Influence of epigenetic modifications on structural and thermodynamic features of DNA

Medved, Nataša (avtor), Plavec, Janez (mentor)

Cytosine methylation is a crucial epigenetic modification that has subsequently been the focus of intense studies, as it plays a significant role in the regulation of gene expression. Despite this considerable attention, its impact on the formation and stability of G-quadruplex structures remains largely unexplored. We deemed it important to focus on the folding of a G-rich sequence and to evaluate its thermodynamic and kinetic aspects as cytosine residues get methylated. In this regard, we studied the impact of introducing a 5-methylcytosine residue into the oligonucleotide sequence 5'-d(GGG CGC GGG AAGGATT GGG C GGG), which folds into the G-quadruplex structure known as bcl2Mid G4. This well-characterized G4 structure forms within a GC-rich region located immediately upstream of the P1 promoter of the B-cell lymphoma 2 (BCL2) gene, which plays a crucial role in regulating its expression. Using solution-state NMR and complementary biophysical techniques, such as UV-Vis, CD spectroscopy and differential scanning calorimetry (DSC), we discovered a previously unknown effect of 5-methylcytosine on G-quadruplex structures. Specifically, the substitution of the cytosine at position C6 with the modified analogue C6$^m$ destabilizes the predominant structure and increases the ratio of the minor G-quadruplex structure. The altered ratio of the major and minor G-quadruplex structures in the equilibrium, coupled to the changes in the rate of DNA (re)folding, highlights an unexpectedly profound effect of a single 5-methylcytosine residue on the folding and thermodynamic stabilities of guanine-rich DNA. Furthermore, by investigating the details of the minor species resolved upon introducing the 5-methylcytosine modification, we obtained insights into a previously unexplored G-quadruplex structure that exhibits an intriguing snapback element at its 5'-end. Additionally, our findings indicate that the zinc finger 3 motif, which is a part of the DNA-binding domain of the Sp1 transcription factor, preferentially binds to the minor G-quadruplex structure, while it does not show specific interactions with the major G-quadruplex structure. Our findings demonstrate that the seemingly modest impacts of 5-methylcytosine modification(s) on the polymorphism of G-quadruplex structures may be of fundamental importance in contexts of modulating DNA interactions with transcription factors and, furthermore, the regulation of gene expression.

• Jezik: Angleški jezik
• Ključne besede: -
• Vrsta gradiva: Doktorsko delo/naloga
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-176635
• COBISS.SI-ID: 268198659
• Datum objave v RUL: 05.12.2025

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Vpliv epigenetskih modifikacij na strukturne in termodinamske lastnosti DNA

Izvleček

Metilacija citozina je ključna epigenetska modifikacija, ki je bila predmet številnih študij zaradi svoje pomembne vloge pri regulaciji izražanja genov. Kljub temu pa vpliv metilacije citozina na tvorbo in stabilnost nekanoničnih terciarnih G-kvadrupleksnih struktur ostaja v veliki meri nepojasnjen. V tej študiji smo se osredotočili na vpliv uvedbe metiliranega citozinskega ostanka v G-bogato zaporedje ter podrobno ovrednotili termodinamske in kinetične vidike njegovega zvijanja. Preučevano oligonukleotidno zaporedje bcl2Mid 5'-d(GGG CGC GGG AAGGATT GGG C GGG) se nahaja znotraj GC-bogatega območju v P1 promotorski regiji gena BCL2 (B-cell lymphoma 2), ki ima ključno vlogo pri regulaciji njegovega izražanja. Tvorba in strukturni detajli G-kvadrupleksov v bcl2Mid in tudi širši regiji so bili predhodno podrobno raziskani, kar smo uporabili kot izhodišče študije vpliva metilacije citozinskih ostankov. Z uporabo NMR spektroskopije in komplementarnih biofizikalnih metod, kot so UV-Vis spektroskopija, CD spektropolarimetrija in diferenčna dinamična kalorimetrija (DSC), smo odkrili nov in doslej nepoznan vpliv 5-metilcitozina na strukture G-kvadrupleksov. S ciljno usmerjenimi substitucijami citozina na položaju C6 s C6$^m$ smo dokazali, da 5-metilcitozin upočasni kinetiko (ra)zvijanja ter vpliva na ravnotežje struktur G-kvadrupleksov. Dokazali smo, da C6$^m$ modifikacija destabilizira prevladujočo in poveča delež manj zastopane zvrsti. Slednjo smo strukturno okarakterizirali in dokazali, da gre za G-kvadrupleks, ki vsebuje nenavadno in redko opaženo "snapback" zanko na 5'-koncu. S primerjalno strukturno karakterizacijo smo ugotovili, da gre za enak tip zvitja, kot ga ima doslej še neraziskana manj zastopana struktura, ki jo tvori nemodificirana DNK. Odkritje, da lahko že en sam 5-metilcitozinski ostanek bistveno spremeni delež G-kvadrupleksnih struktur v ravnotežju, smo postavili v kontekst bioloških procesov in ugotovili, da se motiv cinkovega prsta 3, ki je del DNK-vezavne domene transkripcijskega faktorja Sp1, specifično veže na manj zastopano G-kvadrupleksno strukturo nemodificirane DNK, medtem ko podobnih interakcij s prevladujočo strukturo nismo zaznali. Rezultati naših študij so osvetlili pomen epigenetskih sprememb v kontekstu polimorfizma G-kvadrupleksov in pripomogli k razumevanju, da lahko navidezno majhne in lokalne strukturne spremembe DNK vplivajo na vezavo transkripcijskih faktorjev ter nadalje regulacijo izražanja genov.

• Ključne besede: -