Fig 2: PARP1 inhibitor Olaparib affects the circadian rhythm. A) MDA-MB-231 cells transfected with control shRNA (shSCR) or shRNAs targeting PRMT6 and treated with different doses of Olaparib or Veliparib. Representative images (left), and quantification of colony formation (right), from biological triplicate experiments are shown. B) PER3, FLAG, and PARP1 abundance in MDA-MB-231 cells overexpressing PRMT6 treated with Olaparib or DMSO. C) Western blot analysis of indicated proteins in MDA-MB-231 cells overexpressing PRMT6 following treatment with Olaparib or DMSO. ZT, Zeitgeber Time. D) PER3 and PARP1 abundance in MDA-MB-231 cells transfected with control shRNA (shSCR) or shRNAs targeting PER3 following treatment with Olaparib or DMSO. E) Western blot analysis of indicated proteins in PER3-knockdown MDA-MB-231 cells treated with Olaparib or DMSO. ZT, Zeitgeber Time. F) Cell proliferation curves for control and PRMT6-overexpressing MDA-MB-231 cells treated with Olaparib or DMSO (upper panel). Cell proliferation curves for control and PER3-knockdown MDA-MB-231 cells treated with Olaparib or DMSO (lower panel). G) Colony formation assays for control and PRMT6-overexpressing or -knockdown MDA-MB-231 cells treated with Olaparib or DMSO. H) Transwell invasion assays for control, and PRMT6-overexpressing or -knockdown MDA-MB-231 cells treated with Olaparib or DMSO. I) Western blotting analysis of MDA-MB-231 cells treated with Olaparib and/or EPZ020411 with antibodies against indicated proteins. EPZ, EPZ020411. J) Cell proliferation curves of MDA-MB-231 cells treated with Olaparib and/or EPZ020411. K) Transwell invasion assays of MDA-MB-231 cells treated with Olaparib and/or EPZ020411. L,M) MDA-MB-231 cells were subcutaneously inoculated into the abdominal mammary fat pads of 6 weeks old female NOD-SCID mice (n = 8), and tumor volumes were measured weekly. When the tumor size was ˜100 mm3, treatment with Olaparib or/and EPZ020411 was initiated. N) Western blotting analysis of 12 independent tumor tissues harvested from animals and treated with antibodies against indicated proteins. EPZ, EPZ020411. O) H&E staining of tumor tissues. IHC staining of Ki-67 and PER3 in tumor tissues. Representative photos of three specimens are shown. Scale bars, 100 µm. A,F,G,H,J,K) Bars represent the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). Student's t-test. M) Data are presented as mean ± SEM. (**p < 0.01, ***p < 0.001). Student's t-test.

Fig 3: The PRMT6/PARP1/CRL4B complex exerts transcriptional activity. A) ChIP-seq analysis of PRMT6 and PARP1 chromosome features. B,C) Verification of ChIP-seq results using qChIP analysis of genes identified in MDA-MB-231 cells. Results are represented as fold change over control; GAPDH served as the internal control. D) Binding profiles of PRMT6 and PARP1 on the representative target genes PER1, PER3, CSNK1A1, and BHLHE40. P1, PARP1; P6, PRMT6. E) Western blot analysis of indicated proteins in the stable PRMT6-knockdown, PARP1-knockdown, CUL4B-knockdown, and DDB1-knockdown MDA-MB-231 cells. F) mRNA levels of indicated genes in the PRMT6-knockdown, PARP1-knockdown, CUL4B-knockdown, and DDB1-knockdown MDA-MB-231 cells. G) ChIP and Re-ChIP analysis in MDA-MB-231 cells with indicated antibodies. P1, PARP1; P6, PRMT6; 4B, CUL4B. H) Recruitment of indicated proteins to PER1, PER3, BHLHE40, and CSNK1A1 promoters in MDA-MB-231 cells after transfection with control shRNA (shSCR) or shRNAs targeting PRMT6, PARP1, CUL4B, or DDB1. Purified rabbit IgG was used as a negative control. I) Cell proliferation curves for control, PRMT6-knockdown, PARP1-knockdown, and PER3-knockdown MDA-MB-231 cells. J) Colony formation assays for control, PRMT6-knockdown, PARP1-knockdown, and PER3-knockdown MDA-MB-231 cells. K) Transwell invasion assays for control, PRMT6-knockdown, PARP1-knockdown, and PER3-knockdown MDA-MB-231 cells. B,C,F,H,J,K) Bars represent the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). Student's t-test.

Fig 4: PRMT6 is associated with poor clinical outcomes and represents a prospective cancer biomarker. A) Immunohistochemical staining of PRMT6 and PARP1 in normal breast tissue and breast cancer tumors (histological grades I, II, and III). B,C) Quantification of PRMT6 and PARP1 immunostaining by means of intensity, as calculated using Image J. Total of 46 samples were analyzed. D) Relative level of PRMT6 plotted against that of PARP1. E,F) Immunohistochemical staining of PRMT6 in paired tumor tissues versus adjacent normal tissues. G) Analysis of public datasets (TCGA BRAC, GSE72653, GSE27567, and GSE65194) for PARP1 mRNA expression in normal and breast cancer tissues. H) Representative images from immunofluorescence staining of PRMT6, PARP1, and PER3 in normal breast tissues and breast cancer tumors (histological grades I, II, and III). I) Proportion of PER3, PRMT6/PARP1 positive areas in normal breast tissue and breast cancer tumors (histological grades I, II, and III) analyzed using Image J. Total of 40 samples were analyzed. J) Graphic model as discussed in the text. The proposed regulatory mechanisms of the PRMT6/PARP1/CRL4B complex in regulating the circadian rhythm of breast carcinogenesis. B,F,G,I) Error bars represent the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). Two-tailed unpaired t-test.