In vivo visualization of the i-motif DNA secondary structure in the Bombyx mori testis

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. 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. 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.