Figure 2
From: CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats
Aberrant transcription and formation of DNA:RNA hybrids in ICF mutant cells. (a) The relative expression of pericentromeric satellite-2 repeats and centromeric α-satellite repeats in WT, DNMT3B KO, ZBTB24 KO, CDCA7 KO, and HELLS KO HEK293 cells was examined by RT-qPCR; 18S ribosome RNA was used for normalization. No amplification of RT(–) samples was detected. Experiments were conducted in biological triplicate and technical triplicate. (b) The relative expression of satellite-2 repeats and α-satellite repeats in lymphoblastoid cells from two healthy subjects (L1 and HEV0190), an ICF1 patient (P1), an ICF2 patient (pD), and an ICF4 patient (pU) was examined by RT-qPCR; 18S ribosome RNA was used for normalization. No amplification of RT(–) samples was detected. Experiments were conducted in biological triplicate and technical triplicate. P values were calculated by comparison with HEV0190. (c) Representative images of DNA:RNA hybrids in WT and ICF mutant HEK293 cells detected using the S9.6 antibody. Hoechst 33,342 was used for DNA visualization (c,e,f,g). Scale bars: 10 µm (c,e,f). Experiments were conducted in biological triplicate and more than 300 cells were observed per each sample. (d) Summary of data shown in (c). The total cell number examined (n) is shown in parentheses (each dot represents one image) (d,h). (e) Representative images of DNA:RNA hybrids in CDCA7 KO and HELLS KO HEK293 cells detected using S9.6 antibody with in vitro E. coli RNase H treatment. (f) DNA:RNA hybrids detected by the S9.6 antibody (red) were co-stained with DDX21 (green, a nucleoli marker) in CDCA7 KO and HELLS KO cells (for other cells, see Supplementary Fig. S2). (g) Representative images of DNA:RNA hybrids in lymphoblastoid cells from a healthy female (HEV0190), an ICF1 patient (P1), and two ICF2 siblings (pD and pV) detected using the S9.6 antibody. Scale bars: 5 µm. More than 170 cells were observed per each sample. (h) Summary of data shown in (g). Data are mean ± s.e., and P values were obtained using the Mann–Whitney two-tailed U test (a,b,d,h).
