Fig. 3: FOXM1, MATα2 and MAT2β regulate each other positively at the transcriptional level. | Nature Communications

Fig. 3: FOXM1, MATα2 and MAT2β regulate each other positively at the transcriptional level.

From: The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis

Fig. 3

MAT2A (a), MAT2B (b) and FOXM1 (c) promoter activities in LX-2 cells and primary cholangiocytes ± FDI-6 or siRNA treatment as described in Methods. Effects of mutating FOX elements in LX-2 cells are shown in (d) MAT2A, (e) MAT2B, and (f) FOXM1. Cells transfected with WT and mutant constructs were treated with siRNA against MAT2A, MAT2B, and FOXM1 and reporter activities were measured. Data presented as mean ± SEM (n = 3 per group). For a, Mat2a promoter activities of D-271/ + 60, D-671/ + 60 and D-1329/ + 60 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.0002, p = 0.0052 and p = 0.0948, respectively; SC + DMASO vs. SC + FOXM1 si, p = 0.0001, p = 0.0022 and p = 0.0003, respectively; Mat2a promoter activities of D-271/ + 60 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC+Foxm1si, p = 0.0286 and 0.0149, respectively. For b, Mat2b promoter activities of D-25−/+3, D-713/+3, D-990/+3 and D-1319/+3 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.0051, p = 0.0316, p = 0.016 and p = 0.0034, respectively; SC + DMSO vs. SC + FOXM1 si, p = 0.0106, p = 0.0233, p = 0.0019 and p = 0.0049 respectively; Mat2b promoter activities of D-250/+3 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC + Foxm1 si, p = 0.0002 and 0.023, respectively. For c, Foxm1 promoter activities of D-312/+107 and D-1333/+107 in LX2 cell, SC + DMSO vs. SC + FDI-6, p = 0.5799 and p = 0.0020, respectively; SC + DMSO vs. SC + FOXM1 si, p = 0.7684 and p = 0.0047 respectively; Foxm1 promoter activities of D-1333/ + 107 in cholangiocytes, SC + DMSO vs. SC + FDI-6 or SC + Foxm1 si, p = 0.0163 and 0.0131, respectively. For d, MAT2A promoter activities (−270/+60) of WT and MU, SC vs. SC, p = 0.015 and p = 0.28 respectively; FOXM1si vs. SC, p = 0.0032 and p = 0.042 respectively; MAT2Asi vs. SC, p = 0.0033 and p = 0.066 respectively. For e, MAT2B promoter activities (−250/+3) of WT and MU, SC vs. SC, p = 0.0017 and p = 0.11 respectively; FOXM1si vs. SC, p = 0.0018 and p = 0.022 respectively; MAT2Asi vs. SC, p = 0.0015 and p = 0.026 respectively. For f, FOXM1 promoter activities (−1333/+107) of WT and MU, SC vs. SC, p = 0.000017 and p = 0.0078 respectively; FOXM1si vs. SC, p = 0.000014 and p = 0.0063 respectively; MAT2Asi vs. SC, p = 0.00020 and p = 0.019 respectively. g ChIP assay was performed by spanning two FOX regions of the FOXM1 promoter in LX-2 cells using FOXM1, MATα2 and MAT2β antibodies after treatments that varied the expression of FOXM1, MAT2A or MAT2B in the top three rows. Seq-ChIP with anti-MATα2 and MAT2β antibodies after FOXM1 ChIP was performed as described in Methods. Representative results from three experiments are shown. h qPCR analysis of the ChIP assay from (g). For h, ChIP and Seq-ChIP percentage of input DNA, FOXM1 si vs. SC, MAT2A si vs. SC, MAT2B si vs. SC, FOXM1 OV vs. EV, MAT2A OV vs. EV, and MAT2B OV vs. EV, for FOXM1 ChIP, p = 0.00089, p = 0.011, p = 0.0045, p = 0.0000053, p = 0.00024, and p = 0.000039; for MAT2A seq-ChIP p = 0.0015, p = 0.0023, p = 0.0037, p = 0.000037, p = 0.00076, and p = 0.00032; for MAT2B seq-ChIP p = 0.0036, p = 0.018, p = 0.0092, p = 0.0036, p = 0.0000098, p = 0.0013 respectively. Data presented as mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. SC or EV (n = 3 independent experiments). i EMSA was done using labeled probes containing two FOX binding motifs of the FOXM1 promoter as shown in (f) and 100 ug of nuclear protein from LX-2 cells after treatments that varied FOXM1/MAT2A/MAT2B (n = 3 independent experiments). j Super shifts were done using 100 ng of recombinant proteins of FOXM1, MATα2, MAT2β alone or combined, and antibodies to FOXM1, MATα2 and MAT2β. Probe and IgG only served as negative controls. Results represent three independent experiments. k In vitro pull-down shows direct interaction between MATα2, MAT2β and FOXM1 using recombinant MATα2, MAT2β and FOXM1 proteins (n = 3 independent experiments). l MATα2, MAT2β and FOXM1 interaction in Flox control (WT), Foxm1Hep−/−, with or without BDL was detected by Co-IP and western blotting (n = 3 independent experiments). Statistical significance was determined by using two-tailed, unpaired Student’s t-test. Source data are provided as a Source Data file. Abbreviations: AB antibody, EV empty vector, IP immunoprecipitation, OV overexpression, si siRNA, WT wild type, MU mutants.

Back to article page