Journal: bioRxiv

Article Title: Catalytic pocket of Clr4 (Suv39h) methyltransferase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

doi: 10.1101/2025.08.28.672867

Figure Lengend Snippet: (A) Map of Recombinant Bacmid Vector Encoding CLRC -Clr4 . Diagram of the recombinant bacmid vector used for insect cell expression, encoding all CLRC components except Clr4. Key vector features and regulatory elements are indicated. (B) SEC chromatogram of CLRC complexes using Superose 6 Increase 3.2/300 column (Cytiva). Absorbance at 280 nm (mAU) is shown. The grey line indicated the elution profile of CLRC complex lacking Clr4, with the peak fractions highlighted. The blue line indicated the elution profile of the full CLRC complex with peak fractions highlighted in blue and fractions containing excess unbound Clr4 highlighted in pink. (C) SDS-PAGE analysis of SEC fractions from panel B. Coomassie-stained SDS–PAGE gel showing input (CLRC -Clr4 and Clr4) and eluted fractions from size-exclusion chromatography. Fractions corresponding to the blue peak (1.45–1.55 mL) contain all 5 CLRC complex subunits, while later fractions highlighted in pink (1.65–1.85 mL) contain excess unbound Clr4. *, MBP-Raf1 degradation products. (D) Mass spectrometry analysis of the reconstituted CLRC complex. Quantitative LC-MS/MS analysis of the reconstituted CLRC complex showing normalized spectral counts relative to MBP–Raf1 (set to 1). The calculated molar ratios of 0.66 for Clr4, 0.33 for Rik1, 0.31 for Cul4, and 0.22 for Raf2 are consistent with SDS–PAGE band intensities. (E) Schematic representation of Clr4 truncation constructs used in this study. Domain architecture of Clr4 and its truncation variants, with the chromodomain shown in yellow, the hinge region in green, and the SET domain in blue. (F) SDS-PAGE analysis of in vitro H3 ubiquitination by reconstituted CLRC complex. Ubiquitination reactions were performed using recombinant human E1, E2, HA–ubiquitin (Ub), and the N-terminal peptide of histone H3 (residues 1– 20), in the presence of reconstituted CLRC complexes with or without Clr4. Both CLRC and CLRC -Clr4 efficiently ubiquitinated the H3 peptide, whereas E1 and E2 alone showed no activity. *, MBP-Raf1 degradation products; HO, histone octamer.

Article Snippet: A 5× ubiquitination mix was prepared in reaction buffer [50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 0.5 mM DTT] and contained 500 nM recombinant human E1 (UBE1), 1 μM recombinant E2 (UbcH5c/UBE2D3), 20 mM ATP, and 50 μM recombinant human HA-Ubiquitin (all from R&D Systems, Bio-Techne) together with 100 μM S-adenosylhomocysteine (SAH).

Techniques: Recombinant, Plasmid Preparation, Expressing, SDS Page, Staining, Size-exclusion Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Construct, In Vitro, Ubiquitin Proteomics, Activity Assay