Fig 2: Fkh2 is a resident member of multiple Clr6 complexes and directs Clr6 to nucleation-distal heterochromatin regions.A. Fkh2 co-migrates with medium sized and large Clr6 complexes. Western blot against TAP (red) and MYC (green) on fractions of a sucrose density gradient of whole cells extract containing Fkh2-TAP and Clr6-MYC. Fkh2 migrates as a free protein on top of the gradient and in both major peaks of Clr6. Complex annotation based on [44]. B. Fkh2 migration in sucrose gradients depends on Prw1. Western blot against MYC (green) on fractions of a sucrose density gradient of wild-type (WT) or ?prw1 whole cell extract containing Fkh2:MYC. While the free Fkh2 fraction is not affected by the absence of Prw1, the medium and large peak fractions shift towards the top of the gradient. C.-F. Clr6 chromatin localization in heterochromatin spreading areas and heterochromatin islands depends partially on Fkh2. Clr6:MYC ChIP qPCR in wild-type (Clr6-13XMYC) or ?fkh2 (Clr6-13XMYC ?fkh2) background relative to an untagged (-MYC) control, in MAT ?REIII at the HSS reporter and downstream (C.), heterochromatin islands (D.), tel1L (E.), and act1 (F.). Error bars indicate 1SD of three replicates. G. Model for control of heterochromatin spreading by Clr3, Clr6 and chromatin remodeler complexes. The Clr3- containing SHREC complex is essential for heterochromatin assembly at noncoding RNA-driven nucleation centers, such as cenH, dg/dh repeats or tlh1/2. There, SHREC activity enables normal HP1 and Clr4 H3K9 methylase activity. In the spreading zone, Fkh2-containing Clr6 complexes (complex I? or complex II) are required to propagate H3K9 methylation and gene silencing. Fkh2 recruits Clr6 to nucleation center-distal chromatin. Chromatin remodelers repel spreading via nucleosome destabilization, thus hinder the “guided-state” nucleosome-to-nucleosome spreading mechanism of Clr4.

Fig 3: Scc2 and Mediator physically interact(A) Scc2 and Med14 interact by co-immunoprecipitation independent of DNA. Myc tagged Scc2 was immunoprecipitated from WCE. Co-precipitation of Flag-tagged Med14 was analyzed by immunoblotting. Benzonase treating WCE to remove DNA did not affect the observed co-precipitation.(B) R787G and G1242V mutations do not affect the Scc2-Med14 interaction. Immunoprecipitations from the lysate of Myc tagged wild type, no tag, R787G, and G1242V Scc2 were analyzed for coIP of Med14-Flag by immunoblotting. The ratio of Scc2 to Med14 in the Scc2 mutants is comparable with WT in the Myc pulldowns.(C) Diagram indicating inter- (green) and intra- (purple) protein cross-links of the Scc2-Scc4 complex. Positions of Scc2 domains are marked.(D) Mediator is detected by mass spectrometry of purified Scc2. Cross-linking mass spectrometry was performed on Myc tagged Scc2 purified from WCE with anti-Myc magnetic beads with on bead disuccinimidyl sulfoxide (DSSO) cross-linking and on bead digestion. The heatmap of 1e6 log2 transformed dNSAF values shows enrichment of Mediator subunits in seven MS runs with purified Scc2-Myc compared with six no tag control runs. The loader and cohesin subunits are included as a positive control. Asterisks indicate statistical significance at p < 0.05.See also Table S2 and Figures S4 and S5.

Fig 4: Miro GTPases interact physically with GBF1. (A,B) RPE1 cells were cotransfected with Venus or Venus-tagged GBF1 and either Myc or Myc-Miro1 (A) or Myc-Miro2 (B). Immunoprecipitations were carried out with GBP-beads. Cells lysates (Input) and immunoprecipitated proteins were analyzed by Western blotting using anti-GFP and anti-Myc antibodies. The input lanes represent 15% of lysate used in immunoprecipitation reactions. (C) RPE1 cells were cotransfected with Arf1-T31N-GFP, Arf1-Q71L-GFP or GFP and Myc-Miro2. GFP-tagged proteins were immunoprecipitated with magnetic GFP-Trap®_A. Cells lysates (Input) and immunoprecipitated proteins were analyzed by Western blotting using GFP- and Myc-antibodies. The input lanes represent 10% of lysate used in immunoprecipitation reactions. One representative experiment of three independent experiments is shown. Bands were quantified by a densitometric analysis performed using Image J software. Results are the mean of the ratio between the Myc and the GFP band values ± S.D. of the three independent experiments. *P ≤ 0.05 versus control cells.

Fig 5: Chemical synthesis and ubiquitination of ubiquitin-conjugates.a A cartoon depicting cyclin B1 with its 1–88 aa residue unstructured N-terminal region (NT), and a C-Terminal CDK1 binding region (left). The primary amino acid sequence of the N-terminal region of cyclin B1 that encompasses the Degron and 15 Lysine residues (right). Below are the wild-type (WT) and K0-mutant (all Lysine to Arginine) Cyclin B1-NT sequences used in this study for mammalian cell expression. The K64 HA-Cyclin B1-NT sequence was used for chemical ubiquitination of substrates (bottom). b Schematic illustration of the strategy used to synthesize differentially tagged TetraUbK48-HA-Cyclin B1-NT (Nbz = N-acyl-benzimidazolinones, SR = 3-mercaptopropionic acid). The detailed chemical synthesis protocol is described in Supplementary Methods. c A cartoon representation of a set of four synthetic substrates designed to study proteasome function. Tagged (Myc/Flag) and/or untagged ubiquitin units were attached to lysine64 of the HA-Cyclin B1-NT to obtain ubiquitinated conjugates. The numbers 3, 5, and 7 correspond to the three synthetic substrates in panel (b) (see also Supplementary Methods). d All four synthetic ubiquitinated Cyclin B1-NT conjugates were resolved by SDS-PAGE and stained with Coomassie or probed with the indicated antibodies for immunoblotting (IB). e Human 30S/26S and 20S proteasomes purified from human erythrocytes were resolved by native gel for in-gel activity (left), or for Coomassie (center), or resolved by SDS-PAGE for Coomassie staining. The band marked by * near 90 kDa was identified as HSP90AA1 and HSP90AB1 in MS/MS analysis. All other bands were confirmed to contain primarily 20S (right) or 26S (to its left) subunits. Source data are provided as a Source data file.