Methylation analysis by DNA immunoprecipitation

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)‐based methods to evaluate methylation status of DNA have been instrumental in such genome‐wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome‐wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed. J. Cell. Physiol. 222: 522–531, 2010. © 2009 Wiley‐Liss, Inc.     

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.     

Adipocyte-TSH-receptor-related antibodies in Graves' disease detected by immunoprecipitation

Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of 125I-TSH-receptor complexes and the nature of the antibodies was analyzed. To 125I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50 micrograms of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG. Immunoprecipitation values in 13 patients with Graves' disease were significantly (p less than 0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p less than 0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.     

The degradation of nascent fibrinogen chains is mediated by the ubiquitin proteasome pathway.

Recent studies have shown that ubiquitin-dependent proteolysis by proteasomes plays an essential role in the degradation of ER-retained proteins. We investigated the degradation of individual fibrinogen chains in transfected COS cells which express but do not secrete single chains. In transfected COS cells, the degradation of fibrinogen Bbeta and gamma chain was markedly inhibited by the proteasome inhibitors lactacystin and MG132. These specific proteasome inhibitors also partially affected the degradation of Aalpha chain. In HepG2 cells, which synthesize and secrete fibrinogen, the degradation of intracellular free gamma chain was also inhibited by MG132. We also detected high molecular weight polyubiquitinated forms of fibrinogen chains in transfected COS cells and in HepG2 cells by sequential immunoprecipitation. These results implicate proteasomes in the degradation of fibrinogen chains. In COS cells, gamma chains have a longer half-life than Bbeta chains and Aalpha chains, suggesting that the presence of surplus gamma chains in fibrinogen-producing cells is due to the unequal degradation rate of fibrinogen chains. These results indicate that the ubiquitin-proteasome pathway may be a major system for the degradation of unassembled fibrinogen chains.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.     

Purification of the mRNAs for Ewe alphaS-casein and beta-casein by immunoprecipitation of polysomes.

Specific polysomes synthesizing alphas-casein and beta-casein were immunoprecipitated from total polysomes of the lactating ewe mammary gland. The polysome - anti-casein complex was immunoprecipitated by anti-immunoglobulins. 22%, 32% and 10% of polysomes were immunoprecipitated with saturating amounts of anti-alphas-casein, anti-beta-casein and anti-chi-casein respectively. Poly(U)-Sepharose chromatography of the immunoprecipitated RNAs permitted the isolation of the corresponding poly(A)-containing RNA which migrated as one major band in polyacrylamide gel electrophoresis. As judged by the contamination with the messenger activity for one of the other caseins, the purity of the mRNA for alphas-casein as well as for beta-casein was estimated to be between 75% and 80%.     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Addition of glucosamine and mannose to nascent immunoglobulin heavy chains.

We have investigated the process of protein glycosylation in an attempt to answer the question of whether glucosamine and mannose are added to nascent chains prior to chain completion or only to completed chains after release from the ribosome. The MPC 11 mouse plasmacytoma cell line used in these studies synthesizes a glycosylated gamma2b heavy chain which accounts for 12% of the total protein synthesis. Nascent chains were separated from completed chains by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. Our results indicate that both glucosamine and mannose are incorporated into nascent heavy chains prior to chain completion and release from the ribosome. Gel analysis of specifically immunoprecipitated nascent chains indicates that the carbohydrate moiety can be added to the nascent heavy chains very soon after the presumptive asparaginyl glycosylation site (CH2 domain) is synthesized on the ribosome.     

Purification of messenger ribonucleic acids for fast and slow myosin heavy chains by indirect immunoprecipitation of polysomes from embryonic chick skeletal muscle

Fast and slow myosin heavy chain mRNAs were isolated by indirect immunoprecipitation of polysomes from 14-day-old embryonic chick leg muscle. The antibodies were prepared against myosin heavy chains purified by NaDod-SO4-polyacrylamide gel electrophoresis and were shown to be specific for fast and slow myosin heavy chains. The RNA fractions directed the synthesis of myosin heavy chains in a cell-free translation system from wheat germ. Several smaller peptides were also synthesized in lower concentrations. These probably are partial products of myosin heavy chains, since they are immunoprecipitated with antibodies to myosin heavy chains. Immunoprecipitation of the translation products with the antibodies to fast and slow myosin heavy chains showed the RNA preparations to be approximately 94% enriched for fast myosin heavy chain mRNA and approximately 84% enriched for slow myosin heavy chain mRNA with respect to myosin HC type. Peptides having slightly different mobilities on NaDodSO4-polyacrylamide gels were immunoprecipitated by antibodies to fast and slow myosin heavy chains.     

Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies

Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.