Volume 23, Issue 10, Pages 1261-1270 (October 2016) Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres  Hui-Yun Liu, Qi Zhao, Tian-Peng Zhang, Yue Wu, Yun-Xia Xiong, Shi-Ke Wang, Yuan-Long Ge, Jin-Hui He, Peng Lv, Tian-Miao Ou, Jia- Heng Tan, Ding Li, Lian-Quan Gu, Jian Ren, Yong Zhao, Zhi-Shu Huang  Cell Chemical Biology  Volume 23, Issue 10, Pages 1261-1270 (October 2016) DOI: 10.1016/j.chembiol.2016.08.013 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 1261-1270DOI: (10. 1016/j. chembiol Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Binding Specificity of the D1 Antibody for Parallel G-Quadruplexes (A) Binding selectivity was determined using ELISA and revealed that the D1 antibody had a high affinity for parallel DNA but had undetectable binding to antiparallel G4 DNA, hybrid G4 DNA, double-stranded DNA, random single-stranded DNA, and a DNA hairpin. The error bars represent the SEM calculated from three replicates. (B) Further specificity testing of D1 for parallel G-quadruplex binding was performed using competition ELISA. The error bars represent the SEM calculated from three replicates. (C) ELISA binding curves of D1-DNA interactions were determined. The dissociation constants (Kd) are indicated. The error bars represent the SEM calculated from three replicates. (D) Binding selectivity of D1 for parallel G4 DNA in the cell context was assessed. The arrows indicate the merged foci of D1 and MYC. D1 co-localized with MYC but not with TBA. (E) Quantification of D1 and Cy5-DNA merged foci ratios in the nuclei. In each group, 30 to 50 nuclei were counted. Means with SEM were calculated and presented. Cell Chemical Biology 2016 23, 1261-1270DOI: (10.1016/j.chembiol.2016.08.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Determination of the Consensus Sequence for Parallel G-Quadruplexes Formation in the Genome by ChIP-Seq (A) The top five parallel G4 motifs generated by GLAM2 from the G4 sequences extracted from the top 200 peaks. (B) The statistics of base length distribution between two G tracts. Cell Chemical Biology 2016 23, 1261-1270DOI: (10.1016/j.chembiol.2016.08.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Detection and Determination of Parallel G-Quadruplexes by D1 in Human Telomeres (A) Telomere repeat enrichment analyses. The telomeric fragments were defined as containing more than three TTAGGG/CCCTAA occurrences. (B) Telomeric ChIP on SiHa cells. Dot blots were hybridized with a TTAGGG repeat probe, and the data representing the percent of the TTAGGG repeat DNA recovered in each ChIP were quantified. The average of duplicate signals obtained with 10-fold amplified input DNA samples were used as 100% value for the quantification. The error bars represent the SEM calculated from two replicates. (C) D1 and TRF2 co-staining in SiHa cells with or without RNase-1/DNase treatments. Nuclei were counterstained with DAPI (blue). The dotted outline represents the nuclear borders according to the white light image. Scale bars, 5 μm. (D) Quantification of D1 and TRF2 merged foci number in the nuclei. In each group, 50 to 100 nuclei were counted. Means with SEM were calculated and presented. ***p < 0.001; NS, not significant. (E) Determination of the G4 conformations in human telomeres. Telomeric G4 structures were co-stained with TRF2 and D1 or BG4. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. (F) Quantification of D1/BG4 and TRF2 merged foci numbers in the nucleus. In each group, 50 to 100 nuclei were counted. Means with SEM were calculated and presented. ***p < 0.001; NS, not significant. Cell Chemical Biology 2016 23, 1261-1270DOI: (10.1016/j.chembiol.2016.08.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Regulation of Endogenous Parallel G-Quadruplex Structures in the Telomeres by Parallel G4 Specific Small Molecular Ligands (A) CD spectra of the conformation of 5 μM hTELO in K+ solutions with or without 25 μM NMM/QPB-15e/ber8. (B) ELISA binding curves of D1 for NMM/QPB-15e-induced parallel G-quadruplexes in the telomeres. The error bars represent the SEM calculated from three replicates. (C) Increased D1-TRF2 co-staining in cells treated with NMM/QPB-15e and decreased D1-TRF2 co-staining in cells treated with Ber8 following RNase-1 treatment. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. (D) Quantification of D1 foci number (green), TRF2 foci number (red), and D1-TRF2 merged foci number (yellow) in the nuclei with or without NMM/QPB-15e/Ber8 treatment. Between 50 and 100 nuclei were counted. Means with SEM were calculated and presented. ***p < 0.001; NS, not significant. Cell Chemical Biology 2016 23, 1261-1270DOI: (10.1016/j.chembiol.2016.08.013) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 The Variation in the Abundance of Parallel G-Quadruplexes in the Telomeres as a Function of the Cell-Cycle Stages (A) Dynamic variation of parallel quadruplexes in the telomeres of cell-cycle-synchronized SiHa cells and untreated SiHa cells. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. (B) Quantification of D1 foci numbers (green) and D1-TRF2 merged foci numbers (yellow) in the nuclei of G0/G1, G1/S, S phase, and untreated cells. Between 50 and 100 nuclei were counted. Means with SEM were calculated and presented. ***p < 0.001; NS, not significant. Cell Chemical Biology 2016 23, 1261-1270DOI: (10.1016/j.chembiol.2016.08.013) Copyright © 2016 Elsevier Ltd Terms and Conditions