G-quadruplex DNA structure is a positive regulator of MYC transcription.

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.

5-Hydroxymethyl-, 5-Formyl- and 5-Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks.

The four non-canonical nucleotides in the human genome 5-methyl-, 5-hydroxymethyl-, 5-formyl- and 5-carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten-eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α-ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous. This vagueness is caused by the fact that these oxidized mdC derivatives are also formed as oxidative lesions, resulting in unclear basal levels that are likely to have no epigenetic function. Here, we report the xdC levels in the fungus Amanita muscaria in comparison to murine embryonic stem cells (mESCs), HEK cells and induced pluripotent stem cells (iPSCs), to obtain information about the basal levels of hmdC, fdC and cadC as DNA lesions in the genome.

Active turnover of genomic methylcytosine in pluripotent cells.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of ß-elimination (ßE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and ßE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and ßE-site intermediates.

5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.

5-Formyl-2'-deoxycytosine (fdC) is a recently discovered epigenetic base in the genome of stem cells, with yet unknown functions. Sequencing data show that the base is enriched in CpG islands of promoters and hence likely involved in the regulation of transcription during cellular differentiation. fdC is known to be recognized and excised by the enzyme thymine-DNA-glycosylase (Tdg). As such, fdC is believed to function as an intermediate during active demethylation. In order to understand the function of the new epigenetic base fdC, it is important to analyze its formation and removal at defined genomic sites. Here, we report a new method that combines sequence-specific chemical derivatization of fdC with droplet digital PCR that enables such analysis. We show initial data, indicating that the repair protein Tdg removes only 50 % of the fdCs at a given genomic site, arguing that fdC is a semipermanent base.

An integrated study of the affinities of the Aß16 peptide for Cu(I) and Cu(II): implications for the catalytic production of reactive oxygen species.

A new fluorescent probe Aß16wwa based upon the Aß16 peptide has been developed with two orders of magnitude greater fluorescence intensity for sensitive detection of interactions with Cu(II). In combination with the Cu(I) probe Ferene S, it is confirmed that the Aß16 peptide binds either Cu(I) or Cu(II) with comparable affinities at pH 7.4 (log K = -10.4; log K = -10.0). It follows from this property that the Cu-Aß16 complex is a robust if slow catalyst for the aerial oxidation of ascorbate with H2O2 as primary product (initial rate, ∼0.63 min(-1) for Cu-Aß16 versus >2.5 min(-1) for Cuaq(2+)). An integrated study of variants of this peptide identifies the major ligands and binding modes involved in its copper complexes in solution. The dependence of K upon pH is consistent with a two-coordinate Cu(I) site in which dynamic processes exchange Cu(I) between the three available pairs of imidazole sidechains provided by His6, His13 and His14. The N-terminal amine is not involved in Cu(I) binding but is a key ligand for Cu(II). Acetylation of the N-terminus alters the redox thermodynamic gradient for the Cu centre and suppresses its catalytic activity considerably. The data indicate the presence of dynamic processes that exchange Cu(II) between the three His ligands and backbone amide at physiological pH. His6 is identified as a key ligand for catalysis as its presence minimises the pre-organisation energy required for interchange of the two copper redox sites. These new thermodynamic data strengthen structural interpretations for the Cu-Aß complexes and provide valuable insights into the molecular mechanism by which copper chemistry may induce oxidative stress in Alzheimer's disease.

A study of cyanotetrazole oxides and derivatives thereof.

In this work we report on the syntheses of energetic salts of cyanotetrazolate-1- and -2-oxides; this offers a unique ability to compare the effects of tetrazole 1- versus 2-oxidation. 5-Cyanotetrazolate-2-oxide can be synthesized by oxidation of the 5-cyanotetrazolate anion with Oxone, while the corresponding 1-oxide was synthesized by the rearrangement of azidoaminofurazan. Both chemical (multinuclear NMR, IR, and Raman spectroscopies, mass spectrometry, etc.) as well as explosive (impact, friction, and static sensitivities) properties are reported for these energetic salts. Calculated explosive performances using the EXPLO5 computer code are also reported. We furthermore detail the chemistry of these two anions, and their ability to form tetrazole-carboxamides, dihydrotetrazines, and tetrazines. The ability to hydrolyze cyanotetrazole oxides to their amides was demonstrated by two copper complexes. Several crystal structures of these species are presented in addition to full chemical characterization. Finally, the unique 1,4,-bis(2-N-oxidotetrazolate)-1,2,4,5-tetrazine anion was characterized as an energetic material as its ammonium salt.