Aharon Razin

CpG methylation, chromatin structure and gene silencing—a three-way connection

The three‐way connection between DNA methylation, gene activity and chromatin structure has been known for almost two decades. Nevertheless, the molecular link between methyl groups on the DNA and the positioning of nucleosomes to form an inactive chromatin configuration was missing. This review discusses recent experimental data that may, for the first time, shed light on this molecular link. MeCP2, which is a known methylcytosine‐binding protein, has been shown to possess a transcriptional repressor domain (TRD) that binds the corepressor mSin3A. This corepressor protein constitutes the core of a multiprotein complex that includes histone deacetylases (HDAC1 and HDAC2). Transfection and injection experiments with methylated constructs have revealed that the silenced state of a methylated gene, which is associated with a deacetylated nucleosomal structure, could be relieved by the deacetylase inhibitor, trichostatin A. Thus, methylation plays a pivotal role in establishing and maintaining an inactive state of a gene by rendering the chromatin structure inaccessible to the transcription machinery.

Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal

The mouse insulin-like growth factor type 2 receptor (Igf2r) is imprinted and expressed exclusively from the maternally inherited chromosome. To investigate whether methylation could function as the imprinting signal, we have cloned 130 kb from the Igf2r locus and searched for sequences methylated in a parental-specific manner. Two regions have been identified: region 1 contains the start of transcription and is methylated only on the silent paternal chromosome; region 2 is contained in an intron and is methylated only on the expressed maternal chromosome. Methylation of region 1 is acquired after fertilization, in contrast with the methylation of region 2, which is inherited from the female gamete. Methylation of region 2 may mark the maternal Igf2r locus in a manner that could act as an imprinting signal. These data suggest that the expressed locus carries a potential imprinting signal and imply that methylation is necessary for expression of the Igf2r gene.

The ontogeny of allele-specific methylation associated with imprinted genes in the mouse.

We have investigated the DNA methylation patterns in genomically imprinted genes of the mouse. Both Igf2 and H19 are associated with clear‐cut regions of allele‐specific paternal modification in late embryonic and adult tissues. By using a sensitive PCR assay, it was possible to follow the methylation state of individual HpaII sites in these genes through gametogenesis and embryogenesis. Most of these CpG moieties are not differentially modified in the mature gametes and also become totally demethylated in the early embryo in a manner similar to non‐imprinted endogenous genes. Thus, the overall allele‐specific methylation pattern at these sites must be established later during embryogenesis after the blastula stage. In contrast, sites in an Igf2r gene intron and one CpG residue in the Igf2 upstream region have allele‐specific modification patterns which are established either in the gametes or shortly after fertilization and are preserved throughout pre‐implantation embryogenesis. These studies suggest that only a few DNA modifications at selective positions in imprinted genes may be candidates for playing a role in the maintenance of parental identity during development.

Methylation of CpG sequences in eukaryotic DNA.

An understanding of the function of DNA methylation in eukaryotes will require a clearer, more precise, knowledge of the distribution of methylated bases in the eukaryotic genome. Although it has been known for some time that 5-methylcytosine (m’Cyt) is the only methylated base in eukaryotes, little is known about the sequence specificity of this modification. One important fact which has emerged from base analyses is that >90% of the msCyt residues are found in the sequence CpG [ 11. It was therefore of interest to determine what fraction of this dinucleotide sequence is methylated. The answer to this question would not only contribute to our knowledge of the distribution of msCyt but would also shed light on the factors which make up the recognition signal for eukaryotic methylation. In an attempt to answer this question we have developed a new experimental procedure for detecting msCyt in CpG sequences. This dinucleotide is found to be highly methylated in animal cells and may be the major determinant in the placement of methyl groups on DNA.

DNA Demethylation In Vitro: Involvement of RNA

An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 myoblasts specifically demethylate an alpha-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.

Regulation of the synthesis of 5-phosphoribosyl-I-pyrophosphate in intact red blood cells and in cell-free preparations.

Abstract The metabolic regulation of the synthesis of 5-phosphoribosyl- i -pyrophosphate (PRPP) was investigated in intact erythrocytes and in cell-free enzyme preparations with the aid of specific enzymic assays described in the present communication. The synthesis of PRPP in erythrocytes incubated in saline-glucose medium was strikingly enhanced by increasing the P i content of the medium up to a 60 mM concentration. Incubation of the erythrocytes in the presence of methylene blue or inosine resulted in a marked increase of their steady-state level of Rib-5- P , which showed no relation to the P i concentration in the medium. On the other hand, the stimulation of PRPP formation attending the build-up of the Rib-5- P pool exhibited a strict dependence on an adequately high P 1 level. These results were taken as evidence that the synthesis of PRPP in the erythrocytes is governed primarily by the P i -dependent catalytic capacity of PRPP synthetase, wherease the rate-limiting role of the intracellular supply of Rib-5- P manifest itself only when the optimum requirement of the system for P i had been satisfied. The activity of PRPP synthetase, as measured in cell-free preparations, was found to be about 200-fold higher than the corresponding rate of PRPP synthesis observed in the intact cells. The catalytic rate of the enzyme was strongly inhibited by ADP, GDP and 2,3-diphosphoglycerate at concentrations close to the physiological levels found in erythrocytes. The inhibition was partially relieved by raising the P i concentration in the reaction mixture over and above the optimum level required in the absence of the inhibitors. The interrelations between P i and the inhibitory metabolites, their implications in the control of the intracellular synthesis of PRPP and the allosteric nature of the underlying molecular mechanism are discussed in the light of available data.

The absence of detectable methylated bases in Drosophila melanogaster DNA

Most of the eukaryotic organisms are methylated at specific cytosine residues in their DNA. For more than 2 decades efforts have been made to answer the question of whether the DNA of the fruit fly Drosophila melanogaster is methylated, but the results were inconclusive. The answer to this question is now attracting special interest in light of the fact that in recent years substantial evidence has accumulated, suggesting a correlation between vertebrate DNA methylation and gene expression [1]. Drosophila in particular is an interesting organism in this respect as it goes through severabdefined developmental stages and its genome organization has been extensively investigated. Here, a variety of highly sensitive methods have been used to analyze methylated bases in Drosophila melanogaster DNA. 5-Methyl-cytosine, which is the common methylated base in DNA of eukaryotic organisms, could not be detected in any of the developmental stages of this organism. There is no indication for other modifications of this DNA as well. The welldefined clonally inherited patterns of methylation of the genetic material in mammals [1] and the absence of such methylation patterns in Drosophila DNA suggested here, bring into focus the longstanding enigma of the biological role played by methylation patterns. 2.1. Preparation of DNA DNA of Drosophila melanogaster embryos, larvae, pupae and adults was prepared from isolated nuclei. The nuclei were lysed in 1 mM Tris (pH 8) and after centrifugation at 10 000 x g the chromatin pellet was suspended in a lysis mixture containing 0.5% (w/v) sodium lauryl sulphate; 2.5 mM EDTA; 0.5 M NaC1; 10 mM Tris (pH 8) and 100 ~g proteinase K/ml (Merck Co.). The mixture was incubated for 2 h at 37°C and RNase treated (300/~g pancreatic RNase/ml for l h at 37°C). Phenol extraction was followed by chloroform:isoamyl alcohol (24: 1, v/v) extraction and ethanol precipitation. The DNA was hydrolyzed to free bases for analysis as in [2].

Dna Methylation and its Possible Biological Roles

Publisher Summary The chapter discusses on the DNA methylation and its possible biological roles. The pattern of DNA methylation is species-specific. In the DNA of some prokaryotes, a small fraction of the cytosine residues is methylated, while in others only adenine residues are methylated. A third group of prokaryotic organisms contain both 5-methylcytosine (m5Cyt) and N6-methyladenine (m6Ade) in their DNA. Eukaryotic DNA, in general, is methylated exclusively at cytosine residues. The species-specific patterns of methylation reflect base specificity as well as sequence specificity of the methylases involved. The introduction of restriction enzymes and DNA cloning techniques into this field of research has already proved to be very fruitful, and should provide sufficient tools for the elucidation of the function of methyl groups in DNA. Tissue specificity with respect to the pattern of methylation can be observed when specific methylatable sites in single genes are studied. However, no significant differences are noted when the average methylation of DNA from various tissues is studied. Preliminary results of experiments in bacteria demonstrate that methylase-deficient mutants are hypersensitive to mutagenesis, and have higher rates of DNA recombination in the chapter, are also preliminary indications that DNA methylation is coupled to DNA replication in E. coli. These experiments should be extended to eukaryotic systems.

DNA Methylation from Embryo to Adult

Publisher Summary This chapter discusses the deoxyribonucleic acid (DNA) methylation from embryo to adult. The existence of tissue-specific methylation patterns and the results of the transfection experiments, taken together, clearly prove that once ethylation patterns are formed, they are propagated in somatic tissues. The methyltransferase found in mammalian tissues is suitable for directing such a maintenance activity, as it shows preferential activity, with hemimethylated DNA as a substrate. Embryonic cells are capable of demethylating and de novo methylating their DNA. These activities allow the embryo to erase the methylation patterns inherited from the gametes and create a new methylation pattern. This characteristic of embryonic cells prompted studies aimed at elucidation of the mechanisms involved in the formation of gene-specific methylation patterns during the embryonic development and gametogenesis. The presumed role of methylation in parental imprinting and the recent finding that target mutation of the murine DNA methyltransferase gene results in embryonic lethality, strongly suggest that the dynamic changes in DNA methylation patterns during embryo development play a role in reprogramming the genome in the developing embryo.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.