Fig 1: DAB2 promoter forms euchromatin by dissociation of CRL4A-JARID1A complex during megakaryocytic differentiation. A Chromatin configuration of DAB2 promoter in RepID-deficient K562 cells. ChIP-qPCR was performed using antibodies against H3K4me3 and H3K9me3 in K562 RepID WT or KO cells to examine epigenetic configuration of the DAB2 promoter region. Data are represented as the fold enrichment compared to the IgG control after normalizing with input value. B Correlation between CRL4 components (RepID, CUL4A, CUL4B and DDB1) and histone H3K4 modifiers including methyltransferase and demethylase in all cancer datasets based on CCLE database using CellMinerCDB. Pearson’s correlation R value was presented as heatmap (left panel). Published interactions between CRL4 components and H3K4 modifiers were analyzed using public data from BioGRID database (right panel). C, D JARID1A interacts with CUL4A. A pull-down assay using a CUL4A antibody was performed using whole cell extracts from K562 RepID WT and KO cells and co-precipitated JARID1A, DDB1 and RepID were analyzed by immunoblotting (C). Immunoprecipitation using JARID1A antibodies to pull down CUL4A (D). E CUL4A dissociates from JARID1A during megakaryocytic differentiation. Whole cell extracts from K562 RepID WT and KO cells induced to megakaryocytic differentiation using PMA for 48 h were immunoprecipitated using CUL4A antibodies, and co-precipitated JARID1A was analyzed by immunoblotting and densitometry. F Chromatin binding of JARID1A is highly decreased during megakaryocytic differentiation. The whole cell extracts and chromatin-bound fractions from K562 RepID WT or KO cells incubated with PMA as the indicated times were analyzed by immunoblot analysis using anti-JARID1A, anti-ASH2L, anti-RBBP5 and anti-WDR5 antibodies. Alpha-tubulin and histone H3 was used as the control for loading and fractionation. Quantification of JARID1A levels in whole cell lysates and chromatin-bound fractions after normalizing alpha-tubulin or histone H3, respectively. Quantification was presented under the blots. G Proximity ligation assay for protein interaction between CUL4A and JARID1A in with/without PMA treatment in pre- or no pre-extraction condition. Each red spot represents a single protein–protein interaction and DNA was stained with DAPI. scale bar: 20 µm. Red signal numbers in each cell were quantified by analyzing 315–747 cells randomly, and average numbers for positive red signals per cells are indicated. H, I JARID1A-CUL4A complex is recruited by RepID and dissociates from DAB2 promoter during megakaryopoiesis. ChIP-qPCR of DAB2 promoter region was performed using antibodies against RepID, JARID1A and CUL4A in K562 RepID WT or KO cells treated with PMA to induce megakaryocytic differentiation (H). Re-ChIP-qPCR analysis using RepID antibodies as a first precipitation, followed by second ChIP using rabbit IgG, CUL4A, mouse IgG or JARID1A antibodies (I). All data are presented as the fold enrichment compared to the IgG control after normalizing with input value
Fig 2: ChIP analysis of downstream RBP2 target genes. Chromatin IP from βlox5 cells overexpressing either mRBP2 or RBP2. Antibodies against either mono or tri-methylated H3K4 or RBP2 were used for ChIP. Conventional PCRs was run using primer sequences from the regulatory region of p18 (as a positive control for monomethylation ChIP; PC), p21, p27, p57 or an unrelated region of the genome as a negative control (NC). I–input, m1–H3K4me1 ChIP, m3–H3K4me3 ChIP, R–RBP2 ChIP, W–water control for PCR.
Fig 3: Colony formation with RBP2 overexpression and knockdown. (a) βlox5 cells stably expressing either empty vector (Empty), enzymatically dead mutant RBP2 (mRBP2), or wild-type RBP2 (RBP2) were used for colony formation assays and (b) H727 and (c) QGP-1 cells were transiently transfected with either a scrambled siRNA (Scram) or an RBP2 siRNA (siRBP2) and followed 24 hrs later by colony formation assays. Colonies were stained with crystal violet after 3 weeks and pictures were taken at × 100 magnification or with a standard hand held camera. In addition, colonies present in 25 × 1 cm2 fields were counted and an average number of colonies per cm2 was calculated for each condition. (#P<0.005).
Fig 4: Fbxo22 represses the migration and invasion of TNBC cells by regulation of KDM5A. A Immunoblotting of protein levels of Fbxo22 and KDM5A in MDA-MB-231 and Hs578T cells after Flag-Fbxo22 or HA-KDM5A treatment. B Transwell assay to detect the migration ability of MDA-MB-231 and Hs578T cells after Flag-Fbxo22 or HA-KDM5A treatment. C Transwell assay of MDA-MB-231 and Hs578T cell invasion ability. D Immunoblotting of EMT-related protein levels in MDA-MB-231 and Hs578T cells after Flag-Fbxo22 or HA-KDM5A treatment. E CCK-8 was applied to detect cell viability. F Clone formation assay was used to detect cell clones. * indicates p < 0.05 compared with pCDH group; # indicates p < 0.05 compared with Flag-Fbxo22 group. Measurement data were expressed as mean ± standard deviation and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Cell experiments were repeated three times
Fig 5: p65 is directly and epigenetically downregulated by RBP2. (A, B) qRT-PCR of RBP2 and p65 mRNA level after transfection with RBP2 wild-type or RBP2-mutant (defective in demethylase activity, RBP2 H483A) plasmids in K562 and HL60 cells. (C) Western blot analysis of RBP2 and p65 protein expression levels after transfection with RBP2 wild-type or RBP2 H483A plasmids in K562 and HL60 cells. (D) Schematic illustration of the predicted RBP2 binding sites in the p65 promoter. (E, F) Regulation of p65 wild-type promoter by RBP2, including the first RBP2 binding site (p65 pro1) and the second RBP2 binding site (p65 pro2). HL60 and HEK-293 cells were transfected with p65 wild-type reporters, together with RBP2 expression plasmids, followed by luciferase activity assessment 48 h post-transfection. (G, H) p65 wild-type pro1 and its mutated activity following RBP2 wild-type plasmids transfection in HL60 and HEK-293 cells. (I, J) ChIP assay for RBP2 binding to the p65 promoter in K562 and HL60 cells. (K, L) The binding of RBP2 and H3K4me3/2 to p65 promoter after RBP2 plasmid transfection in K562 and HL60 cells. The results are from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not statistically significant.
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