G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders

Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.

A polymorphic GGC repeat in the NPAS2 gene and its association with melanoma

Circadian clock regulation in mammals is controlled by feedback loops of a set of circadian genes. One of these circadian genes, NPAS2, encodes for a member of the bHLH-PAS class of transcription factors and is expressed in the forebrain and in some peripheral organs such as liver and skin. Other biological processes are also regulated by circadian genes. For example, NPAS2 is involved in cell proliferation, DNA damage repair and malignant transformation. Aberrant expression of clock genes has been previously observed in melanoma which led to our effort to sequence the NPAS2 promoter region in this cancer type. The NPAS2 putative promoter and 5′ untranslated region of ninety-three melanoma patients and ninety-six control subjects were sequenced and several variants were identified. Among these is a novel microsatellite comprising a GGC repeat with different alleles ranging from 7 to 13 repeats located in the 5′ untranslated exon. Homozygosity of an allele with nine repeats (9/9) was more prevalent in melanoma than in control subjects (22.6% and 13.5%, respectively, P: 0.0206) suggesting that some NPAS2 variants might contribute to melanoma susceptibility. Impact statement This report describes a variable microsatellite repeat sequence located in the 5′ untranslated exon of NSPAS2, a gene encoding a clock transcription factor. Significantly, this study is the first to show that a variant copy number GGC repeat sequence in the NPAS2 clock gene associates with melanoma risk and which may be useful in the assessment of melanoma predisposition.

Allele-specific DNA methylation reinforces PEAR1 enhancer activity

Key Points Rs12041331 is the first functional CpG-SNP related to platelet function whose regulatory mechanism depends on DNA methylation. Rs12041331 marks allele-specific methylation at the CpG island encompassing the first untranslated exon during megakaryopoiesis.

The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression

ABSTRACT The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110α through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110α gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110α was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110α expression and hyperactivation of the PI3K/Akt pathway.