MAF1 represses CDKN1A through a Pol III-dependent mechanism
- National Yang-Ming University, Taiwan
Figures
Figure 1 with 1 supplement
MAF1 knockdown strongly upregulates CDKN1A expression and arrests MCF-7 cells at the G0/G1 phase.
Analysis of MAF1 expression after MAF1 knockdown using three different siRNAs in MCF-7 cells by quantitative RT-PCR (A) and immunoblot analysis (B). The immunoblot results were quantified (left …
Figure 1—figure supplement 1
MAF1 knockdown upregulates CDKN1A and GDF15 expression in HCT116p53+/+ (wild-type), HCT116p53−/− (p53-null), MCF-10A, and MDA-MB-231 cell lines.
(A) Immunoblot analysis of p53 expression in wild-type and p53-null HCT116. Quantitative RT-PCR of genes in HCT116 wild-type (B) and HCT116p53−/− (C) cells subjected to siRNA knockdown for 72 hr. (D)…
Figure 2
MAF1 knockdown upregulates CDKN1A at the transcriptional level.
(A) For run-on assay, MCF-7 cells were subjected to siRNA knockdown of MAF1 (KD MAF) or simultaneous knockdown of MAF1 and Pol III for 72 hr (KD P/M). Nuclei were prepared, and a run-on reaction was …
Figure 3 with 1 supplement
MAF1 knockdown enhanced binding of Pol III and Pol II at the CDKN1A promoter.
ChIP was performed in MCF-7 cells subjected to siRNA knockdown for 72 hr. DNA isolated from immunoprecipitated chromatin was subjected to qPCR and calculated as indicated in the ‘Materials and …
Figure 3—figure supplement 1
Expression and promoter activity of CDKN1A transcripts in the MCF-7 cell line.
(A) Representative diagram of NM_001220777 (CDKN1A-L, long form) and NM_001220778 (CDKN1A-S, short form). (B) cDNA samples used in Figure 1C were used to analyze CDKN1A transcript expression. …
Figure 4
In vitro binding and transcription assays demonstrate MAF1-regulated Pol III-mediated activation of Pol II-regulated genes.
(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 …
Figure 5 with 1 supplement
MAF1 knockdown induces Pol II initiation, active histone marks (H3K4me3, H3K9Ace, and H3K27Ace), and binding of CFP1, p300, PCAF, TBP, and POLR2E at the CDKN1A promoter.
(A) Diagram of the CDKN1A promoter, including locations of exon 1 (Ex1), SINEs (AluSx and MIR3), and ChIP qPCR amplicons (p21-L, p1, p2, and p3). (B) Knockdown coupled with ChIP assays with …
Figure 5—figure supplement 1
Enhanced gene expression by MAF1 knockdown is abolished by simultaneous knockdown of MAF1 with TBP or CFP1.
(A) Quantitative RT-PCR of genes in MCF-7 cells subjected to siRNA knockdown of MAF1, CFP1, or simultaneous knockdown of both for 72 hr. CDKN1A expression was upregulated after MAF1 knockdown, and …
Figure 6
Pol III is required for chromatin looping at the CDKN1A promoter after MAF1 knockdown.
(A) Schematic diagram of CDKN1A with the orientation of 3C primers (arrows: 5r, 4r, 3r, 2r, and 2f) and location of exon 1 (long form: L-Ex1; short form: Ex1). (B) MCF-7 cells were subjected to …
Figure 7
Pol III is required for chromatin looping at the GDF15 promoter after MAF1 knockdown.
(A) Schematic diagram of GDF15 with ChIP–qPCR amplicons (AluSx, 3C, MIR, p1, p2, and p3), the orientation of 3C primers (arrows: 3C-3r, 3C-2r, and 3C-1f), and locations of exons (Ex1 and Ex2). (B) …
Figure 8
Demonstration of MAF1- and Pol III-mediated transcription regulation using a reporter gene assay.
(A) Promoter regions of Pol II genes were constructed and cloned into pGL3-basic reporter plasmids, as indicated in the ‘Materials and methods’. Luciferase reporter assays were performed in MCF-7 …
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