Volume 26, Issue 3, Pages 367-379 (May 2007) Mammalian Maf1 Is a Negative Regulator of Transcription by All Three Nuclear RNA Polymerases  Sandra S. Johnson, Cheng Zhang, Jody Fromm, Ian M. Willis, Deborah L. Johnson  Molecular Cell  Volume 26, Issue 3, Pages 367-379 (May 2007) DOI: 10.1016/j.molcel.2007.03.021 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Increased Maf1 Expression Represses Both RNA Pol I- and RNA Pol III-Dependent Gene Activity (A) Transient expression of Maf1 in U87 cells represses transcription of rRNA gene and tRNA gene promoters. The U87 glioblastoma cell line was transiently cotransfected with promoter-reporter plasmids, pArg-maxigene containing a tRNAArg gene, PrHuCAT containing an rRNA gene promoter, or c-fos-Luc, and empty vector or HA-Maf1 expression vector. Total RNA was isolated, and an RNase protection assay (to determine RNA Pol III promoter activity) or a primer extension assay (to determine RNA Pol I promoter activity) was performed. Representative autoradiographs are shown. Protein lysates were isolated for luciferase assays (to determine RNA Pol II promoter activity). Each graph represents at least three independent determinations (average ± standard error [SE]). (B) Transient expression of Maf1 in LN18 cells represses transcription of rRNA gene and tRNA gene promoters. Experiments were performed as indicated in (A) using a LN18 glioblastoma cell line. (C) Expression of HA-Maf1 in U87 and LN18 cells. Immunoblot analysis was performed using protein lysates derived from the transient transfected cells in (A) and (B) using antibodies against HA or β-actin. Representative immunoblots are shown. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Decreased Maf1 Expression Enhances Both RNA Pol I- and RNA Pol III-Dependent Gene Activity (A) Decreased Maf1 expression in U87 cells increases transcription of rRNA and tRNA genes. U87 cells were transiently cotransfected with promoter-reporter plasmids pArg-maxigene, PrHuCAT (RNA Pol I), or c-fos-Luc and empty vector or Maf1 shRNA expression vector. Total RNA was isolated for RNase protection or primer extension assays,and protein lysates were isolated for luciferase assays. Representative autoradiographs are shown. The data represent at least three independent determinations (average ± SE). (B) Decreased Maf1 expression in LN18 cells increases transcription of rRNA and tRNA genes. Experiments were performed as indicated in (A) using the LN18 cell line. (C) Expression of Maf1 in U87 and LN18 cells stably expressing Maf1 shRNA. Cell lines were stably transfected with empty vector or Maf1 shRNA expression vector. Immunoblot analysis was performed using total protein lysates and Maf1 or β-actin antibody. Representative immunoblots are shown. (D) Reducing Maf1 expression enhances endogenous 45S rRNA, tRNA, and 7SL RNA transcription. Total RNA was isolated from U87 cells stably expressing Maf1 shRNA and S1 nuclease protection assay performed to measure precursor 45S rRNA (left panel). Total RNA isolated from U87 cells infected with lentivirus expressing Maf1 shRNA (+) or mismatch RNA (−) was reverse transcribed, and cDNAs were subjected to quantitative real-time PCR measuring precursor tRNALeu (middle panel) or 7SL RNA (right panel). Both tRNALeu and 7SL RNA values were each normalized to values obtained for GAPDH transcripts. Bars represent the average ± SE of at least two independent determinations. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Decreasing Maf1 Expression Selectively Enhances TBP Protein Levels Total cell protein lysates were isolated from U87 cells stably transfected to express Maf1 shRNA or empty vector. Immunoblot analysis was performed and membranes were probed with the indicated antibodies. Representative blots are shown. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Maf1 Represses TBP Promoter Activity through an Elk-1-Binding Site (A) Alterations in Maf1 expression in U87 cells regulate TBP promoter activity. U87 cells were transiently cotransfected with the p4500/+66 hTBP promoter-luciferase plasmid and empty vector or two different concentrations of Maf1-HA expression vector (left) or Maf1 shRNA expression vector (right). The fold change was calculated based on luciferase activity in cells transfected with empty vector for each promoter construct shown (white bars). Bars represent the mean ± SE of at least three independent determinations. (B) Alterations in Maf1 expression in LN18 cells regulate TBP promoter activity. Experiments were performed as indicated in (A) using LN18 cells. (C) Maf1 repression of the TBP promoter requires an Elk-1-binding site. U87 cells were transiently cotransfected with human TBP promoter constructs and either plasmids expressing Maf1-HA, Maf1 shRNA, Maf1 mismatch shRNA, or empty vector. Bars represent the mean relative luciferase units/mg protein/1000 ± SE of at least three independent determinations. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Maf1 Occupies the TBP Promoter and Decreases Elk-1 Occupancy (A) Alterations in Maf1 expression are reciprocal with Elk-1 occupancy at the TBP promoter. Stable U87 cells expressing Maf1-HA (+) or empty vector (−) (left), or infected with lentivirus expressing Maf1 shRNA (+) or mismatch RNA (−) (right) are shown. ChIP analysis was performed with qPCR using primers designed to amplify sequences between +5 and −119 or between −3315 and −3158 relative to the transcription start site as designated in schematic (top). The results shown are the percent of input where immunoprecipitations were performed using either control IgG, Maf1, or Elk-1 antibodies. Bars represent mean ± SE of at least three independent determinations from two separate chromatin preparations. (B) Altering Maf1 expression does not change Elk-1 expression. U87 cells in (A) were subjected to immunoblot analysis with antibodies indicated. A representative blot is shown. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Maf1 Differentially Regulates Elk-1-Dependent Promoters (A) Alterations in Maf1 expression change TBP and egr-1 expression, but not c-fos and β-actin expression. Total RNA was isolated from U87 cells expressing Maf1-HA (black bars), vector alone (white bars), or Maf1 shRNA (gray bars), or mismatch shRNA (white bars) was subjected to RT-qPCR. Values for each transcript were normalized to values for GAPDH transcript, and the fold change was calculated relative to the expression in control cells. Bars represent the average ± SE of more than three independent determinations. (B) Maf1 occupies the egr-1 promoter and decreases Elk-1 occupancy. ChIP analysis was performed using U87 cells expressing Maf1-HA (black bars), vector alone (white bars), or Maf1 shRNA (gray bars) or mismatch shRNA (white bars) using qPCR with egr-1 promoter-specific primers. Bars represent mean ± SE of at least three independent determinations from two separate chromatin preparations. (C) Maf1 expression does not alter Elk-1 occupancy on the c-fos promoter. ChIP analysis was performed using qPCR with c-fos promoter-specific primers as in (B). (D) Maf1 expression alters egr-1 but not c-fos or actin protein expression. Cell protein lysates derived from the stable U87 cell lines were subjected to immunoblot analysis with antibodies indicated. A representative blot is shown. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Effects of TBP Overexpression and ChIP Analysis on Pol I and Pol III Genes in Cells with Altered Maf1 Expression (A) Increased expression of TBP suppresses the inhibitory effect of Maf1 on Pol I transcription. U87 cells were transiently cotransfected with promoter-reporter plasmids pArg-maxigene or PrHuCAT and Maf1-HA expression vector and/or TBP expression vector. Total RNA was isolated and an RNase protection or a primer extension assay was performed to measure Pol III or Pol I transcription, respectively. Representative autoradiographs are shown. (B) Maf1 does not bind to the rRNA gene promoter. U87 cells expressing Maf1 shRNA, mismatch shRNA, Maf1-HA, or vector alone were subjected to ChIP analysis using qPCR where immunoprecipitations were performed using either control IgG, Maf1, or Elk-1 antibodies. The results shown are the percent of input where bars represent mean ± SE of at least three independent determinations from three separate chromatin preparations. (C) Maf1 occupancy of a tRNA gene is inversely correlated with the occupancy of TFIIIB and Pol III subunits. ChIP analysis of Maf1, Pol III (Rpc155), TFIIIC90, Brf1, and Bdp1 occupancy at the tRNALeu gene promoter in U87 cells expressing Maf1 shRNA (+) or mismatch shRNA (−). Input and IgG control products are shown. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 8 Altered Maf1 Expression Induces Changes in the Transforming and Morphological Properties of U87 Cells (A) Increased Maf1 expression decreases U87 anchorage-independent growth. U87 cells were stably transfected with vector or expression plasmids containing HA- or Flag-epitope-tagged Maf1 cDNA. Immunoblot analysis of U87 stable cells lines was performed using total protein lysates and Maf1, HA, Flag, or β-actin antibody (left panel). To determine accumulation rates (center panel), cells were plated and then harvested daily to determine viable cell numbers by trypan blue exclusion. Values are the mean ± SEM of at least two independent determinations. The U87-Maf1 stable cell lines were analyzed for growth in soft agar (right panel). Colonies greater than 50 μm in diameter were counted 14 days after plating. Values are the mean ± SEM of four independent determinations per cell line. (B) Decreased expression of Maf1 alters cellular morphology. Rhodamine phalloidin (a and c) and α-tubulin (b and d) staining of U87 cells infected with lentivirus expressing mismatch (mm) RNA (left) or Maf1 shRNA (right). Nuclei were stained with DAPI. Scale bar, 50 μm. (C) Decreased Maf1 expression increases cell surface area but does not alter cell volume. U87 cells infected with lentiviral expression constructs containing Maf1 shRNA or a mismatched control shRNA were costained with rhodamine phalloidin and α-tubulin-FITC to delineate the cell margins. Surface area was analyzed using MetaMorph Imaging Software. At least 65 images in 25 fields were analyzed (left). Forward scatter distribution in FACS histograms was utilized to determine relative volumes of U87 cells infected with lentiviral constructs expressing either Maf1 shRNA or a mismatched control shRNA. The histogram is representative of three independent determinations for each cell line. Molecular Cell 2007 26, 367-379DOI: (10.1016/j.molcel.2007.03.021) Copyright © 2007 Elsevier Inc. Terms and Conditions