DNA methylation is one of the best-characterized epigenetic modifications 1–4. While the enzymes that catalyze DNA methylation have been characterized, enzymes responsible for the reversal process have been elusive 5. A recent study indicates that the human Tet1 protein could catalyze the conversion of 5-methyl-C (5mC) of DNA to 5-hydroxyl-methyl-C (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process 6. Here we extended this study by demonstrating that all three mouse Tet proteins can also catalyze a similar reaction. Interestingly, Tet1 plays an important role in mouse ES cell maintenance through maintaining the expression of Nanog in ES cells. Importantly, Tet1 knockdown-mediated down-regulation of Nanog correlated with its promoter methylation, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in preimplantation embryos resulted in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and ICM cell specification.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.

Cytosine methylation in CpG dinucleotides is believed to be important in gene regulation, and is generally associated with reduced levels of transcription. Methylation-mediated gene silencing involves a series of DNA-protein and protein-protein interactions that begins with the binding of methyl-CpG binding proteins (MBPs) followed by the recruitment of histone-modifying enzymes that together promote chromatin condensation and inactivation. It is widely known that alterations in methylation patterns, and associated gene activities, are often found in human tumors. However, the mechanisms by which methylation patterns are altered are not currently understood. In this paper, we investigate the impact of oxidative damage to a methyl-CpG site on MBP binding by the selective placement of 8-oxoguanine (8-oxoG) and 5-hydroxymethylcytosine (HmC) in a MBP recognition sequence. Duplexes containing these specific modifications were assayed for binding to the methyl-CpG binding domain (MBD) of one member of the MBP family, methyl-CpG binding protein 2 (MeCP2). Our results reveal that oxidation of either a single guanine to 8-oxoG or of a single 5mC to HmC, significantly inhibits binding of the MBD to the oligonucleotide duplex, reducing the binding affinity by at least an order of magnitude. Oxidative damage to DNA could therefore result in heritable, epigenetic changes in chromatin organization.

Alterations in cytosine methylation patterns are usually observed in human tumors. The consequences of altered cytosine methylation patterns include both inappropriate activation of transforming genes and silencing of tumor suppressor genes. Despite the biological effect of methylation changes, little is known about how such changes are caused. The heritability of cytosine methylation patterns from parent to progeny cells is attributed to the fidelity of the methylation-sensitive human maintenance methyltransferase DNMT1, which methylates with high specificity the unmethylated strand of a hemimethylated CpG sequence following DNA replication. We have been studying DNA damage that might alter the specificity of DNMT1, either inhibiting the methylation of hemimethylated sites or triggering the inappropriate methylation of previously unmethylated sites. Here, we show that known forms of endogenous DNA damage can cause either hypermethylation or hypomethylation. Inflammation-induced 5-halogenated cytosine damage products, including 5-chlorocytosine, mimic 5-methylcytosine and induce inappropriate DNMT1 methylation within a CpG sequence. In contrast, oxidation damage of the methyl group of 5-methylcytosine, with the formation of 5-hydroxymethylcytosine, prevents DNMT1 methylation of the target cytosine. We propose that reduced DNMT1 selectivity resulting from DNA damage could cause heritable changes in cytosine methylation patterns, resulting in human tumor formation. These data may provide a mechanistic link for the associations documented between inflammation and cancer. [Cancer Res 2007;67(3):946-50]

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (APT) or incorrect (G-T, COT, T-T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon disso- energies of dissociation of correct and incorrect base pairs account for nucleotide insertion fidelity? To address these questions, a thermodynamic analysis (3) is made of melting data for oligonucleotide duplexes containing matched and mismatched template-primer termini. The thermodynamic measurements are compared with enzyme kinetic data obtained with the same DNA sequences under the same conditions, for right and wrong nucleotide insertions (4), and for elongation from matched and mismatched template-primer termini. MATERIALS AND METHODSPurified Drosophila DNA polymerase a holoenzyme (5) was a generous gift of I. R. Lehman (Stanford University, Stanford, CA). Four versions of a 20-base DNA primer (5'-TGATATTCACAACGAATGGN-3'), where N = A, C, G, or T), complementary in sequence (except for terminal base N) to bases 2242-2222 in wild-type M13 DNA (6), were synthesized by conventional solid-phase methods. The template was single-stranded DNA isolated from wild-type M13 phage grown in Escherichia coli strain JM103. Each primer was labeled at the 5' end with 32P using [y-32P]ATP (4500 Ci/ mmol; 1 Ci = 37 GBq) purchased from ICN Radiochemicals and T4 polynucleotide kinase from United States Biochemicals, Cleveland, OH. Procedures for primer 5'-end-labeling and hybridizing to template were the same as described (4).Synthetic DNA duplexes used in melting experiments, representing the last 9 base pairs in the primer-template complexes and differing only in the terminal base pair (NOT), were prepared by annealing equimolar amounts of the component 9-base strands synthesized in the same way as primers.DNA Polymerase Reactions. To measure extension rates at primer 3' ends (N opposite T), with dTTP as substrate for addition of T opposite A, reactions were carried out in the same way with each of the four 5'-end-labeled primers hybridized to M13 template as illustrated in Fig. lb

Oxidation of uracil (U) and thymine (5-Me-U) are believed to play a role in genetic instability because of the changes these oxidations cause in the ionization constants (pK a values), which in turn affects the base pairing and hence coding. However, interpretation of the experimental evidence for the changes of pK a with substitution at U has been complicated by the presence of two sites (N1 and N3) for ionization. We show that a procedure using first principles quantum mechanics (density functional theory with generalized gradient approximation, B3LYP, in combination with the Poisson-Boltzmann continuum-solvation model) predicts such pK a values for a series of 5-substituted uracil derivatives in excellent correlation with experiment. In particular, this successfully resolves which cases prefer ionization at N1 and N3. Such first principles predictions of ionization constant should be useful for predicting and interpreting pK a for other systems.

It has long been postulated that rare tautomeric or ionized forms of DNA bases may play a role in mispair formation. To investigate the role this phenomenon plays in the mispairing of guanine and to develop a calculation methodology that can be extended to mutagenic DNA damage products, we used first principles quantum mechanics (density functional theory (B3LYP) with the Poisson-Boltzmann continuum-solvation model) to calculate the relative stabilities of tautomers of guanine in various environments and their pK a values in aqueous solution. This method allows us to calculate site specific pK a values s information that is experimentally inaccessible s as well as overall pK a values for each stage, wherein our numbers are in agreement with experimental values. We find that neutral guanine exists in aqueous phase as a mixture of two major keto tautomers, the N 9 H form (1) and a N 7 H form (3). These keto forms are also major species present in the gas phase, as well as the O 6 H enol tautomer (7a). These results show that tautomeric configurations can be drastically different depending on the environment. Here, we discuss the reasons for this environmental variability and suggest some possible implications. Finally, we estimate that the relative population of deprotonated guanine is 0.2-2% in the range of pH 7-8, a significant enough population to potentially play a role in mispair formation.