Fig 1: PADI4 promoter undergoes demethylation during ATRA-induced differentiation(A) Expression of PAD4 at mRNA (top) and protein (bottom) levels in HL-60 (left) and NB4 (right) cells before and after treatment with 1.0 mM DAC for 72 h or 200 nM TSA for 24 h. (B) MSP was applied to examine the methylation status in the PADI4 promoter in HL-60 (top) and NB4 (bottom) at indicated timepoints during ATRA-induced differentiation. A PCR band in lane M indicates a methylated PADI4 gene; a band in lane U indicates an unmethylated PADI4 gene. (C) After DAC treatment for 72 h, changes of DNA methylation in the promoter of PADI4 was assayed by MSP. (D) Relative methylation levels in the PADI4 promoter region was analyzed with MeDIP-qPCR. (E) ChIP analysis was performed to determine the changes of the enrichments of DNMT1 in the PADI4 promoter. Pre-cleared lysate (1%) was taken before immunoprecipitation and used as the input control. *P < 0.05, **P < 0.01. Data are means of biological triplicates (± standard error) and all data are representative of triplicate experiments.
Fig 2: A putative model illustrating the role of PAD4 in the abnormal differentiation of APL cellsDemethylation in PADI4 and a simultaneous restoration of its expression occurred during ATRA-induced differentiation. Furthermore, ATRA stimulation facilitates the nuclear translocation of PAD4, and in turn, PAD4could promote the differentiation of APL cells in the presence of ATRA. Mechanistically, PAD4 could directly regulate SOX4 expression via citrullinating histone3, thereby antagonizing the methylation of H3Arg17. In addition, PU.1 is validated as the direct target of SOX4, and PAD4 could function to promote differentiation or regulate PU.1 expression in a SOX4-dependent manner, suggesting a functional pathway may form among PAD4, SOX4, and PU.1 in the pathological mechanism of APL.
Fig 3: SOX4 mediates the regulation of PU.1 by PAD4(A–C). qRT-PCRand Western blot were performed to detect the expression of SOX4 and PU.1 in HL-60 cells after overexpressing SOX4 in a gradually increasing manner mediated by electroporation for 36 h. (D) Expression of PU.1 in HL-60 cells with siRNA-mediated SOX4 knockdown. (E) The binding of SOX4 to SPI1 promoter was analyzed by ChIP. (F) Expression of SOX4 was determined after indicated treatment and GAPDH was applied as the loading control. (G) Schematic representation of the promoter region of SOX4. P1–P6 represents different regions of SOX4 promoter as shown in the color map above. (H) The pGL3-basic control or various SOX4 constructs were co-transfected with the PADI4 plasmid into HEK293 cells. 24 hours after transfection, cells were harvested for the luciferase reporter assay. (I) ChIP-qPCR was applied to detect the enrichment of PADI4 on the SOX4 promoter. Upon ChIP with an anti-PADI4 antibody, PCR with primers at different positions of the SOX4 promoter were performed. I, II, III, IV and V represent various primers amplifying regions of different colors as indicated in the map (H). *P < 0.05, **P < 0.01. Data are means of biological triplicates (± standard error) and representative of triplicate experiments.
Fig 4: PAD4 promotes leukemia cell differentiation and ATRA treatment facilitates its nucleus translocation(A) Efficiency of siRNA transfection targeting PAD4 was validated by Western blot in the absence or presence of ATRA exposure. (B) The differentiation of HL-60 cells after silencing B1–B6 or over-expressing B7–B12 were detected by Flow cytometry. The effect of PAD4 inhibitor, Cl-amidine, on the differentiation of HL-60 cells were also detected by FCM B13–B16. *P < 0.05, **P < 0.01. Data are means of biological triplicates (± standard error). (C) After treatment with ATRA for various times, the PAD4 expression in cytoplasm (left) and nucleus (right) at protein level was detected by Western blot. Tubulin and Lamin B were applied as loading controls, respectively. (D) PAD4 immunostaining (visualized in green) in HL-60 before and after ATRA treatment was assayed by immuno-fluorescent method. All data are representative of triplicate experiments.
Fig 5: PAD4 regulates SOX4 expression through citrullination and functions in a SOX4-dependent manner(A–C) ChIP analysis was applied to detect the binding of PAD4, H3Cit, and H3R17me to the SOX4 promoter upon ATRA-induced granulocyte differentiation. Positive controls represent the known anti-H3R17Me-positive and anti-H3cit-positive region in the p21 promoter. Negative control was IgG. (D–F) The same assay was also applied to detect the above parameters in the presence of siRNA targeting PAD4. (G) and (H) FCM analysis of the differentiation of HL-60 after indicated treatment. Ectopic PAD4 or SOX4 expression was induced by electroporation. (I) Western blot analysis of protein expression of PAD4, SOX4, and PU.1 in HL-60 cells after electroporation-induced addition of exogenous PAD4, SOX4, or both. (J–L) qRT-PCR assays were performed to quantify the expression levels of PADI4, SOX4, and SPI1 in clinical samples of APL (n = 12). Correlation between PADI4 and SOX4 J., PADI4 and SPI1 K., as well as SOX4 and SPI1 L. by Pearson correlation analysis. *P < 0.05, **P < 0.01. Data are means of biological triplicates (± standard error). All assays were performed with at least three independent preparations and measured in triplicate.
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