Fig 1: WHSC1 interacts with histone H1 in nuclear extracts of UD-SCC-2 cells and directly mono-methylates it at lysine K85 in vitro and in vivo. A. Western blotting of endogenous immunoprecipitates for WHSC1 from 700 µg of nuclear extract (Active Motif kit) using the anti-WHSC1 mouse Abcam 75359 at 5 µg. Supernatants were denaturated with 5× sample buffer and heating of immunoprecipitate at 95 °C for 5 min. Normal mouse IgG was used as control for IP. B. Silver staining of nuclear extracts from UD-SCC-2 cells. Band indicated with arrow was analyzed by liquid chromatography-mass spectrometry. C. In vitro methyltransferase assay of recombinant WHSC1 with linker histone H1. Recombinant histone H1 (37 kD) was incubated with WHSC1 (130 kD) in the presence of 3H-SAM. Bovine serum albumin (BSA, 66 kD) was used as a negative control and recombinant histone H3 (17 kD) as a positive control. The reactants were analyzed by SDS–PAGE followed by fluorography for 3 days (left panel). The PVDF transfer membrane was then stained with MemCode reversible protein stain to visualize the total protein (right panel). D. The MS/MS spectrum corresponding to the mono-methylated histone H1 fragment IKLGLK (79–85). The mono-methylation corresponds to lysine (K) at position 85 within the globular domain of histone H1. MS/MS score, Mascot ion score and Expectation value in Mascot Database search results are shown. E. Amino acid sequence alignment of human linker histone H1.4. The IKLGLK sequence which includes lysine K85 is located within the globular domain of H1 and is preserved from Homo sapiens to Mus musculus. F. Evaluation of the specificity of anti-mono-methylated K85 H1.4 antibody using enzyme-linked immunosorbent assay (ELISA). Y-axis represents ELISA optical density (OD) units read at 492 nm. ELISA plates coated with the mono-methylated K85 H1.4 peptide versus the unmodified H1.4 peptide were incubated with the primary rabbit antisera for 16 h at 4 °C (ST0812, Sigma-Aldrich, Japan). Detection was performed using a secondary rabbit antibody conjugated with horseradish peroxidase. Primary rabbit antisera were examined after dual selection against the modified versus the unmodified peptides. G. HeLa cells co-transfected with HA-Mock and FLAG-H1.4-WT versus HA-WHSC1 and FLAG-H1.4-WT or HA-WHSC1 and FLAG-H1.4K85A. FLAG-immunoprecipitation was performed using an anti-FLAG antibody and immunoprecipitates were blotted with the H1.4K85me1 and anti-FLAG antibodies. The input was blotted with anti-FLAG, anti-HA and anti-H3 antibodies.
Fig 2: Mono-methylated H1.4K85 is enriched and is associated with increased binding of WHSC1 to the OCT4 gene body region in SCC-35 cells. A. CHIP-assay for FLAG-H1.4-WT versus FLAG-H1.4K85A using an anti-FLAG antibody (F3165) followed by RT-PCR for OCT4. SCC-35 cells were transiently transfected with FLAG-H1.4-WT versus FLAG-H1.4K85A vectors. Nuclear extraction was conducted and chromatin immunoprecipitation followed by RT-PCR for OCT4 was performed *p-value <0.05, Student t-test, data represented as mean ± SEM of triplicates). B. CHIP-assay for WHSC1 in SCC-35 cells stably transfected to express FLAG-H1.4-WT versus FLAG-H1.4K85A using a WHSC1 antibody (Abcam 75359) in nuclear extracts, followed by RT-PCR for OCT4 *p-value <0.05, Student t-test, data represented as mean ± SEM of triplicates).
Fig 3: OCT4 is regulated by H1.4K85 mono-methylation and WHSC1 in SCC-35 cells. A. Differential gene expression analysis in SCC-35 cells stably expressing FLAG-H1.4-WT versus FLAG-H1.4K85A. Y-axis and X-axis represent log2-transformed gene expression levels in FLAG-H1.4K85A and FLAG-H1.4-WT expressing cells respectively. Each circle represents a gene. A total of 11,470 protein-coding genes are shown. Genes that are upregulated (n = 74) or downregulated (n = 370) by at least 1.5-fold in FLAG-H1.4K85A expressing cells relative to FLAG-H1.4-WT cells are shown in red and blue circles, respectively. The rest of the genes of lesser fold change between the two conditions are shown as grey circles. The size of the circles represents the relative expression fold of change between the two groups. B. Ingenuity canonical pathways enriched in the SCC-35 cells stably transfected with FLAG-H1.4K85A compared to cells transfected with FLAG-H1.4-WT. Data shown for pathways filtered by FDR-corrected p < 0.20. C. RT-PCR (left panel) and Western blotting of 8ug of nuclear extract (right panel) for OCT4 in SCC-35 cells stably transfected with FLAG-H1.4-WT versus FLAG-H1.4K85A (*p < 0.05, Student t-test, data represented as mean ± SEM of triplicates). Relative densitometry values are also shown. D. RT-PCR for WHSC1 (left panel) and OCT4 (middle panel), and Western blotting (right panel) for WHSC1 (enzymatically active isoform shown, molecular weight 152kD) and OCT4 in 10ug of nuclear extract from parental SCC-35 cells transfected with WHSC1-specific siRNA (RT-PCR in far left panel) *p < 0.05, Student t-test, data represented as mean ± SEM of triplicates). Relative densitometry values are also shown. Similar results were obtained in two separate biological replicates.
Fig 4: H1.4K85 mono-methylation increases sphere formation and enhances cell proliferation of SCCHN cells. A. Sphere formation assay of stably transfected SCC-35 SCCHN cells expressing FLAG-H1.4-WT versus FLAG-H1.4K85A constructs. 5000 cells were seeded in 24-well low-attachment plates in serum-free medium supplemented by epidermal growth factor (20 ng/ml). Cells were followed for 7 days and number of spheres was counted in 3 separate wells per condition under the microscope and the mean number of spheres was calculated. Left panel: representative images from one well in each condition. Right panel: histogram of the mean number of spheres from 3 individuals wells in each condition *p = 0.001, Student t-test, data represented as mean ± SEM). Similar results were obtained in two different experiments. B. MTT proliferation assays in SCC-35 cells stably expressing FLAG-H1.4-WT versus FLAG-H1.4K85A constructs. 2,000 cells were seeded in 24-well plates in quadruples for each condition. CCK-8 assay was performed at each indicated time point. FLAG-H1.4-WT expressing SCC-35 cells show a significantly higher proliferation rate compared to FLAG-H1.4K85A expressing SCC-35 cells *p < 0.05, Student t-test, data represented as mean ± SEM of quadruple values for each time point).
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