G-quadruplex forming sequences modulate basal transcription of a yeast reporter and impact the transactivation by P53 family proteins

abstrakt

angličtina

Local DNA structures play an important role in various biological processes. A very thermodynamically stable four-way structure formed in G-rich sequences G-quadruples (G4) is increasingly being revealed as critical players in DNA metabolism, including the accessibility and activity of promoters. Exploiting a well-defined transcriptional reporter system in yeast, we systematically investigated the impact of various G4 prone sequences on basal and induced transcription. A 22-mer G4-prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate the characteristic guanine stretches were placed upstream of a minimal promoter and adjacent to a P53 response element (RE) in otherwise isogenic strains of luciferase reporter. The group of G4 sequences was analyzed by the thioflavin T binding test and circular dichroism in vitro and then we analyzed the impact of G4 sequences on the yeast reporter system. We have measured the basal activity of the reporter as well as the expression induced by transactivation of the p53 family protein. Wild-type P53, P73 and wild-type P63 isoforms, nine cancer-associated P53 missense mutants and three ectodermal dysplasia P63 alleles were tested. The results show that G4-prone sequences increase basal transcriptional activity proportional to their relative propensity to adopt a G4 structure. Furthermore, G4-prone sequences can cooperate with weaker wild-type P63 and P73 proteins but not with P53. Finally, G4-prone sequences increase the ability to transactivate mutant alleles from the partial function P53 family, indicating the importance of including an evaluation of local DNA structural motifs within promoters to predict the functional impact of disease-associated TP53 and TP63 mutations. These results point to the possibility of transcriptional regulation by G4-associated transcription factors and by induction of G4 structure formation within yeast promoters.

P53 family proteins, G-quarduplex, DNA-protein interactions, transcription activity, Yeasts one-hybrid isogenic system

BRÁZDA, V.; KRATOCHVILOVÁ, L.; VOJSOVIČ, M.; VALKOVÁ, N.; ŠISLEROVÁ, L.; EL RASHED, Z.; MONTI, P.; INGA, A.

20. 12. 2023

2564-615X

The EuroBiotech Journal

7 (2023)

s1

Italská republika

15

15

1

10.2478/ebtj-2023-0019