Crosstalk between G-Quadruplexes and Dnmt3a-Mediated Methylation of the c-MYC Oncogene Promoter.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

G-Quadruplex Recognition by DARPIns through Epitope/Paratope Analogy.

We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4 s specific sensors. To this end we carried out all-atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human-telomeric (h-telo), Bcl-2, and c-Myc, with different peptides, forming a DARPin/G4 complex. By comparing the sequences of DARPin with that of a peptide known for its high affinity for c-Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three-dimensional arrangement of the molecular fragments involved: the α-helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.

Effects of G-Quadruplex-Binding Plant Secondary Metabolites on c-MYC Expression.

Guanine-rich DNA sequences tending to adopt noncanonical G-quadruplex (G4) structures are over-represented in promoter regions of oncogenes. Ligands recognizing G4 were shown to stabilize these DNA structures and drive their formation regulating expression of corresponding genes. We studied the interaction of several plant secondary metabolites (PSMs) with G4s and their effects on gene expression in a cellular context. The binding of PSMs with G4s formed by the sequences of well-studied oncogene promoters and telomeric repeats was evaluated using a fluorescent indicator displacement assay. c-MYC G4 folding topology and thermal stability, as well as the PMS influence on these parameters, were demonstrated by UV-spectroscopy and circular dichroism. The effects of promising PSMs on c-MYC expression were assessed using luciferase reporter assay and qPR-PCR in cancer and immortalized cultured cells. The ability of PMS to multi-targeting cell signaling pathways was analyzed by the pathway-focused gene expression profiling with qRT-PCR. The multi-target activity of a number of PSMs was demonstrated by their interaction with a set of G4s mimicking those formed in the human genome. We have shown a direct G4-mediated down regulation of c-MYC expression by sanguinarine, quercetin, kaempferol, and thymoquinone; these effects being modulated by PSM's indirect influence via cell signaling pathways.

Enhanced antioxidant and cytotoxic activity of ferrocenyl-substituted curcumin via stabilization of promoter c-MYC silencer element.

We are reporting a successful attachment of ferrocenyl moiety at the active methylene carbon atom of ß-diketone of curcumin via Knoevenagel condensation reaction, to utilize the optimum selectivity toward biological targets. The formation of ferrocenyl curcumin (i.e., Fc-cur) has been confirmed by 1H NMR, 13C NMR, and FT-IR spectra analysis. Further, circular dichroism (CD) spectroscopy, thermal denaturation, absorption, and fluorescence spectroscopy have been used to understand the association of ligand (i.e., Fc-cur) with G-quadruplex. Based on these analysis, the binding mechanism of the ligand i.e., Fc-cur to the parallel and hybrid topology present in different G-quadruplex has been proposed. Further, the binding and modes of the interaction of Fc-cur with Pu27 c-MYC silencer element and H-telo G-quadruplex have unravelled selective and stronger binding via intercalation with the parallel topology of c-MYC G-quadruplex rather than the hybrid topology of H-telo quadruplex. The manifestation of better antioxidant activity of Fc-cur has been demonstrated by showing a stronger radical scavenging capability than pristine curcumin. The cytotoxicity analysis of the proposed ligand i.e., Fc-cur against Vero and HeLa cells have clearly reflected the nontoxicity toward Vero cells and quite effective against the HeLa cells which reduces the cancer cells more effectively than the already reported for curcumin.Communicated by Ramaswamy H. Sarma.

HIF­1α and MBP1 are associated with the progression of breast cancer cells by repressing ß­catenin transcription.

Breast cancer (BC) is a common type of tumor. Numerous patients are diagnosed and treated in the early stages of the disease; however, the recurrence rate remains high. Therefore, identifying sensitive and specific tumor markers to prevent and treat BC is essential. c­Myc promoter binding protein 1 (MBP1) is a regulatory molecule located in the cell nucleus. It targets and regulates the expression of various cell proliferation­, apoptosis­ and tumor­associated genes. MBP1 expression in BC tissues was detected using immunohistochemistry and further validated in BC and normal human cell lines using RT­qPCR and western blot analysis. Low MBP1 expression, in clinical samples of BC, was associated with a poor prognosis of BC (n=50). MBP1 overexpression effectively inhibited the growth and metastasis of xenograft tumors in vivo. Cell counting kit­8 assays confirmed that the proliferation of the BC cell lines was significantly increased following knockdown of MBP1 expression, while overexpression of MBP1 could significantly inhibit the proliferation of the BC cell lines. Mechanistically, a dual­luciferase assay was used to confirm that MBP1 was the key transcriptional regulator of ß­catenin. In addition, MBP1 transcription and hypoxia­inducible factor (HIF­1α) induction were associated. By regulating the hypoxic microenvironmental state in the MDA231 and MCF7 cell lines, it was demonstrated that MBP1 served as a hypoxia­responsive factor and could be a new target for tumor therapy. Taken together, these results suggested that MBP1, as a potential tumor marker associated with prognosis of BC and may serve as a therapeutic target for BC. Moreover, MBP1 plays a critical role in inhibiting the growth and progression of BC cell lines.

Progranulin (granulin/epithelin precursor) and its constituent granulin repeats repress transcription from cellular promoters.

Progranulin (also known as granulin/epithelin precursor, GEP) is composed of seven granulin/epithelin repeats (granulins) and functions both as a full-length protein and as individual granulins. It is a secretory protein but a substantial amount of GEP is found inside cells, some in complexes with positive transcription elongation factor b (P-TEFb). GEP and certain granulins interact with the cyclin T1 subunit of P-TEFb, and with its HIV-1 Tat co-factor, leading to repression of transcription from the HIV promoter. We show that GEP lacking the signal peptide (GEPspm) remains inside cells and, like wild-type GEP, interacts with cyclin T1 and Tat. GEPspm represses transcription from the HIV-1 promoter at the RNA level. Granulins that bind cyclin T1 are phosphorylated by P-TEFb in vivo and in vitro on serine residues. GEPspm and those granulins that interact with cyclin T1 also inhibit transcription from cellular cad and c-myc promoters, which are highly dependent on P-TEFb, but not from the PCNA promoter. In addition, GEPspm and granulins repress transcriptional activation by VP16 or c-Myc, proteins that bind and recruit P-TEFb to responsive promoters. These data suggest that intracellular GEP is a promoter-specific transcriptional repressor that modulates the function of cellular and viral transcription factors.