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. 2021 Nov 8;10:e67952. doi: 10.7554/eLife.67952

Figure 1. Sumoylation inhibits p300-mediated H4 acetylation in octamer and mononucleosome substrates.

(A) Synthetic scheme for H4K12su. (i) An H4(1–14)K12aux peptide was ligated with a SUMO-3 (2–91) C47S α-thioester. (ii) The sumoylated H4(1–14) peptidyl hydrazide containing the auxiliary was converted to a C-terminal α-thioester and ligated with H4(15–102) A15C. The auxiliary was then reductively cleaved from the ligation product. Cys15 in the final ligation product was desulfurized to the native Ala15 to yield site-specifically sumoylated H4K12su. (B) C4 analytical RP-HPLC trace of purified H4K12su (top). ESI-MS of purified H4K12su (bottom). Calculated mass 21,596.7 Da. Observed, 21,594.2 ± 3.4 Da. (C) Coomassie-stained 15% SDS-PAGE of reconstituted octamers containing wild-type (wt) H4 or H4K12su. (D) Ethidium bromide stained 5% TBE gel of mononucleosomes containing wt H4 or H4K12su. (E) Western blots of p300 assay products with octamer substrates containing wt H4 or H4K12su, probed with a site-independent pan-acetyllysine antibody (top) and an H4K16ac-specific antibody (bottom). An asterisk indicates assays with heat-inactivated p300 to exclude non-enzymatic acetylation. (F) Fluorogram of p300 assay products with [3H]-acetyl-CoA as the co-factor and mononucleosome substrates containing wt H4 or H4K12su.

Figure 1—source data 1. Unedited intact SDS-PAGE gels for all gel images shown in Figure 1.

Figure 1.

Figure 1—figure supplement 1. Histone octamer and mononucleosome acetylation by p300.

Figure 1—figure supplement 1.

(A) Scheme outlining the p300 histone acetyltransferase assay with octamer and mononucleosome substrates. (B) Cartoon representation of SUMO-3 showing all surface-exposed lysine residues in stick representation. The dashed line represents N-terminal residues not observed in the structure. PDB code 1U4A. (C) Coomassie-stained SDS-PAGE of purified catalytic domain of SENP2, cat.SENP2, consisting of residues 365–590. (D) Coomassie-stained SDS-PAGE of purified p300-FLAG from HEK293T cells. (E) Coomassie-stained SDS-PAGE corresponding to the HAT assay shown in Figure 1E. Asterisk indicates heat-inactivated p300 was used. (F) Histone acetylation assay with octamers containing wild-type (wt) H4 or H4K12su. Autoacetylation of p300 was observed with a pan-acetyllysine antibody. (G) Histone acetylation assay with p300 and wt H4 containing octamers and mononucleosomes. No cat.SENP2 was used in this assay. The asterisk indicates pre-incubation of p300-FLAG with acetyl-CoA for 1 hr to allow for the build-up of activating p300-autoacetylation prior to the addition of mononucleosome substrate (Thompson et al., 2004) (H) Histone acetylation assay with wt H4 octamer with or without equimolar amounts of 147 bp Widom 601 double-stranded DNA (dsDNA).
Figure 1—figure supplement 1—source data 1. Unedited intact SDS-PAGE gels and western blot membranes for all gel and western blot images shown in Figure 1—figure supplement 1.