G-quadruplexes as novel cis-elements controlling transcription during embryonic development

Abstract

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

Transcriptional control by G-quadruplexes: In vivo roles and perspectives for specific intervention

“…Several reports suggest that G-quadruplexes act as transcriptional repressors by impeding transcription factor binding to duplex-DNA or stalling the progression of RNA polymerase, mostly when they are located downstream the transcription start site in the template strand. 2,[20][21][22] Conversely, other reports showed that G-quadruplex may enhance the transcription of particular genes by favoring the binding of specific transcription factors 2,12,22 or by holding the DNA molecule open thus facilitating the re-initiation of transcription 2,7,14,22 (Fig. 1B).…”

“…In this context, a novel strategy consisting of the use of short antisense DNA sequences or oligonucleotides (ASO) blocking the formation of a specific G-quadruplex has been recently reported. [12][13][14] Numerous studies performed in cellulo have demonstrated the influence of different PQS patterns and loop lengths, 15,16 the effect of ions, 17,18 and the action of specific G-quadruplex ligands 8,9,19 on G-quadruplex stability and the transcriptional process. Mostly, these studies consisted in assessing the effect of specific G-quadruplex ligands on the transcriptional expression of reporter genes governed by promoter elements containing PQS.…”

“…23 However, such study was unable to conclusively demonstrate that phenotypes were due to the tested ligand on the Cdh5 G-quadruplex. In view of the high number of PQS present in zebrafish genome, 14 the possibility of non-specific or pleiotropic effects of such ligands could not be ruled out.…”

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA

“…The stability of these sequence-dependent dynamic structures of DNA are highly dependent on the immediate ionic microenvironment 12 . For example, certain G-rich DNA sequences have been shown to fold into noncanonical four-stranded structures called G-quadruplexes (G4, which are stabilized by cations) in the regulatory regions of the human genome and are proven to regulate transcription, recombination and replication [12][13][14] . To determine whether exposure of cells to GNRs can result in structural alterations of genomic DNA, we evaluated alterations in G4 foci in cells exposed to GNRs and GNPs (CTAB-and MTAB-coated), compared to PS NPs, MTAB only, CTAB only, chloro(triphenylphosphine) gold(i) (PPh 3 .AuCl) treated and untreated cells.…”

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

“…On one hand, they can perform regulatory functions in the cell, inhibit oncogene expression, block undesired elongation of telomeric DNA, control the level of negative supercoil ing in the genome, and serve as targets of antitumor ther apeutics. Recently, the role of G quadruplexes in embryo development, which is the most controlled process in ver tebrate biology, was established [426]. On the other hand, G4 formation causes genome instability (genomic inver sions, recombination, mutations, deletions, and others) associated with oncological diseases and neurological dis orders.…”