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.

Background: A large number of in vitro experiments have confirmed that DNA molecules can form i-motif advanced structure when multiple cytosines exist in the sequence. However, whether these structures are present in vivo environment still lacks sufficient experimental evidence. Results: In this paper, we report the in vivo visualization of i-motif structures in the nuclei and chromosomes of the testis of the invertebrate Bombyx mori using immunofluorescence staining with an antibody specifically recognizing the endogenous transcription factor BmILF, which binds i-motif structure with high specificity. The number of i-motif structures observed in the genome increased when the pH was changed from basic to acidic and decreased under treatment with an i-motif inhibitor, the porphyrin compound TMPyP4. The pH change affected the transcription of genes that contain i-motif sequences. Moreover, there were more i-motif structures observed in the testis cells in interphase than in any other cell cycle stage. Conclusions: In this study, the i-motif structures in invertebrates were detected for the first time at the cell and organ levels. The formation of the structures depended on cell cycle and pH and affected gene expression.