Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis -Element in Response to 5-Formylcytosine and 5-Carboxycytosine

Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2′-deoxy-(5-fC/5-caC) or their BER-resistant 2′-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis -element (5′-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5′-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches

Analysis of an Active Deformylation Mechanism of 5‐Formyl‐deoxycytidine (fdC) in Stem Cells

The removal of 5‐methyl‐deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5‐hydroxymethyl‐deoxycytidine (hmdC) and further on to 5‐formyl‐deoxycytidine (fdC) and 5‐carboxy‐deoxycytidine (cadC) with the help of α‐ketoglutarate‐dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC. A second pathway is proposed to involve C−C bond cleavage that converts fdC directly into dC. A 6‐aza‐5‐formyl‐deoxycytidine (a‐fdC) probe molecule was synthesized and fed to various somatic cell lines and induced mouse embryonic stem cells, together with a 2′‐fluorinated fdC analogue (F‐fdC). While deformylation of F‐fdC was clearly observed in vivo, it did not occur with a‐fdC, thus suggesting that the C−C bond‐cleaving deformylation is initiated by nucleophilic activation

Synthesis of 2'-fluorinated-2'-deoxycytidine derivatives to investigate a direct DNA demethylation pathway in stem cells

This master’s thesis is divided into a literature review and an experimental part. The literature review starts with an introduction of nucleic acids and epigenetics, covering both chemical- and biological aspects. These chemical- and biological processes are explained at a level that is needed to understand the aim of the experimental project and the purpose of the synthesized compounds. The main focus of this thesis is the 2’-deoxyribonucleic acid, especially 2’-deoxycytidine derivatives being in the leading role. The roles and functions of other components, like purine bases and histones, are not included in this thesis. The latter part of the literature review, concerning the chemistry of 2’-deoxy pyrimidines and respective 2’-fluorinated derivatives, introduces their chemical properties. Based on the published data, this review will summarize different chemical approaches to introduce a variety of functional groups to the C5-postion of the pyrimidine ring to obtain C5 functionalized- 2’-deoxyuridine and 2’- deoxycytidine derivatives. In the experimental part, the main objective was to synthesize 5-nitro-2’-fluoro2’-deoxycytidine and 5-(butyl-4-acetoxy-benzoate)-2’-fluoro-2’-deoxycytidine to study their roles in the active demethylation process that occurs via C–C bond cleavage. The last steps of the latter mentioned compound requires further optimization and therefore remains under work. 5-Nitro-2’-fluoro-2’-deoxycytidine was further studied by feeding it to cultured mammalian cells. For the detection of its potential incorporation into DNA, UHPLC-MS/MS was used to provide quantitative data. The analysis showed no incorporation occurred into the DNA, however the nucleoside was found in the soluble pool and therefore, could have other biological implications. This thesis gives a brief insight into the challenging field of chemical biology that is not studied during the master courses but is explained in the understandable manner for the students on master level. It will be of interest to an audience of multidisciplinary researchers in organic- and biological chemistry and it can be helpful for entering chemists to understand the chemical aspects concerning epigenetics and nucleoside chemistry as well as their connection to biology.Tämä opinnäytetyö jakautuu kirjallisuuskatsaukseen ja kokeelliseen osioon. Kirjallisuuskatsaus alkaa tutustumisella nukleiinihappoihin ja epigenetiikkaan kemiallisesta ja biologisesta näkökulmasta. Biologinen tausta on selitetty tasolla, mikä on tarpeellista kokeellisen osion ymmärtämiseksi, sekä ymmärtääkseen mikä on syntetisoitujen yhdisteiden tarkoitus. Tämän kirjallisuuskatsauksen pääroolissa ovat 2’-deoksyribonukleiinihapot, erityisesti 2’-deoksysytidiini johdannaiset. Muiden komponenttien, kuten puriini emästen ja histonien rooli ei sisälly tähän tutkielmaan. Toisessa osiossa kohteena ovat 2’-deoksipyrimidiinien ja niiden 2’-fluorinoitujen johdannaisten kemia, jossa esitellään näiden yhdisteiden kemiallisia ominaisuuksia. Perustuen julkaistuihin tutkimustuloksiin, tämä kirjallisuustutkielma kokoaa erilaisia lähestymistapoja lisätä eri funktionaalisia ryhmiä C5 asemaan pyrimidiini emäksissä. Kokeellisen osion pääkohteena oli syntetisoida 5-nitro-2’-fluoro-deoksisytidiini ja 5-(butyyli-4-asetoksi-bentsoaatti)-2’-fluoro-deoksisytidiini työkaluiksi aktiivisen demetylaation tutkimiseen, joka tapahtuu C–C sidoksen katkeamisen kautta. Jälkimmäisenä mainitun yhdisteen viimeiset vaiheet ovat vielä työnalla. 5-nitro2’-fluoro-deoksisytidiini syötettiin nisäkkään kantasoluille, joissa sen toivottiin yhdistyvän niiden genomiin. Potentiaalinen DNA:han liittyminen analysoitiin ultra korkean erotuskyvyn nestekromatografialla, joka oli kytketty tandem massaspektrometriin. Analysiin perusteella voidaan sanoa, ettei syötetty nukleosidi liittynyt DNA:han, mutta löydettiin solulimaan liukenevasta osasta. Tämä löydös vaatii lisätutkimusta, jotta yhdisteen biologinen merkitys kantasoluissa saadaan selville. Tämä opinnäytetyö on suunnattu poikkitieteelliselle orgaanisen- ja biologisen kemian aloista kiinnostuneille. Se helpottaa alalle tulevia uusia kemistejä ymmärtämään epigenetiikaa kemiallisesta näkökulmasta ja sen yhteydestä biologiaan

Unified Description of Ultrafast Excited State Decay Processes in Epigenetic Deoxycytidine Derivatives

none8siEpigenetic DNA modifications play a fundamental role in modulating gene expression and regulating cellular and developmental biological processes, thereby forming a second layer of information in DNA. The epigenetic 2′-deoxycytidine modification 5-methyl-2′-deoxycytidine, together with its enzymatic oxidation products (5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine, and 5-carboxyl-2′-deox- ycytidine), are closely related to deactivation and reactivation of DNA transcription. Here, we combine sub-30-fs transient absorption spectroscopy with high-level correlated multiconfigurational CASPT2/MM computational methods, explicitly including the solvent, to obtain a unified picture of the photophysics of deoxycytidine-derived epigenetic DNA nucleosides. We assign all the observed time constants and identify the excited state relaxation pathways, including the competition of intersystem crossing and internal conversion for 5-formyl-2′-deoxycytidine and ballistic decay to the ground state for 5-carboxy-2′-deoxycytidine. Our work contributes to shed light on the role of epigenetic derivatives in DNA photodamage as well as on their possible therapeutic use.openKabaciński, Piotr; Romanelli, Marco; Ponkkonen, Eveliina; Jaiswal, Vishal Kumar; Carell, Thomas; Garavelli, Marco; Cerullo, Giulio; Conti, IreneKabaciński, Piotr; Romanelli, Marco; Ponkkonen, Eveliina; Jaiswal, Vishal Kumar; Carell, Thomas; Garavelli, Marco; Cerullo, Giulio; Conti, Iren