Fig. 2: MITF stimulates GREB1 Is4 gene expression.
From: GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation
A Scatter plots between the GREB1 gene (x-axis) and ESR1, AXIN2, MITF, or MLANA (y-axis) from TCGA Skin-SKCM (N = 470). The solid line indicates a linear fit. R and P represent the Pearson’s correlation coefficient and P-value, respectively. B GREB1 Is4, MLANA and PMEL mRNA expression in control and MITF KO Colo679 clones. The qPCR data are shown as the fold to control and presented as the mean ± s.d. from four independent clones (***P < 0.001, Student’s t test). Lysates from control vector and MITF KO Colo679 clones were probed with the indicated antibodies. HSP90 was used as a loading control. C Left panel, 293T cell lysate expressing WT MITF or MITFE318K was probed with the indicated antibodies. Right panels, MITF, GREB1 Is4, PMEL, and MLANA mRNA expression in 293T cells expressing WT MITF or MITFE318K. The qPCR data are shown as the fold to control and presented as the mean ± s.d. from three independent experiments (**P < 0.01; ***P < 0.001, Student’s t test). D Top panel, schematic representation of the putative GREB1 Is4 promoter region. TSS and MITF binding sites were predicted as described in Materials and Methods. The dashed arrows indicate the primer set of ChIP. Bottom panels, ChIP qPCR by anti-MITF or H3K27ac antibody of Is4 promoter (from −55 to +47) or control region (exon 24) at GREB1 locus, or PMEL promoter (Intron 1-2) at PMEL locus in Colo679 and A375 cells. The signals were presented as percentage of inputs. E. Top panel, schematic representation of the GREB1 Is4 promoter constructs for transcriptional reporter assay. The WT and Mut constructs of MITF-binding E-box CANNTG motif are shown. Bottom panels, WT/WT, WT/Mut, Mut/WT, or Mut/Mut GREB1 Is4 promoter (-82-0) activity without or with MITF. The luciferase activities are shown as the fold-change relative to the pGL4 vector control and presented as the mean ± s.d. from two independent experiments. F. RNA- and ATAC-seq from Japanese PDX, and ChIP-seq of H3K27ac, MITF, Pol II, and H3K4me3 from public ChIP data in the putative GREB1 Is4 promoter. The closed triangles or red box show exons 1 and 19 or putative GREB1 Is4 promoter, respectively.
