(A) Diagrams of Pol II promoters (CDKN1A, ACTB, RPPH1, and GDF15) with locations of exon 1, SINEs (red), and constructed DNA template (green arrow) for the in vitro MAF1 binding assay. (B) An in vitro DNA binding assay was performed as described in the ‘Materials and methods’. In brief, DNA template, MAF1 protein (His-tagged), and Anti-6× His tag antibody were added to the binding reaction (Protein + Ab). A negative control was performed by substituting IgG antibody for Anti-6× His tag antibody (Protein + IgG) or with only the Anti-6× His tag antibody for the MAF1 protein (Ab only). DNA isolated from the immunoprecipitated protein–DNA complex was subjected to qPCR. Deletion of a SINE in the CDKN1A template as well as deletion or mutation of the Pol III A-box element in the CDKN1A and GDF15 template depleted MAF1 binding. Binding of MAF1 to RPPH1 or ACTB promoters was not detected. Data shown are the mean ± SD, n ≥ 3, **p < 0.01, ***p < 0.001 (t-test). (C) An in vitro DNA–protein binding assay was performed using a colorimetric assay kit (ab117139). The assayed DNA template ‘p21’ (DNA template with a Pol III A-box element obtained from CDKN1A) was labeled with biotin (a probe). Purified MAF1 protein (His tag) (80R-1955, Fitzgerald) was used for the binding assay. Different competitors (described below) were added to the mixture to demonstrate the specificity of binding of MAF1 at the Pol III promoter element. Competitors: ‘self’ indicates the same DNA template without the biotin label, ‘GDF’ indicates the non-labeled DNA template that contained the Pol III promoter element obtained from the GDF15 promoter, and ‘Mut’ indicates the Pol III A-box element was mutated in the DNA template. A blank control was performed without the addition of protein, and the degree of enrichment was calculated by subtracting the value of the blank control. MAF1 directly bound to the Pol III promoter element, but the mutant form did not. Data shown are the mean ± SD, n = 3, ***p < 0.001 (t-test). (D) In vitro transcription assays were performed on CDKN1A and TAF5 using the HeLaScribeR Nuclear Extract in vitro Transcription System (Promega), as indicated in the ‘Materials and methods’. Inhibition of Pol II transcription was performed by addition of α-amantin during in vitro transcription of CDKN1A and TAF5. The MAF1 protein was pre-incubated with template DNA before addition of nuclear extract to enable binding of MAF1 to the template DNA. (E) Different antibodies, as indicated, were pre-incubated with nuclear extract before adding template DNA to perform in vitro transcription to deplete the target protein of interest. For the control, no antibody was added prior to in vitro transcription. In vitro transcription performed on Pol III-transcribed RPPH1 and Pol II-transcribed TAF5 served as controls. In vitro transcription performed on CDKN1A and GDF15 revealed that removal of MAF1 promoted transcription, whereas A-box-deleted GDF15, denoted as ‘GDF15 (Del)’, did not. The degree of enrichment of all performed in vitro transcription was calculated relative to the ratio of signals obtained from the input RNA after subtraction of the negative control (no biotin labeling). All data shown represent the mean ± s.e.m., n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001 (t-test).