Figure 4.
Figure 4. RNF8-mediated histone H3-K4 ubiquitylation promotes nucleosome disassembly. Immunoblotting analyses were performed with the indicated antibodies. WB, Western blot. (A) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R or with RNF8 depletion and reconstituted expression of WT rRNF8 or rRNF8 FHA-M mutant were treated with or without 100 ng/ml EGF for 24 h. LS-soluble chromatin extracts were prepared. (B) RNF8+/+ and RNF8−/− MEFs were treated with or without 100 ng/ml EGF for 24 h. LS-soluble chromatin extracts were prepared for detection of H2A, H2B, H4, and PCNA control. Total cell lysates were used to determine the expression levels of c-Myc, cyclinD1, and actin control. (C) U251 cells with RNF8 depletion and reconstituted expression of WT rFlag-RNF8 or rFlag-RNF8 ΔRING mutant were treated with 100 ng/ml EGF for 8 h, followed by formaldehyde fixation, micrococcal nuclease digestion, ChIP with an anti-Flag antibody, and electron microscopy analyses. The red arrows point to the chromatin regions with disassembled nucleosomes. Two samples were prepared, and ∼60 pictures were taken for each group. Two representative pictures are shown for WT rFlag-RNF8 or rFlag-RNF8 ΔRING mutant accordingly. (D) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R mutant were treated with or without 100 ng/ml EGF for 8 h. ChIP assay with an anti-H3 antibody and quantitative PCR analyses with the primers against promoters of MYC and CCND1 were performed. (E) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R mutant or with RNF8 depletion and reconstituted expression of WT rRNF8 or rRNF8 FHA-M mutant were treated with or without 100 ng/ml EGF for 8 h. ChIP assay with an anti–RNA Pol II antibody and quantitative PCR analyses with the primers against promoters of MYC and CCND1 were performed. (D and E) The data represent the means ± SD of three independent experiments. **, P < 0.001 by Student’s t test. The experiments shown in A and B were repeated three times.

RNF8-mediated histone H3-K4 ubiquitylation promotes nucleosome disassembly. Immunoblotting analyses were performed with the indicated antibodies. WB, Western blot. (A) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R or with RNF8 depletion and reconstituted expression of WT rRNF8 or rRNF8 FHA-M mutant were treated with or without 100 ng/ml EGF for 24 h. LS-soluble chromatin extracts were prepared. (B) RNF8+/+ and RNF8−/− MEFs were treated with or without 100 ng/ml EGF for 24 h. LS-soluble chromatin extracts were prepared for detection of H2A, H2B, H4, and PCNA control. Total cell lysates were used to determine the expression levels of c-Myc, cyclinD1, and actin control. (C) U251 cells with RNF8 depletion and reconstituted expression of WT rFlag-RNF8 or rFlag-RNF8 ΔRING mutant were treated with 100 ng/ml EGF for 8 h, followed by formaldehyde fixation, micrococcal nuclease digestion, ChIP with an anti-Flag antibody, and electron microscopy analyses. The red arrows point to the chromatin regions with disassembled nucleosomes. Two samples were prepared, and ∼60 pictures were taken for each group. Two representative pictures are shown for WT rFlag-RNF8 or rFlag-RNF8 ΔRING mutant accordingly. (D) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R mutant were treated with or without 100 ng/ml EGF for 8 h. ChIP assay with an anti-H3 antibody and quantitative PCR analyses with the primers against promoters of MYC and CCND1 were performed. (E) U251 cells with histone H3.3 depletion and reconstituted expression of WT histone rH3.3B or rH3.3B-K4R mutant or with RNF8 depletion and reconstituted expression of WT rRNF8 or rRNF8 FHA-M mutant were treated with or without 100 ng/ml EGF for 8 h. ChIP assay with an anti–RNA Pol II antibody and quantitative PCR analyses with the primers against promoters of MYC and CCND1 were performed. (D and E) The data represent the means ± SD of three independent experiments. **, P < 0.001 by Student’s t test. The experiments shown in A and B were repeated three times.

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