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. 2020 Mar 5;13(1):12.
doi: 10.1186/s13072-020-00334-y.

In Vivo Visualization of the I-Motif DNA Secondary Structure in the Bombyx Mori Testis

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Free PMC article

In Vivo Visualization of the I-Motif DNA Secondary Structure in the Bombyx Mori Testis

Wenhuan Tang et al. Epigenetics Chromatin. .
Free PMC article

Abstract

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.

Keywords: Chromosome; DNA secondary structure; Epigenetic regulation; In vivo detection; i-motif.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CD analysis of the effect of pH on the formation of i-motif structures. aBmPOUM2 wild-type; bBmPOUM2 mutant; cBGIBMGA003213 wild-type; dBGIBMGA003213 mutant. The sequences of these DNA fragments are listed in Table 1. DNA oligonucleotide sequences were folded in Tris–TAE buffer at pH 5.00, 6.02, 7.13 and 8.00 before CD scanning from 200 to 360 nm. The wild-type and mutated sequences are shown in Table 1
Fig. 2
Fig. 2
EMSA for the specific binding of BmILF to the i-motif structure. The i-motif probe was synthesized and refolded into an i-motif structure at pH 4.0, 6.0 and 8.0. The ssDNA is the unfolded sequence. The cold probe is the un-labeled i-motif probe. The sequence of the mutated probe is shown in Table 1. The linear free probe is the same DNA fragment that did not form an advanced structure during the annealing cooling process. EMSA for the binding of recombinant BmILF to the i-motif probe of BmPOUM2a and BGIBMGA003213b at pH 4.00 in the 1 × binding solution (20 μl, 2.5% glycerol, 0.05% NP-40, 5 mM MgCl2, 4 mM EDTA, recombinant BmILF protein and biotinylated end-labeled probe) at room temperature for 20 min. The running buffer contained 0.04 M Tris, 0.04 M H3BO3, 0.001 M EDTA-2Na and was filtered with 0.22-µm pore-size filter. EMSA for the binding of recombinant BmILF to the i-motif structure of BmPOUM2 (c) and BGIBMGA003213 (d) or the linear ssDNA probe at pH 4.0, 6.0 and 8.0. EMSA for the binding of recombinant BmILF to different DNA motifs on BmPOUM2 (e) and BGIBMGA003213 (f). The positions of the labeled i-motif-containing probe, labeled ssDNA probe, labeled bound i-motif and BmILF are shown by the arrows. g CD analysis of the complex of BmILF and i-motif at pH 4.0. The sequences of all the probes used in this figure are listed in Table 1
Fig. 3
Fig. 3
The binding affinity of BmILF to i-motif structures. EMSA for the binding of the recombinant BmILF to the i-motif probes of BmPOUM2 (A) and BGIBMGA003213 (B) at different concentrations of the probe (Aa, Ba) or protein (Ab, Bb). Quantitative measurement of the binding band of BmILF and i-motif of BmPOUM2 (Ac) and BGIBMGA003213 (Bc). Hill curves and the Kd values of the binding of BmILF and i-motif probes of BmPOUM2 (Ad) and BGIBMGA003213 (Bd). MST analysis for the binding affinity of the recombinant BmILF with the i-motif of BmPOUM2 (C) and BGIBMGA003213 (D) at different protein concentrations
Fig. 4
Fig. 4
Immunofluorescence visualization of i-motif structure in the B. mori testis. a Testes were isolated from the 5th instar larvae and treated as described in “Materials and Methods” section. b Schematic diagram of i-motif immunofluorescence detection. The primary antibody was an anti-BmILF antibody at a 1:2000 dilution; the secondary antibody was Alexa Fluor™ 594-conjugated goat anti-rabbit IgG at a 1:400 dilution. The red fluorescence shows the binding of BmILF to the i-motif structure in the nuclei of testis cells. c BmILF antibody recognizes the endogenous BmILF-bound i-motif structure in the nucleus, without the addition of exogenous BmILF. d Increased i-motif staining after incubation with exogenous recombinant BmILF. e Immunofluorescence staining of the i-motif after treatment with DNase I. The dotted lines show the boundaries of the nuclei; f the white light and fluorescence merged image of e, showing the position of the nuclei. g Immunofluorescence staining of the i-motif after the pre-incubation of BmILF with pre-folded C-quadruplex oligonucleotides. Nuclei were counterstained with DAPI (blue). h Quantification of i-motif signals for 50 nuclei in five random observation regions on the slide. The data are represented as the mean ± SEM of three replicates
Fig. 5
Fig. 5
Immunofluorescence detection of i-motif structures in B. mori chromosomes during the cell cycle in testis. Testes were isolated from the 4th instar larvae and treated as described in “Materials and Methods” section. ac Staining of the i-motif (red) was observed in the interphase, prophase and metaphase of the cell cycle. d Quantification of the average number of i-motif staining signals for nuclei of testis cells. The data are represented as the mean ± SEM of three replicates. Chromosomes were counterstained with DAPI (blue)
Fig. 6
Fig. 6
Effects of porphyrin compounds on i-motif formation in B. mori testis cells. Testes were isolated from 5th instar larvae and treated as described in “Materials and Methods” section. The porphyrin compound TMPyP4 (a, c, e, h and j) or TMPyP2 (b, d, f, i and k) at 1 (h, i), 5 (j, k), 30 (a, b), 50 (c, d) or 70 µM (e, f) was added to the testis culture medium before immunofluorescence analysis. The red fluorescence signals show the binding of BmILF to i-motif structures in the nuclei. The nuclei were counterstained with DAPI (blue). g, l Quantification of i-motif staining signals for 50 nuclei in five randomly selected observation regions on the slide. The data are represented as the mean ± SEM of three replicates
Fig. 7
Fig. 7
Effects of different pH values on i-motif formation in the B. mori testis. Testes were isolated from 5th instar larvae and treated as described in “Materials and Methods” section. a Analysis of the intracellular pHrodo Red fluorescence intensity in testis cells cultured in media with different pH values. A higher fluorescence intensity indicates a lower pH value. be The pH values of the testis cell culture medium were adjusted with HCl and NaOH to 5.00, 6.02, 7.13 and 8.00 before immunofluorescence detection. The red fluorescence signals show the binding of BmILF to i-motif structure. Nuclei were counterstained with DAPI (blue). f Quantification of i-motif staining signals for 50 nuclei in five randomly selected observation regions on the slide. The data are represented as the mean ± SEM of three replicates. g qRT-PCR analysis of the pH effects on the expression of BmPOUM2. h qRT-PCR analysis of the pH effects on the expression of BGIBMGA003213

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