Maf1 represses transcription from type I, II, and III pol III promoters <i>in vitro.</i>

<p>A) Maf1 represses transcription from the 5S, VAI, and 7SL pol III promoters, but not the Ad2 ML pol II promoter, <i>in vitro</i>. 40, 80, 400, and 800 ng of bacterially produced Maf1 (lanes 3–6) or similar amounts of Brf2 (lanes 7–10) were added to transcription reactions identical to that shown in lane 2. Lane 1 shows unprogrammed transcription extract. B) SDS-polyacrylamide gel stained with coomassie blue indicating the amounts of recombinant Maf1 and Brf2 used in the <i>in vitro</i> transcription experiment described in A. C) Maf1 represses transcription from the U6 promoter <i>in vitro</i>. The lanes are as in A, except that the HeLa cell extract was programmed with the U6 promoter and that increasing amounts of GST rather than Brf2 were added as control. D) SDS-polyacrylamide gel stained with coomassie blue indicating the amounts of recombinant Maf1 and GST used in the <i>in vitro</i> transcription experiment described in C. E) Maf1 associates with pol III in the transcription extract. <i>In vitro</i> transcription reactions containing either no (lane 2) or 400 ng (lane 3) of recombinant His-tagged Maf1 were incubated with Ni-NTA beads, and the beads were then washed several times with D100 buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000134#pone.0000134-Dignam1" target="_blank">[40]</a>. The affinity-purified complex was analysed by SDS-PAGE followed by immunoblotting with anti-RPC1 or anti-His tag antibodies.</p

Maf1 associates with pol III, the individual pol III subunits RPC1 and RPAC2, and Brf1.

<p>A) Association of endogenous Maf1 with pol III and Brf1. Whole cell extract derived from the HeLa cell line 9–8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000134#pone.0000134-Hu1" target="_blank">[41]</a> was used for immunoprecipitations with anti-Maf1 or anti-GAPDH (Abcam) antibodies. The beads were washed, bound material was then eluted by boiling and used for immunoblotting with an anti-RPC1 antibody (CS377) (upper panel), an anti-Brf1 antibody (CS146) (middle panel) or an anti-Maf1 antibody (SZ2793) (lower panel). Lane 1 shows 1/20 of the input material, lanes 2, 3, and 4 show 1/30 of the flow through, 1/30 of the last wash, and 1/5 of the eluted material, respectively. Quantification of the signal shows that 6% of the RPC1 protein was co-immunoprecipitated with Maf1. B) Association of Maf1 with individual pol III subunits. GST-Maf1 or just GST were expressed in E. coli and immobilized on glutathione sepharose beads. Pol III subunits were synthesized by coupled <i>in vitro</i> transcription/translation in the presence of [³⁵S] methionine, and incubated with GST-Maf1 or GST beads. The beads were washed extensively and the bound material was analysed by SDS–PAGE and autoradiography. The first lane (input) shows 1/10 of the <i>in vitro</i> translated material added to the beads. C) Association of Maf1 with RPC1 and RPAC2 is not mediated by RNA nor DNA. Before the binding reaction, GST, GST-Maf1, and <i>in vitro</i> translated RPC1 and RPAC2 were subjected to RNase A or DNase I treatment as indicated above the lanes. D and E) Association of Maf1 with TFIIIB components and SNAP_c subunits, respectively. The experiments were performed as in B but with the <i>in vitro</i> translated proteins indicated. F) Pol III subunits and Brf1 associate with different Maf1 regions. On top, a schematic representation of full-length and truncated versions of human Maf1 are shown, with the A, B, and C conserved regions indicated. The experiment shown in the three bottom panels was performed as in B but with the GST fusion proteins indicated and <i>in vitro</i> translated GST, GST-Maf1^1-82, or GST-Maf1^1-142, as indicated.</p

Human Maf1 is phosphorylated and becomes dephosphorylated after rapamycin and MMS treatment.

<p>A) Nuclear extract from a HEK 293 cell line expressing HA-tagged Maf1 either untreated (upper panel), or treated with rapamycin (middle panel) or MMS (lower panel) was incubated with nothing, CIAP with or without phosphatase inhibitors, or phosphatase inhibitors alone, as indicated above the lanes. Tagged Maf1 was visualized with an antibody directed against the HA tag. Lane 5 shows bacterially expressed Maf1, which was loaded on a non-adjacent lane of the same gel. B) Nuclear extract from IMR-90Tert cells transiently expressing HA-tagged Maf1 was prepared in the presence of phosphatase inhibitors prior to immunoprecipitation with affinity-purified anti-RPC1 antibody. Tagged Maf1 was visualized with an antibody directed against the tag (α-HA antibody). Only the fastest migrating form of Maf1 bound to pol III. Arrows: phosphorylated Maf1. The negative control was a mock immunoprecipitation performed with protein A sepharose beads without antibody. Lane 1 shows 1/20 of the input material.</p

Knock-down of endogenous Maf1 increases the levels of unstable tRNA precursors.

<p>A) Knock-down of endogenous Maf1 in untransformed cells. Exponentially growing IMR-90Tert cells were either left untransfected, or transfected with either siRNAs #2 or #3 against MAF1, or with a control siRNA, as indicated on top of the panel. RNA was then isolated from untransfected or transfected cells and 2 µg of total RNA used for random-primed reverse transcription. The resulting cDNA was analyzed by real-time quantitative PCR (qPCR) with primers corresponding to either Maf1 (black bars), tRNA^Tyr precursor (yellow bars), or GAPDH as a control (blue bars). B) and C) Knock-down of endogenous Maf1 in transformed cells. In B, the levels of either α-tubulin as a loading control (upper panel) or Maf1 (lower panel) were analyzed by western-blot in extracts from either untransfected 293 cells (lane 1) or 293 cells transfected with the siRNAs indicated above the lanes (lanes 2–5). In C, 293 cells were either left untransfected (lane 1) or transfected with the siRNAs indicated above the lanes. Cellular RNA was then collected and analyzed by northern blot with a probe detecting U2 snRNA precursors as a control (upper panel), or intron-containing tRNA^Leu (middle panel) and tRNA^Tyr (bottom panel) precursors.</p

<i>RNU1</i>, <i>RNU2</i>, and <i>RNU6</i> transcription and factor recruitment during mitosis to G1 phase transition.

<p>(A) Time course of U1 and U6 reporter transcript and U2 and pre-U2 snRNA accumulation after mitosis release. The 5.8S RNA served as an internal control. The time after mitosis release is indicated above each panel. (B) Time course analysis of transcription factor recruitment on various promoter regions. ChIPs were performed at the times indicated (x axis) after mitosis release with antibodies directed against the factors indicated on top of each panel, and analyzed by real time PCR. The analyzed regions are indicated at the upper right of each panel. The control region (Ctrl) is 2 kb upstream of <i>RNU1</i>. The results are expressed relative to input DNA. Two sets of <i>RNU1</i> primers were used: set U1A recognizes <i>U1-1, U1-2, U1-3, U1-8, U1-like-3</i> loci and was used in the top panel; set U1B recognizes <i>U1-2</i> and <i>U1-3</i> loci and was used in the 3 lower panels. The <i>RNU2</i> primers are specific for the <i>RNU2</i> cluster in chr17_unknown, and the <i>RNU6</i> primers for the <i>U6-1</i> locus. (C) Real time PCR analysis of <i>RNU1</i> (top panel, U1A primer set for POLR2B, GTF2B, SNAPC1 and POLR3D ChIPs; and U1B primer set for the other ChIPs) and <i>RNU6</i> (bottom panel) promoters pulled down after ChIP with antibodies against the factors indicated below the panels either at mitosis (1 h after release) or in mid-G1 (7 h after release). The results are expressed relative to mitosis values, which were set at 1 for each factor. Means and error bars were calculated over triplicate PCR analyses. Each experiment was performed at least twice.</p

SNAP_c subunits occupancy and proximal promoter motifs.

<p>(A) UCSC browser views of a pol III (<i>tRNAU1</i>) and a pol II (<i>RNU4atac</i>) gene showing occupancy by the factors indicated on the left. The chromosome coordinates are shown on top, the genes present in the region and their orientation at bottom. The y axis shows tag counts. (B) Two examples of non-annotated genomic regions showing occupancy by SNAP_c subunits, GTF2B, and POLR2B. (C) Box plot of BRF2, GTF2B, and SNAP_c subunit positions. For each gene, the position of the peak summit for each SNAP_c subunit relative to the TSS (set at 0) was determined. A median position (black bars in boxes, number in brackets on the y axis) was calculated. For the pol II genes, only the upper two tertiles of each SNAP_c subunit and GTF2B scores were included. The position for each gene is represented by a circle. (D) LOGOs of PSE and TATA box generated by WebLogo with the motifs identified with MEME (alignments in Figures S4 and S5). The top panel shows the PSE LOGO for pol II snRNA genes, the middle panel shows the PSE LOGO for pol III genes, and the bottom panel shows the TATA box LOGO for pol III genes.</p

Depletion of endogenous ZNF143 reduces transcription factor recruitment on the U1 promoter in mid-G1.

<p>(A) Immunoblot showing ZNF143 and Tubulin (control) levels during mitosis and mid-G1 phase after treatment with siRNA against Luciferase (Luc, control siRNA) or ZNF143. (B) Real time PCR analysis of <i>RNU1</i> promoter pulled down after ChIP with antibodies against the factors indicated below the panel either after treatment of the cells with siRNA against Luciferase (siLuc, control siRNA) or siRNA against ZNF143 (siZNF143). The values obtained with the siZNF143 treatment are shown relative to those obtained with the siLuc treatment, which were set at 100%. Means and error bars were calculated over triplicate PCR analyses. Each experiment was performed at least twice. The U1A primer set was used for the POLR2B, GTF2B and SNAPC1 ChIPs, the U1B primer set for the other ChIPs.</p

Pol II and III occupancy of snRNA genes.

<p>(A) Bar graph showing POLR2B (dark blue), GTF2B (light blue), POLR3D (red), and BRF2 (orange) ChIP-seq scores (y axis) on SNAP_c-occupied genes and the few snRNA genes devoid of SNAP_c (x axis). Genes are ordered by decreasing POLR2B scores for the pol II and <i>RPPH1</i> genes followed by increasing POLR3D scores for the pol III genes. (B) UCSC browser view of <i>RPPH1</i> gene showing POLR2B, POLR3D, GTF2B, and BRF2 occupancy. Y axis: tag counts. (C) POLR2B (light grey) or POLR3D (dark grey) occupancy in cells not treated or treated with 50 µg/ml α-amanitin for 2 or 6 h, as indicated on the x axis. Upper two panels: results are shown as % of input. Lower two panels: POLR2B and POLR3D occupancy without α-amanitin was set at 1.</p

Activator occupancy and distal promoter motifs.

<p>(A) UCSC browser view of three pol II (<i>RNU4atac</i>, <i>U1-like-5</i>, and <i>Unknown-6</i>) and one pol III (<i>tRNAU1</i>) gene showing occupancy by the factors indicated on the right of each panel. The chromosome coordinates are shown on top, the genes present in the region and their orientation at bottom. The y axis shows tag counts. (B) Promoter region (−400 to +1) of the four genes depicted in (A) with the positions of the GABPA (GA-motif), ZNF143 (SBS), and POU2F1 (octamer) binding sites found by MEME or MAST indicated. The positions of the PSE and TATA box are also shown, and the promoters were aligned according to the PSE position. The crossed-out motifs have either no corresponding peak of occupancy or are not the closest to the peak summit. The orientation of each motif is indicated with an arrow. (C) LOGOs of the ZNF143, POU2F1 (octamer) and GABP binding motifs generated by WebLogo with the motifs located closest to the corresponding factor peak summits (see alignments in Figures S9, S10, S11).</p

Table1_Contrasting effects of whole-body and hepatocyte-specific deletion of the RNA polymerase III repressor Maf1 in the mouse.XLSX

MAF1 is a nutrient-sensitive, TORC1-regulated repressor of RNA polymerase III (Pol III). MAF1 downregulation leads to increased lipogenesis in Drosophila melanogaster, Caenorhabditis elegans, and mice. However, Maf1−/− mice are lean as increased lipogenesis is counterbalanced by futile pre-tRNA synthesis and degradation, resulting in increased energy expenditure. We compared Chow-fed Maf1−/− mice with Chow- or High Fat (HF)-fed Maf1hep−/− mice that lack MAF1 specifically in hepatocytes. Unlike Maf1−/− mice, Maf1hep−/− mice become heavier and fattier than control mice with old age and much earlier under a HF diet. Liver ChIPseq, RNAseq and proteomics analyses indicate increased Pol III occupancy at Pol III genes, very few differences in mRNA accumulation, and protein accumulation changes consistent with increased lipogenesis. Futile pre-tRNA synthesis and degradation in the liver, as likely occurs in Maf1hep−/− mice, thus seems insufficient to counteract increased lipogenesis. Indeed, RNAseq and metabolite profiling indicate that liver phenotypes of Maf1−/− mice are strongly influenced by systemic inter-organ communication. Among common changes in the three phenotypically distinct cohorts, Angiogenin downregulation is likely linked to increased Pol III occupancy of tRNA genes in the Angiogenin promoter.</p