PPARγ S82A and S82D mutations do not affect SUMOylation.

<p>The indicated PPARγ K33R, K77R, S82A, S82D, K33R/S82A, K33R/S82D, K77R/S82A and K77R/S82D mutants were transfected in HEK293 cells and analyzed for SUMO modification in the absence and presence of ligands as outlined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1</a>. (A) The phosphorylation blocking S82A and the phosphorylation mimicking S82D mutations did not significantly affect SUMOylation of PPARγ at K33 and K77. (B) PPARγ S82A and S82D mutations did not affect rosiglitazone-induced reduction of PPARγ SUMOylation at K33.</p

Transrepression activity of PPARγ mutants.

<p>(A) RAW264.7 macrophages were transfected with the <i>iNOS</i> luciferase reporter plasmid along with PPARγ mutants. Forty-two hours after transfection, cells were treated for 6 hours with 1 µg/ml LPS and 1 µM rosiglitazone (Rosi) as indicated. The reporter activities in the presence of LPS were set to 100% promoter activity. (B) Hela cells were transfected with the <i>3xNF-κB</i> luciferase reporter plasmid along with PPARγ mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi). Four hours prior lysis, 10 ng/ml interleukin-1β (IL-1ß) was added as indicated. The reporter activities obtained by interleukin-1ß stimulation were set to 100% promoter activity. Error bars are mean +/− SD. Statistics was performed using Student´s t-test. *, p<0.05; **, p<0.005.</p

Lysine 365 within the LBD is essential for ligand-induced reduction of PPARγ SUMOylation.

<p><b>(</b>A) (B) and (C) The PPARγ K365R (A) and PPARγ K77/365R (B) mutants were transfected in HEK293 cells and analyzed for His-SUMO2 and His-SUMO1 modification in the absence and presence of ligands as outlined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1</a>. The blot shown in the upper left panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g004" target="_blank">figure 4B</a> was re-probed with an anti-His antibody to control for loading. (C) Summary of quantitative Western blot analysis. SUMOylation of the PPARγ K365R mutant in the absence or presence of rosiglitazone or GW1929 was analyzed by an independent quantitative Western blot analysis using fluorescence-labeled secondary antibodies. The values obtained for SUMOylated PPARγ K365R relative to the input signal in the absence of ligands were arbitrarily set to 1.</p

Ligand binding to the C-terminal LBD reduces SUMOylation of lysine 33 within the N-terminal AF1 domain.

<p>PPARγ mutants were transfected in HEK293 cells and analyzed for His-SUMO2 or His-SUMO1 modification in the absence and presence of ligands as outlined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1</a>. <b>(</b>A) SUMOylation of wild type PPARγ and of the PPARγ K77R mutant in the absence and presence of 1 µM rosiglitazone (Rosi). (B) Summary of quantitative Western blot analyses. SUMOylation of wild type PPARγ by His-SUMO2 and of the PPARγ K77R mutant by His-SUMO2 or His-SUMO1 in the absence and presence of rosiglitazone (Rosi) or GW1929 (GW) was analyzed by imager quantification using fluorescence-labeled secondary antibodies. Wild type PPARγ and the PPARγ K77R mutant were analyzed separately. The values obtained for SUMOylated wild type PPARγ or for the PPARγ K77R mutant relative to the input signal in the absence of ligands were arbitrarily set to 1. (C) Analysis of the N-terminal (amino acid 1-256) and the C-terminal domain (amino acid 247-475) of PPARγ for modification by His-SUMO1 or His-SUMO2. (D) Analysis of the PPARγ Δ1-68 K77R mutant for SUMOylation by His-SUMO1 or His-SUMO2 in the absence or presence of rosiglitazone. (E) Analysis of the PPARγ mutants PPARγ K33R, PPARγ K77R, PPARγ K33/77R and PPARγ K33/64/68/77R for modification by His-SUMO2. (F) Analysis of the PPARγ K33/64/68/77R mutant for modification by His-SUMO2 in the absence or presence of rosiglitazone. (G) Analysis of the PPARγ K77R and PPARγ K33/64/68R mutants for modification by His-SUMO2 in the absence or presence of rosiglitazone. <b>(</b>H) Model depicting interdomain communication regulating SUMOylation of PPARγ. Ligands reduce SUMOylation of K33 and potentially of K64 and K68 but not of K77.</p

Analyzing SUMOylation of PPARγ.

<p>(A) PPARγ domain structure. PPARγ2 differs from PPARγ1 by a 30 amino acid extension at the N-terminus. The activation function 1 and 2 domains (AF1 and AF2), the DNA-binding domain (DBD) and the ligand-binding domain (LBD) are indicated. Positions of lysines (K) and serines (S) refer to PPARγ2 and PPARγ1, respectively. (B) Schematic outline of the experimental procedure for detecting SUMOylated PPARγ1. HA-PPARγ1 was transfected along with untagged SUMO1, His-SUMO1 or His-SUMO2 in HEK293 or HeLa cells. His-SUMO-conjugated proteins were subsequently purified from cell lysates by Ni-NTA affinity chromatography. SUMOylated HA-PPARγ1 was detected by immunoblotting for the HA-tag. (C) SUMOylation of PPARγ was analyzed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1B</a>. PPARγ is SUMOylated by His-SUMO1 and His-SUMO2. (D) SUMOylation of PPARγ by His-SUMO1 in HEK293 or HeLa cells was analyzed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1B</a> in the absence and presence of 1 µM rosiglitazone. (E) Upper panel: SUMOylation of PPARγ by His-SUMO2 in HEK293 cells was analyzed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066947#pone-0066947-g001" target="_blank">Figure 1B</a> in the absence and presence of 1 µM GW1929 or 1 µM rosiglitazone. The asterisk indicates a cross-reacting protein. Lower panel: To control for loading, the blot was re-probed with an anti His antibody. S1, untagged SUMO1, His-S1 and His-S2, His-tagged SUMO1 and His-tagged SUMO2; I, Input: 1% of cell lysate; P, Ni-pulldown: 90% of cell lysate.</p

Transcriptional activity of PPARγ mutants.

<p>(A) HeLa (top) and RAW264.7 (bottom) cells were transfected with the <i>Aox-tk</i> luciferase reporter construct along with the indicated PPARγ1 lysine mutants. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (+) or the vehicle (-), and incubated for additional 24 hours. The reporter activity in the absence of PPARγ was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by PPARγ mutants compared to wild type PPARγ in the absence (*) or presence (⁺) of rosiglitazone was calculated using the Student´s t-test. * and ⁺, p<0.05; ** and ⁺⁺, p<0.005. (B) HEK293 cells were transfected with a <i>5</i>×<i>UAS</i>-driven luciferase reporter along with expression constructs for Gal4 or Gal4-PPARγ-LBD fusions as indicated. Twenty-four hours after transfection, cells were treated with 1 µM rosiglitazone (Rosi) or 1 µM GW1929 for additional 24 hours. The reporter activity in the absence of Gal4 fusions was arbitrarily set to 1. Error bars are mean +/− SD. Statistical significance of activation by Gal4-LBD and Gal4-LBD-K365R compared to Gal4 was calculated by the Student´s t-test. *, p<0.05; **, p<0.005.</p

MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

<div><p>Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.</p></div

Mga, L3mbtl2 and Pcgf6 colocalize in mouse ESCs and repress genes involved in differentiation.

<p>(A) Venn diagrams representing the overlap of Mga, L3mbtl2 and Pcgf6 peaks in mouse ESCs. The total number of filtered (≥30 tags and ≥3-fold enrichment over IgG control) ChIP-seq peaks and their overlap is shown. (B) A heat map view of the distribution of the top 8000 union Mga-L3mbtl2-Pcgf6 peaks in mouse ES cells at +/- 2 kb regions centred over the MGA peaks. (C) Representative genome browser screenshot of a 100 kb region of chromosome 1 showing co-binding of Mga, L3mbtl2 and Pcgf6 to four promoter regions. (D) Sequence motifs enriched in Mga-L3mbtl2-Pcgf6 binding regions in mouse ESCs. Top, logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union Mga-L3mbtl2-Pcgf6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Bottom, local motif enrichment analysis (CentriMo) showing central enrichment of the Mga/Max bHLH domain E-box binding motif and the motif that identified MEME Tomtom as a T-box as well as a E2f6 recognition sequence. The Nrf1 motif was not centrally enriched within the 300 bp peak regions. (E) Distribution of Mga, L3mbtl2 and Pcgf6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (F) Middle panel, Venn diagram illustrating the overlap of PRC1.6-bound genes and genes up-regulated in Pcgf6<i>ko</i> cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref026" target="_blank">26</a>] and in L3mbtl2<i>ko</i> cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref013" target="_blank">13</a>]. Left panel, GO analyses of biological functions of PRC1.6-bound genes that were de-repressed ≥2-fold in Pcgf6<i>ko</i> cells. Right panel, GO analyses of biological functions of PRC1.6-bound genes that were de-repressed ≥2-fold in L3mbtl2<i>ko</i> cells. Enriched GO terms were retrieved using DAVID 6.8. (GOTERM_BP_DIRECT, Functional Annotation Chart). Benjamini values are plotted in log10 scale.</p

Primers for RT-qPCR analyses.

<p>Primers for RT-qPCR analyses.</p

PRC1.6 binding sites partially overlap with cPRC1, PRC2 and ncPRC1.1 binding sites.

<p>(A) ChIP-seq heatmaps of Pcgf6, IgG control, Ring1b (GSM1041372) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Rybp (GSM1041375) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Cbx6-HA (GSM2610616) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Cbx7 (GSM2610619) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref033" target="_blank">33</a>], Pcgf2 (GSM1657387) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref056" target="_blank">56</a>], Suz12 (GSM1041374) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref034" target="_blank">34</a>], Kdm2b (GSM1003594) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref006" target="_blank">6</a>] and H3K27me3 (GSM1341951) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007193#pgen.1007193.ref010" target="_blank">10</a>] peaks in mESCs at +/- 2 kb regions centred over the Mga-L3mbtl2-Pcgf6 peaks. (B) Venn diagrams showing the overlap of high confidence Pcgf6 target genes (location of binding sites between -2.5 kb of TSS and TES) with those of Cbx7 (cPRC1), Suz12 and H3K27me3 (PRC2) and Kdm2b (ncPRC1.1). (C) Genome browser screenshots of ChIP-seq tracks at promoters of representative meiosis-related genes (<i>Dazl</i>, <i>Sycp3</i>, <i>Stk31 and Mei1</i>) (D) Genome browser screenshots of ChIP-seq tracks at cPRC1 target genes (<i>Nkx2-4</i> and <i>Hoxa7)</i>.</p