Mmi1 interacts with Erh1 in a 2:2 stoichiometry. a Shown is a schematic representation of the domain architecture of Mmi1. The disorder score from DISOPRED3 Server and the conservation score are shown below. The conservation score is generated by a Protein Residue Conservation Prediction Server from the sequence alignment of Mmi1 orthologues from S. pombe, S. cryophilus, S. octosporus, and S. japonicus. b–d Interactions of GST tagged Mmi1 peptides (GST-Mmi1) with Erh1 visualized by Coomassie blue staining. The indicated GST-Mmi1 fusion proteins or GST alone were incubated with Erh1. The complexes were collected with glutathione-agarose resin and bound proteins were eluted and then subjected to SDS-PAGE. GST or GST-Mmi1 fusion proteins without Erh1 are shown as a negative control. Source data are provided as a file. e The raw ITC titration data of Erh1 with SUMO tagged Mmi1^95–122 and its fitting curve are shown. K_D dissociation constant, DP differential power, N binding stoichiometry, ΔH binding enthalpy. f ITC fitting curves of Erh1 using SUMO tagged Mmi1^95–122 (magenta), Mmi1^106–122 (blue) and Mmi1^95–111 (green) are shown. g Size-exclusion analysis of Erh1 (magenta) and Erh1-Mmi1^95–122 (blue). Marker sizes are 62.9 KDa (Albumin), 46.7 KDa (Ovalbumin), 20.3 KDa (Chymotrypsinogen), 14.6 KDa (Ribonuclease A). Also shown are theoretical monomer sizes (kDa) of Erh1 protein and Mmi1^95–122 peptide

Structure of Erh1 in complex with Mmi1^95–122 peptide. a Ribbon representations of Erh1 bound to Mmi1^95–122 peptide. One monomer of the Erh1 homodimer is colored in aquamarine, while the other is colored in wheat. The two bound Mmi1^95–122 peptides are colored in magenta. b The two Mmi1^95–122 peptides are represented as sticks on the molecular face of the Erh1 homodimer. Red and blue colors denote negative and positive surface charge, respectively. c Ribbon representation of the interface of the Erh1 dimer. The residues at the dimer interface are shown as sticks. The hydrogen bonds formed at the dimer interface are depicted as black dashed lines

Molecular interface between Erh1 and Mmi1^95–122. a Sequence alignment of S. pombe Erh1 and the enhancer of rudimentary homolog (ERH) proteins from H. sapiens (Human), C. elegans (Worm), and D. melanogaster (Fly). The alignment was generated by ESPript3 with CLUSTALW. The secondary structures of Erh1, as determined by DSSP, are shown above the sequences. The conserved residues at the ERH dimer interface are emphasized by up-pointing triangles below the sequences. The key residues for Mmi1^Trp1112 interaction are emphasized by down-pointing triangles above the sequences. Aligned amino acids in red share similar biophysical properties, those in white with red highlight are perfectly conserved, and blue boxes denote conserved clusters of amino acids. Black arrows denote hydrophobic residues, blue arrows denote residues involved in hydrogen bond interaction. b Sequence alignment of Mmi1^95–122 of S. pombe and the corresponding regions from S. cryophilus, S. octosporus, and S. japonicus. The secondary structures are also shown above the sequences. c Interactions of Mmi^Trp112, α-helix H2, 3₁₀ helix H1, and N-terminal loop with Erh1 dimer. Erh1 (aquamarine and wheat) and Mmi1^95–122 (magenta) are shown as ribbons with selected side-chain and main-chain atoms as sticks. Hydrogen bonds are shown as black dashed lines. The Y97 and F99 subpanel shows the binding pocket for Mmi1^Tyr97 and Mmi1^Phe99; the Hydrogen bonding panel shows the hydrogen bonding network between Mmi1 and the Erh1 dimer; the H1 subpanel shows the interactions between the 3₁₀ helix H1 of Mmi1 EIM peptide and Erh1 dimer; the W112 subpanel shows the hydrophobic pocket of the Erh1 dimer for Mmi1^Trp112 recognition; the H2 subpanel shows the interactions between the α-helix H2 Mmi1 of the EIM peptide and the Erh1 dimer. d GST pull-down assay showing the diminished interaction between the Mmi1^W112A mutant and Erh1.  are provided as a  file. e ITC fitting curves of Erh1 using SUMO tagged Mmi1^95–122 (magenta) and Mmi1^W112A (blue) are shown

Mmi1^W112A perturbs EMC functions in vivo. a Western blot analysis of FLAG-Mmi1 WT and FLAG-Mmi1^W112A protein expression levels. Cdc2 serves as loading control. Also shown is a linear schematic of mmi1^W112A mutant.  are provided as a  file. b Coimmunoprecipitation of Erh1-GFP and FLAG-Mmi1 WT or FLAG-Mmi1^W112A.  are provided as a  file. c Spotting assays at 32, 37, or 18 °C on non-selective media. The mmi1-ts6 strain was included as a control for 37 °C growth. d Erh1 localization in WT or mmi1^W112A cells. WT and mutant cells expressing Erh1-GFP were imaged using a DeltaVision Elite fluorescence microscope (Applied Precision, GE Healthcare). Scale bar (white) represents 5 μm. e Western blot analysis depicting Erh1 protein levels in wild-type or mmi1^W112A cells. Ponceau S stain serves as a loading control.  are provided as a  file. f ChIP-qPCR analyses of Erh1-GFP enrichment in WT and mmi1^W112A cells at indicated loci. Fold enrichment values plotted were calculated relative to the control leu1 locus. Shown are mean±SD for two experiments

Mmi1^W112A affects EMC-dependent facultative heterochromatin islands. a Genome-wide ChIP-seq profiles showing enrichment of histone H3 dimethylation (H3K9me2) in WT and mmi1^W112A cells. Examples of several individual heterochromatin islands are indicated. b H3K9me2 enrichment ChIP-seq profiles of EMC-dependent islands. c H3K9me2 enrichment ChIP-seq profiles of EMC-independent islands. The color scheme used in all plots shown is WT (black), mmi1^W112A (red), and erh1∆ (blue)

Mmi1^W112A affects a subset of the Mmi1 regulon silenced by EMC. a Heatmap of genes upregulated by ≥2-fold in at least one mutant as compared to WT. Cluster 1 contains genes that are derepressed in mmi1∆, mmi1^W112A, and erh1∆. Cluster 2 contains genes that are derepressed in mmi1∆ and mmi1^W112A or erh1∆. Cluster 3 contains genes that are derepressed in mmi1∆ only. In total, there are 531 genes considered here as upregulated out of a possible 7019.  are provided as a  file. b RNA-seq profiles of 9 representative genes whose repression requires Erh1-Mmi1 interaction. The color scheme used is WT (black), mmi1^W112A (red), and erh1∆ (blue). Shown is normalized RPKM expression. c RNA-seq profiles of 3 representative loci showing derepression in mmi1∆ but not in mmi1^W112A and erh1∆ cells

EMC assembly is critical for nuclear retention of gametogenic gene transcripts in mitotic cells. a Representative images of the EMC target ssm4 mRNA (red) detected by Single molecule RNA Fluorescence In-Situ Hybridization (smFISH). DNA was stained with DAPI (blue). Images are shown as the maximum-intensity projections of Z-stacks. Dotted lines indicate the outline of cells. Scale bar (white) represents 5 μm. b Quantification of nuclear and cytoplasmic localization of ssm4 mRNA. The upper panel shows the mean ± SEM from more than 140 cells and the lower panel indicates the distribution of the percentages of ssm4 mRNA spots/cell

Mmi1^W112A uncouples EMC-dependent and -independent Mmi1 functions. a 3′ RACE of ssm4 and leu1 (control) loci. Red triangles indicate termination sites as determined by sequencing of 3′ RACE products. The black arrow indicates the gene-specific forward primer used, which can be found in Supplementary Table . b RNA-seq expression profiles of prt-pho1 loci and nam1-byr2 in WT, mmi1^W112A, erh1∆, and mmi1∆. Shown is normalized RPKM expression. c Northern blot analysis at the prt-pho1 locus in indicated strains using a probe targeting pho1 mRNA (top and bottom panels). d Northern blot analysis at the nam1-byr2 locus in indicated strains using a probe targeting nam1 ncRNA.  are provided as a  file