Primers for RT-qPCR analyses.

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

Reduced splicing efficiency of the <i>rbf1</i>-gene leads to impaired function of the Rbf1 master regulator.

<p>(<b>A</b>) Plot depicting splicing efficiency of the <i>rbf1</i>-gene. Plotted are the FPKM values (fragments per kilobase of sequence per million fragments mapped) across the genomic region indicated (coordinates in nucleotides) of three independent RNA-Seq experiments for AB31 wild-type (blue lines) and AB31<i>Δnum1</i> (red lines), respectively. Exons (E) and introns (I) are indicated. All four introns show increased intron retention rates in AB31<i>Δnum1</i>. (<b>B</b>) Western analysis showing abundance of Rbf1:3×HA and α-tubulin (loading control) from AB31 wild-type and Δ<i>num1</i>-deletion strains. In AB31<i>Δnum1</i>, Rbf1 is reduced to 30% of wild-type-level (Quantification: ImageJ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046-Abramoff1" target="_blank">[109]</a>). (<b>C, D</b>) Gene expression analyses of <i>b</i>- and <i>rbf1</i>-genes (<b>C</b>) as well as <i>rbf1</i>-target genes (<b>D</b>) using qRT-PCR. RNA samples were isolated from strains AB31 and AB31<i>Δnum1</i> eight hours after induction of the bE1/bW2-heterodimer. Gene expression is shown relative to the highest expression value, using <i>actin</i> and <i>eIF2b</i> for normalization. Shown are the mean values of three biological and two technical replicates. Error bars represent the SD. (<b>E</b>) Venn diagram depicting the total number of genes repressed in AB31<i>Δnum1</i> and AB31<i>Δrbf1</i>. * RNA-Seq analysis, this study; ** Microarray analysis conducted five hours after induction of the bE1/bW2-heterodimer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046-Heimel1" target="_blank">[2]</a>.</p

Num1 and Kin1 are functionally connected.

<p>(<b>A</b>) The <i>num1</i> deletion leads to aberrant vacuole morphology. Vacuoles were visualized by CellTracker Blue (7-amino-4-chloromethyl-coumarin, CMAC) staining. Both <i>num1</i> and <i>kin1</i> deletion strains contain more, but smaller vacuoles in comparison to wild-type sporidia. Scale bars: 5 µm. Right panel: Quantification of the average number of vacuoles of strains indicated. N represents the number of individual cells analyzed. Mean values of three independent experiments are shown. (<b>B</b>) The <i>num1</i> deletion leads to an aberrant distribution of early endosomes. For visualization of early endosomes, a Yup1:eGFP fusion protein <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046-Lenz1" target="_blank">[9]</a> was expressed under control of the constitutively active <i>P_otef</i>-promoter in strains AB33 and AB33<i>Δnum1</i>, which harbor <i>b</i>-genes under control of the nitrate-inducible <i>P_nar1</i>-promoter. Endosomes were analyzed 14 hours after <i>bE1/bW2</i>-induction. Septa (arrowhead) were visualized by Calcofluor White staining. Yup1-labelled early endosomes accumulate at basal and apical parts of the hyphae as well as around delocalized septa in AB33<i>Δnum1</i> in contrast to wild-type hyphae. Scale bars: 10 µm. Movies detecting Yup1:eGFP-fluorescence of the depicted hyphae are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046.s023" target="_blank">Videos S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046.s024" target="_blank">S2</a>. Shown below are the corresponding kymographs (time and distance as indicated) reflecting the motility of early endosomes. In AB33 hyphae, early endosomes travel continuously towards the hyphal tip (anterograde) and reverse direction to travel back towards the septum (retrograde). Pausing organelles are reflected by a vertical line. In contrast, endosomal motility is drastically reduced in AB33<i>Δnum1</i> hyphae containing additional septa, where the majority of the Yup1:eGFP-signal is non-motile. An arrowhead indicates the septum. (<b>C</b>) The localization of Dynein is dependent on Num1. In AB33 filaments 3eGFP:Dyn2 fusion proteins form comet-like motile structures that accumulate at the hyphal tip and from there move into retrograde direction <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004046#pgen.1004046-Lenz1" target="_blank">[9]</a>. In filaments of AB33<i>Δnum1</i> strains the apical accumulation is significantly reduced (p = 0.002); instead a stronger signal is observed in the cytoplasm. Dynein localization was monitored 14 hours after induction of the bE1/bW2 heterodimer. Scale bars: 10 µm. Right panel: Quantification of the apical accumulation of 3eGFP:Dyn2 fusion proteins. N represents the number of hyphae analyzed. Mean values of three independent experiments are shown.</p

Commercially available antibodies used in this study.

<p>Commercially available antibodies used in this study.</p

Num1 is required for polar growth and septum formation.

<p>Filament formation of AB31 wild-type and AB31<i>Δnum1</i> deletion strains was monitored 12–14 hours upon induction of the bE1/bW2 heterodimer. (<b>A</b>) AB31 wild-type cells grow as long, straight filaments; only the tip compartment is filled with cytoplasm, separated from highly vacuolated distal compartments by a septum (arrowhead). (<b>B</b>) AB31<i>Δnum1</i> hyphae grow shorter and more curved. In AB31<i>Δnum1</i>, bipolar (<b>C</b>) and branched (<b>D</b>) hyphae are observed frequently. (<b>E</b>) Septa of AB31<i>Δnum1</i> are often formed within the compartment filled with cytoplasm (arrowheads denote delocalized septum; in lower panel, Calcofluor White staining was used to visualize the septum). (<b>F</b>) In approx. 2.5% of hyphae delocalized septa (arrowhead) lead to empty tip compartments. Scale bars: 10 µm.</p

MGA promotes binding of PRC1.6 by DNA-binding-dependent and DNA-binding-independent mechanisms.

<p>(A) Expression of wild type MGA in MGA<i>ko</i> cells rescues binding of PRC1.6. ChIP-qPCR data showing binding of transiently expressed MGA and of endogenous L3MBTL2, E2F6, PCGF6, RING2 and MAX to representative PRC1.6 target promoters. The level of the H2AK119ub1 was not affected. Percent of input values represent the mean of at least three independent experiments +/- SD. (B) Schematic representation of the MGA ΔTbox and bHLH mutants. (C) Western blot analysis of wild type MGA and of the DNA-binding-deficient MGA mutants (ΔT-Box, bHLHmut and ΔTbHLHmut) expressed in MGA<i>ko</i> cells. The anti-Tubulin blot served as a loading control. (D) ChIP-qPCR analyses of MGA and L3MBTL2 binding to selected PRC1.6 target promoters in MGA<i>ko</i> cells and in MGA<i>ko</i> cells re-expressing wild type MGA (MGA WT) or DNA-binding-deficient MGA mutants (MGA ΔT-Box, MGA bHLHmut or MGA ΔTbHLHmut). The error bars denote SD; n = 3.</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

The role of PRC1.6 in HEK293 cell function.

<p>(A) Reduced proliferation of MGA<i>ko</i>, L3MBTL2<i>ko</i> and E2F6<i>ko</i> cells. Shown are growth curves of wildtype, MGA<i>ko</i>, L3MBTL2<i>ko</i>, E2F6<i>ko</i> and PCGF6<i>ko</i> HEK293 cells. Cells were seed at 3x10⁵, and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA<i>ko</i> cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA<i>ko</i> cells; right circle, genes with ≥2-fold increased transcript levels in MGA<i>ko</i> cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the <i>CNTD1</i> and <i>SMC1B</i> promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA<i>ko</i> cell clones (MGA<i>ko</i>_cl26, MGA<i>ko</i>_cl27 and MGA<i>ko</i>_cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA<i>ko</i>, E2F6<i>ko</i>, L3MBTL2<i>ko</i> and PCGF6<i>ko</i> cells. Transcript levels were normalized to <i>B2M</i> transcript levels, and are depicted relative to transcript levels in wild type cells.</p

Reduced endosomal motility in <i>num1</i>-deletion strains.

*<p>t-test p = 0.005.</p