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.

G-quadruplex (G4) structures have been predicted in the genomes of many organisms and proven to play regulatory roles in diverse cellular activities. However, there is little information on the evolutionary history and distribution characteristics of G4s. Here, whole-genome characteristics of potential G4s were studied in 37 evolutionarily representative species. During evolution, the number, length, and density of G4s generally increased. Immunofluorescence in seven species confirmed G4s’ presence and evolutionary pattern. G4s tended to cluster in chromosomes and were enriched in genetic regions. Short-loop G4s were conserved in most species, while loop-length diversity also existed, especially in mammals. The proportion of G4-bearing genes and orthologue genes, which appeared to be increasingly enriched in transcription factors, gradually increased. The antagonistic relationship between G4s and DNA methylation sites was detected. These findings imply that organisms may have evolutionarily developed G4 into a novel reversible and elaborate transcriptional regulatory mechanism benefiting multiple physiological activities of higher organisms.

Double-stranded DNAs are usually present in the form of linear B-form double-helix with the base pairs of adenine (A) and thymine (T) or cytosine (C) and guanine (G), but G-rich DNA can form four-stranded G-quadruplex (G4) structures, which plays important roles in transcription, replication, translation and protection of telomeres. In this study, a RNA recognition motif (RRM)-containing protein, BmLARK, was identified and demonstrated to bind G4 structures in the promoters of a transcription factor BmPOUM2 and other three unidentified genes of Bombyx mori , as well as three well-defined G4 structures in the human genes. Homologous LARKs from Bombyx mori, Drosophila melanogaster, Mus musculus and Homo sapiens bound G4 structures in BmPOUM2 and other genes in B. mori and H. sapiens . Upon binding, LARK facilitated the formation and stability of the G4 structure, enhancing the transcription of target genes. The G4 structure was visualized in vivo in cells and testis from invertebrate B. mori and vertebrate Chinese hamster ovary (CHO) cells. The results of this study strongly suggest that LARK is a novel and conserved G4-binding protein and that the G4 structure may have developed into an elaborate epigenetic mechanism of gene transcription regulation during evolution.

Insulin-like growth factors (Igfs) are implicated in a wide variety of physiological roles in teleost gonadal development and reproduction. In the present study, igf3 mRNA expression in the tilapia ovary was found to be higher than in the testis from 5 to 40 days after hatching (dah) but was lower than that in testis from 50 to 70 dah. Consistently, Igf3 protein signal was detected in the somatic cells of XX and XY gonads from 10 dah until adulthood by immunohistochemistry, using a specific Igf3 polyclonal antibody. Incubation of ovarian and testicular cells in primary culture with recombinant Igf3 significantly increased nr5a1, foxl2, dmrt1, cyp19a1a, cyp11a1, cyp11b2, hsd3b2 , and cyp17a1 expression in a time- and dose-dependent manner. Promoter analysis using luciferase assays in HEK293 cells revealed that igf3 promoter activity was directly activated by Nr5a1 (Sf1) and further enhanced by Foxl2, Nr0b1a (Dax1), and Nr0b1b (Dax2) but repressed by Dmrt1 and estrogen receptor (Esr1, Esr2a, or Esr2b) along with 17beta-estradiol treatment. In addition, igf3 promoter activity was increased slightly by forskolin treatment alone but synergistically up-regulated by transfection with nr5a1. These in vitro results correlated well with the expression profile of igf3 during early gonad differentiation. Our results indicated that igf3 is involved in fish gonad steroidogenesis because of its ability to regulate the expression of foxl2, dmrt1, and nr5a1 and steroidogenic enzymes. The expression of igf3 is in turn regulated by transcription factors Foxl2, Dmrt1, and Nr5a1, as well as by 17beta-estradiol treatment.

Investigations on callus cultures of Securinega suffruticosa indicated that the cell line of S. suffruticosa callus was able to accumulate four known Securinega alkaloids with dextro rotation but not levo rotation as reported before: virosecurinine (1), viroallosecurinine (2), 14,15-dihydrovirosecurinine (3) and ent-phyllanthidine (4). Time course studies on the growth of callus cultures were carried out. The effects of different plant growth regulators, sucrose concentrations on callus growth and virosecurinine production were also reported.