrate of mammalian maintenance MTases.