Fig. 3
Monoubiquitination of cysteines in Suv39h1. a Suv39h1 from 293F cells transfected with SirT6-HA affinity-purified with myc resin and analyzed by mass spectrometry rendered peptides of Suv39h1 and ubiquitin. b MS/MS spectra corresponding to ubiquitinated peptides detected. Left panel: ubiquitinated peptide K.AGQPIYECNSR.C precursor m/z 676.3091 containing the Gly–Gly modification in cysteine 222. Right panel: ubiquitinated peptide R.CCCGYDCPNR.V precursor m/z 738,254 containing the Gly–Gly modification in cysteines 226 and 228. Corresponding MS/MS spectra of the non-ubiquitinated precursors and tables are shown in Figure 2. Additional data are shown in Supplementary Data Set 1. c Schematic representation of the monoubiquitination identified in Suv39h1. Primary sequence of the PRE-SET domain is indicated. The nine conserved cysteines that define the PRE-SET domain are shown underlined. d Western blotting of Myc-Suv39h1-containing extracts incubated in the presence or absence of β-mercaptoethanol at pH11 (see Online Methods). e Schematic representations of Suv39h1 mutants 3C and 8C in the PRE-SET domain used in f–h. The vertical bars correspond to the nine conserved cysteines (in red). The cysteines mutated to alanine are shown as blue bars. f Global H3K9me3 levels in Suv39h1/2 double KO MEFs (Suv39h KO) cells rescued with empty vector, or equivalent levels of Myc-Suv39h1 full-length (FL), 3C, or 8C (lanes 2–5, respectively). WT MEFs were included as positive control (lane 1). Total histone H3 is used as control. g Immunofluorescence analysis of DAPI, H3K9me3, and Myc in the same rescue experiment performed in f. A representative 5 μm scale bar is included in DAPI WT image. h Quantitative RT-PCR analysis (n = 5) of the mRNA levels of minor and major satellites and LINE L1 in the same cells used in (f) (T-test; SEM, *p < 0.05, **p < 0.01)
