HRASis silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property

Abstract

The HRAS promoter contains immediately upstream of the transcription start site two neighboring G-elements, each capable of folding into a G-quadruplex structure. We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1. In the present study we have examined the interaction between the HRAS promoter and MAZ, demonstrating for the first time that the protein unfolds the G-quadruplex structures. We also demonstrate that MAZ-GST, in the presence of the complementary strands, promotes a rapid transformation of the two HRAS quadruplexes into duplexes. By a mutational analysis of the HRAS G-elements, we dissected the MAZ-binding sites from the quadruplex-forming motifs, finding that the two neighboring G-quadruplexes bring about a dramatic repression of transcription, in a synergistic manner. We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules. We found that a cell-penetrating anthratiophenedione (ATPD-1), which binds tightly to the G-quadruplexes (ΔT > 15°C), promotes the total extinction of HRAS transcription. In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%. Our study provides relevant information for the rationale design of targeted therapy drugs specific for the HRAS oncogene.

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

“…The LTR G4 system displays regulating features similar to those described for the c-myc4344, HRAS26 and KRAS oncogene-promoters30454647. As in these eukaryotic G4-modulated promoters, the HIV-1 LTR promoter is processed by G4 stabilizing (nucleolin)22 and destabilizing proteins (hnRNP A2/B1).…”

“…R was calculated from FRET efficiency values as , where R 0 (Förster distance) is the distance at which energy transfer is 50% of the maximum value. Between FAM and TAMRA fluorophores, R 0 is assumed to be 50Ǻ26. F-test (F) and the probability (P) values were calculated using R statistical environment (v. 3.3.2)31.…”

“…Besides the shelterin complex proteins that are involved in telomere homeostasis24, G4 interacting proteins either stabilize (e.g. nucleolin, MAZ and nucleophosmin) or unfold (the helicase and heterogeneous nuclear ribonucleoprotein (hnRNP) families)2526 the G4 conformation to allow for a tightly controlled modulation of the epigenetic G4 switch.…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…The proteins recognizing G4s belong to different classes and perform different functions in the cell: regula tion of telomere length, ribosome assembly [272], tran scription, translation, splicing, and alternative splicing [20, 269, 273 276], DNA replication and reparation [277,278], replicative bypass base damaged sites [279], reverse transcription [206], conjugation of homologous chromosomes during meiosis, DNA duplex unwinding, changing DNA topology, and other processes of nucleic acid metabolism. The G4 binding proteins are classified into three functional groups: (i) telomere related pro teins, such as proteins of shelterin complex [267,280,281]; (ii) G4 unwindig or cleaving enzymes, such as DNA and RNA helicases (molecular motors that unwind structured nucleic acids and thus are involved in numerous biological processes), nucleases [273,282,283]; (iii) proteins that stabilize G quadruplexes and prone their folding (molecular chaperons), for example, nucleolin (multifunctional nuclear protein) and nucleo phosmin [269,276,284]. The proteins and enzymes with Fig.…”

High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

“…The biological functions of G4 DNA are largely dependent on the protein factors that modulate the G4-conformation and/or serve as a bridge to recruit additional protein regulators [23]. These G4 binding proteins can be classified into three functional groups: 1) telomererelated proteins, such as the shelterin complex, human CST (CTC1-STN1-TEN1), and yeast RAP1 and Est1 [24][25][26]; 2) proteins that unfold and/or process the G4 structure, such as the helicases including RecQ family helicases hBLM, hWRN, ySgs1, and yPif1 [27]; and 3) proteins that stabilize G4 structures including MAZ and nucleophosmin [28,29]. Mutations in some of these G4-interacting proteins have been linked to genetic diseases such as Werner syndrome, Fanconi anemia, and cancer [27,[30][31][32].…”