Fig 1: PHF6 reads H2BK12Ac and writes mono-ubiquitination of H2BK120. (A) Far-western blotting analysis of histone proteins with either GST or GST-PHF6 proteins. Assays were performed on histone extracts obtained from ZHBTc4 ESCs. Histone extracts were separated by H3, H2B, H2A and H4 according to size on the SDS-PAGE gel. (B) Top five ranked histone modifications that show the highest affinity with GST-PHF6. The screening was performed by using a histone peptide array kit. (C) Histone peptide pulldown analysis was performed with GST-PHF6 for binding of the top five modified histone peptide candidates and nearby modifications. (D) Amino acid sequences of the extended PHD1 (ePHD1) and extended PHD2 domains (ePHD2) of PHF6 orthologues in diverse species are aligned. The amino acids with red characters indicate the negatively charged region in the ePHD2. The amino acid alignment was performed the ClustalX. (E) In vitro peptide binding assay was performed with GST-PHF6 WT or E223S MT. (F) MST binding curves of H2B peptides (1:20) (non-modified and K12Ac) with GST-PHF6 WT and E223S mutant (left axis) and GST (right axis) as a negative control. Error bars represent the standard deviation of three independent experiments. The measured KD value is shown for each binding curve. * N.B. represents no apparent binding. (G) Immunoblot analysis conducted using the indicated antibodies in WT and Phf6 KO ESCs with DOX treatment. (H) Immunoblot analysis performed using the indicated antibodies in ESCs in the absence or presence of siRNA against p300 or CBP after DOX treatment. (I) Comparison of correlation between K12Ac and K120ub using H2B K12R / K120R MTs. MNase digestion was performed to elute mono-nucleosomes containing these H2B WT and MTs, and the modification states of these ectopic-H2B containing mono-nucleosomes were confirmed by immunoblot analysis. (J) RNF20 and RNF40 protein levels by immunoblot between WT and Phf6 KO ESCs with DOX treatment. (K) Co-immunoprecipitation assay was performed to detect the interaction between the endogenous PHF6 with USF44, RNF20, or RNF40 in ESCs with or without DOX treatment. (L) Immunoblot of H2BK120ub levels between knockdown of Phf6, Rnf20, and Rnf40 with or without DOX treatment. (M) qRT-PCR analysis of Cdx2, and Gata2 in Phf6, Rnf20 and Rnf40 knockdown ESCs with DOX treatment. mRNA levels of each gene were determined as relative values for Gapdh and relatively compared based on shNS + DOX. Statistical significance was calculated by ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001). (N) ChIP assays were performed on the promoters of Cdx2 and Gata2 using anti-H2BK120ub antibody in Phf6, Rnf20, and Rnf40 knockdown ESCs with DOX treatment. Statistical significance was calculated by ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 2: Physical and functional interaction of Egr2 and Rnf40/Rnf20. (A) Venn diagram depicting the overlap of Egr2 target genes with lost H2Bub1 marks in Rnf40ΔSC nerves. (B, C) GO analysis (B) and list (C) of Egr2 target genes with reduced H2Bub1 marks in Rnf40ΔSC nerves. (D) Co-immunoprecipitation (IP) of Rnf40 and Rnf20 with antibodies directed against Egr2 and pre-immune serum (PI) from extracts of HEK293T cells that were transfected with various expression plasmids as indicated below the panels. Western blot was used to detect precipitated Rnf40 (upper panel), Rnf20 (middle panel) and Egr2 (lower panel). Numbers on the right indicate position of co-electrophoresed size markers in kDa. (E) Scheme of Egr2 proteins and their ability to interact with myc-tagged Rnf40 in extracts from transfected HEK293T cells. DUF3446, domain of unknown function. (F) Scheme of myc-tagged Rnf40 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. (G) Scheme of HA-tagged Rnf20 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. (H, I) Co-immunoprecipitation (IP) of Egr2 with antibodies directed against Rnf40 (H), Rnf20 (I) and IgG control (H, I) from extracts of differentiating SC cultures. Western blot was used to detect precipitated Egr2 (upper panels), Rnf40 (lower panel, H) and Rnf20 (lower panel, I). Experiments were repeated three times. Uncropped western blots for D–I are presented as Supplementary Figures S9 and S10. (J–M) Determination of endogenous expression levels of Mbp (J), Mpz (K), Mag (L) and Prx (M) by qRT-PCR in Neuro2a cells transfected with various combinations of expression plasmids for Egr2, Rnf40-specific shRNA (shRnf40), scrambled shRNA (shScr) or various combinations of these as indicated below the lanes after FACS. Transcript levels in cells transfected with empty expression vectors (ctrl) were set to 1 and all other values were expressed in relation to it (n = 3; mean values ± SEM). Statistical significance was determined by one-way ANOVA with Bonferroni's multiple comparisons test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Exact values are provided in the Supplementary Tables. (N) Proposed model for the action of the Rnf40/Rnf20 E3 ligase and H2Bub1 modification in SC development.
Fig 3: Peripheral neuropathy resulting from Rnf40 deletion and H2Bub1 loss in SCs. (A–E) Temporal occurrence of Rnf40 and H2Bub1 in SCs of spinal (A–C) and sciatic (D, E) nerves from control mice at E12.5 (A), E15.5 (B), E18.5 (C), P5 (D) and P56 (E) as determined by co-immunofluorescence studies with antibodies against Rnf40 (red), H2Bub1 (cyan) and Sox10 (green). Nerves are demarcated by dotted lines. (F–H) Stage-specific occurrence of H2Bub1 in SCs as determined by co-immunofluorescence studies with antibodies against Sox2 (F, P0), Oct6 (G, P0) and Egr2 (H, P14). (I–L) Deletion kinetics of Rnf40 (I, J) and H2Bub1 (K, L) in SCs from spinal nerves of Rnf40ΔSC mice as determined by immunofluorescence studies with antibodies against Rnf40 (red in I, J), H2bub1 (red in K, L), Sox10 (green) and Oct6 (cyan) at E12.5 (I), E15.5 (J, K) and E16.5 (L). (M) Quantification of the relative numbers of Rnf40- and H2Bub1-positive SCs in sciatic nerves of control (Ctrl, white bars) and Rnf40ΔSC (gray bars) mice at P0 following immunohistochemistry. SCs were identified by Sox10 staining (n = 3; mean values ± SEM). (N) Survival curve of Rnf40ΔSC mice (red line, n = 20) during the first 6 months of life. All controls stayed alive during the analyzed period. (O–R) Hindlimb clasping phenotype (O, Q) and sciatic nerve hypomyelination (P, R) in control (O, P) as compared to Rnf40ΔSC (Q, R) mice at P28. Scale bars: 15 μm. Statistical significance was determined by unpaired, two-tailed Student's t-test (***P ≤ 0.001). Exact values are provided in the Supplementary Tables.
Fig 4: H2Bub1 positively modulated the expression of BMAL1 at the transcript level(A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
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