BmILF and i-motif structure are involved in transcriptional regulation of BmPOUM2 in Bombyx mori

Abstract

Guanine-rich and cytosine-rich DNA can form four-stranded DNA secondary structures called G-quadruplex (G4) and i-motif, respectively. These structures widely exist in genomes and play important roles in transcription, replication, translation and protection of telomeres. In this study, G4 and i-motif structures were identified in the promoter of the transcription factor gene BmPOUM2, which regulates the expression of the wing disc cuticle protein gene (BmWCP4) during metamorphosis. Disruption of the i-motif structure by base mutation, anti-sense oligonucleotides (ASOs) or inhibitory ligands resulted in significant decrease in the activity of the BmPOUM2 promoter. A novel i-motif binding protein (BmILF) was identified by pull-down experiment. BmILF specifically bound to the i-motif and activated the transcription of BmPOUM2. The promoter activity of BmPOUM2 was enhanced when BmILF was over-expressed and decreased when BmILF was knocked-down by RNA interference. This study for the first time demonstrated that BmILF and the i-motif structure participated in the regulation of gene transcription in insect metamorphosis and provides new insights into the molecular mechanism of the secondary structures in epigenetic regulation of gene transcription.

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

“…The reason for choosing cyanine and ATTO dyes was their high applicability in detecting low‐abundance nucleic acids and effective FRET . Oligonucleotides were purchased from IDT (HPLC purified) and were used as received.…”

“…Given that they are covalently linked to the terminal positions of DNA probes, fluorophores interact with both the environment and the nucleic acid helix, which results in enthalpy‐driven stabilisation of up to 12 °C . In particular, terminally attached cyanine dyes have a strong stabilisation of the DNA helix that has been studied by UV melting at a concentration of 1 μ m . In particular, the positive molecular charge of a fluorophore reduces the electrostatic repulsion that stabilises the DNA helix …”

“…Overall, the stability of the control ON7+ON8 duplex decreased by lowering the concentration of the oligonucleotides, whereas the duplexes with Cy3 and Cy5 showed a constant T m value within the studied concentration range. The control ON7+ON8 duplex showed stability of 57 °C at a 1 μ m concentration, which was higher than that previously reported . Upon diluting the duplex to 0.25 μ m , the T m value decreased to 54.5 °C.…”

“…In this way, denaturation is associated with a decrease in fluorescence emission by the acceptor and an increase in fluorescence emission by the donor, and this can be monitored separately by using a spectrofluorometer with different channels. Mergny investigated the secondary structure of cytosine‐rich oligodeoxynucleotides with fluorescent probes labelled with a FRET pair and was able to follow the intramolecular folding into an i‐DNA motif (a four‐stranded DNA secondary structure) at concentrations as low as 50 p m .…”

Structural Characterization of i‐Motif Structure in the Human Acetyl‐CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression

“…The physiological relevance of i‐motif structures has remained elusive, primarily because of the lack of specific regulatory protein(s) that bind to this motif. However, a nuclear transcription factor in Bombax mori , BmILF, which regulates transcription of POUM2 gene, acts through the i‐motif structure . Additionally, heterogeneous nuclear ribonucleoprotein K (hnRNP K), ASF/SF2 and single‐strand DNA‐binding protein bind efficiently to C‐rich sequences .…”