Fig 1: FBXO42 mediates global chromatin remodeling in an RBPJ-dependent manner.(A) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding epigenetically modified proteins. Then, cell lysates were incubated with S-protein beads and blotted with antibodies against FLAG- or MYC-epitope tags. (B) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding SWI/SNF complex proteins. Then, the cells were harvested and analyzed as described in (A). (C) Heatmap showing the differential interaction between chromatin factors and RBPJ in WT and FBXO42 KO cells as identified by MS. (D) Enrichment analysis of the differentially interacting proteins of heterochromatin components is shown on the basis of GO annotation. (E) Immunofluorescence detection of HP1α foci in WT and FBXO42 KO cells. Scale bars, 10 μm. (F) HP1α foci number and percentage of HP1α foci area were calculated using ImageJ software. (G) WT and FBXO42 KO cells were digested with micrococcal nuclease (MNase) for 3 min, and chromatin relaxation was monitored by the release of nucleosomes. (H) Chromatin association of the SWI/SNF subunits SMARCA2, SMARCA4, and SMARCC2 in WT and FBXO42 KO cells was analyzed using Western blotting after chromatin isolation. ORC2 served as the loading control. (I) WT and FBXO42 KO cells were digested with deoxyribonuclease (DNase) for 3 min and followed with agarose gel electrophoresis analysis. (J) Chromatin from WT and FBXO42 KO cells was isolated, and DNase I was digested and used as substrate for accessibility assay. (K and L) The heatmap view for ATAC-seq signal intensity at TSSs in WT and FBXO42 KO JURKAT cells. (M) ATAC-seq peaks; H3K4m1, H3K4m3, and H3K27ac ChIP-seq peaks; and DNase-seq peaks downloaded from ENCODE database at MYC locus were analyzed. (A), (B), and (E) to (J), n = 3. Quantitative data are presented as means ± SEM. P values were calculated using two-tailed Student’s t tests. *P < 0.05 and **P < 0.01.
Fig 2: Validation of established EBNA3C HDmut and EBNA3C W227S LCLs. (A) Cell proliferation assay at day 36 after infection of primary B cells with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), or EBNA3C RBPJ BM (3C RBPJ BM) recombinant virus. Live cells were analyzed for proliferation by measuring EdU incorporation and DNA content (by use of FxCycle Far Red DNA stain). Gates show the populations of cells in the sub-G1, G1, S, and G2/M phases. Data are representative of three independent infections. (B) Expression of Epstein-Barr virus latency-associated proteins EBNA3A, EBNA3B, EBNA3C, EBNA1, EBNA2, LMP1, and EBNA-LP, as well as RBPJ and γ-tubulin, was demonstrated by Western blotting of extracts of LCLs established from primary B cell infections with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), and EBNA3C RBPJ BM (3C RBPJ BM) EBVs used for panel A. (C) Immunoprecipitation (IP) of RBPJ or an antibody isotype control (IgG) in the WT, 3C HDmut, 3C W227S, and 3C RBPJ BM LCLs and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT).
Fig 3: MAML2 could activate LINC01152 expression via modulating Notch pathway in GBM cells.A–C Expression of p21 and HES-1 was tested via qRT-PCR and western blot when MAML2 or LINC01152 was silenced in T98G and U343 cells. D Luciferase activity of RBPJ was detected via luciferase reporter assay in T98G and U343 cells when MAML2 or LINC01152 was silenced. E CoIP assay searched the binding of MAML2 to RBPJ and NICD. F, G JASPAR (http://jaspar.genereg.net/) database was used to search the DNA motif of RBPJ and possible binding sites between RBPJ and LINC01152 promoter. H qRT-PCR probed the expression of LINC01152 when MAML2 and RBPJ were silenced. I, J The binding of RBPJ and MAML2 to LINC01152 promoter was verified via ChIP assay in T98G and U343 cells. K, L Luciferase activity of LINC01152 promoter (wild-type or mutant) was tested via luciferase reporter assay when RBPJ or MAML2 was overexpressed. **P < 0.01.
Fig 4: FBXO42 regulates RBPJ chromatin association and transcriptional activity.(A) WT and FBXO42 KO HEK293T cells were harvested and subjected to subcellular fractionation. The nuclear, cytoplasm, chromatin, and soluble fractions were isolated, and immunoblot analysis was performed. (B) HEK293T cells overexpressing RBPJ WT or K175R were harvested, the chromatin and soluble fractions were isolated, and the immunoblot analyses were performed. (C) WT and FBXO42 KO HEK293T cells were cotransfected with cSFB-RBPJ and Myc-p300. Cell lysates were collected, incubated with S-protein beads, and blotted with antibodies against FLAG- or MYC-epitope tags. (D) WT and FBXO42 KO HEK293T cells were transfected with cSFB-RBPJ and used for S-protein bead pull-down assay, followed by immunoblotting with endogenous antibodies as indicated. (E) HEK293T cells expressing RBPJ WT or its K175R mutant were harvested and used for S-protein bead pull-down assay, followed by immunoblotting with endogenous antibodies as indicated. (F) WT and FBXO42 KO HEK293T cells were transfected with cSFB-RBPJ and Myc-FBXO42 WT or ΔF mutant. Then, the histone modifications were evaluated with Western blotting using antibodies against H3K4m1 and H3K27ac. H3 and actin served as the loading controls. (A) to (F), n = 3. (G) WT and FBXO42 KO HEK293T cells were collected for a CUT&Tag assay. Classical RBPJ motif identified in CUT&Tag assay was shown. (H) Representative images of RBPJ-binding sites in known Notch pathway target genes HES1, HES4, and MYC were shown. HSP90AA1 was used as the negative control. (I) Heatmap showing RBPJ CUT&Tag read densities of WT and FBXO42 KO HEK293T cells. TSS, transcription start site. (J) Signaling pathway enrichment of genes with differential RBPJ-binding affinities based on GO annotation. The size of the dots represents the number of genes associated with the GO term, and the color of the dots represents the adjusted P values. AMPK, adenosine monophosphate–activated protein kinase; mTOR, mammalian target of rapamycin. (K) Heatmap showing CUT&Tag read densities of RBPJ WT and K175R mutant.
Fig 5: FBXO42 promotes RBPJ K63-linked polyubiquitination and positively regulates Notch signaling.(A and B) Endogenous RBPJ ubiquitination was measured under FBXO42 overexpression (A) or FBXO42 depletion (B) after IP with antibody against RBPJ and immunoblotted for ubiquitination. (C) cSFB-RBPJ, hemagglutinin (HA)–ubiquitin, and Myc-FBXO42 WT or ΔF mutant were cotransfected into HEK293T cells and analyzed for RBPJ polyubiquitination. (D) RBPJ ubiquitination change under NICD1 overexpression. (E) Determination of RBPJ ubiquitination chain linkage. (F) Sequences of the predicted and identified peptides (the respective lysine residues are indicated) in MS are shown. (G) Evaluating ubiquitination intensity of five cSFB-RBPJ lysine mutants. (H) Sequence alignment of RBPJ in different species showed the conservation of the K175 site. (I) HEK293T cells transfected with cSFB-RBPJ WT or K175R mutant were treated with cycloheximide (CHX) for the indicated times, and the lysates were analyzed by Western blotting. (J) Quantitation of the results shown in (I). (K) Effect of MLN4924- on FBXO42-mediated RBPJ ubiquitination. (L to N) Quantitative polymerase chain reaction (qPCR) analysis of the Notch target genes in HEK293T cells overexpressing FBXO42, RBPJ WT, or K175R mutant. (O) mRNA expression of Notch target genes in cells treated with MLN4924. (P) Efficiency of RBPJ short interfering RNAs (siRNAs). (Q) qPCR analysis of Notch target genes in RBPJ knockdown cells. (R) HEK293T cells were transfected with si-Scramble or si-RBPJ and overexpressed with Myc-FBXO42 and then subjected to qPCR analysis of Notch target gene expression. (A) to (G) and (I) to (R), n = 3. Quantitative data are presented as means ± SEM. P values were calculated using two-tailed Student’s t tests or analysis of variance (ANOVA) for multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, and ##P < 0.01 versus FBXO42.
Supplier Page from Abcam for Anti-RBPJK antibody