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. 2020 Dec 29;22(2):e50967. doi: 10.15252/embr.202050967

Figure 2. HAT1 is a novel histone succinyltransferase.

Figure 2

  1. Histones were extracted from HepG2 (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells. The total histone succinylation and acetylation levels were analyzed by Western blot analysis. Total histone levels were measured by GelCode Blue staining. The efficiency of HAT1 knockout was measured by Western blot analysis.
  2. Histone H3 was immunoprecipitated from HepG2 cells with or without endogenous HAT1 depletion (by knockout). The levels of succinylation, acetylation, histone H3, HAT1, and β‐actin were analyzed by Western blot analysis in the cells.
  3. HAT1‐catalyzed histone H3 succinylation was determined by mixing purified HAT1, histone H3, and succinyl‐CoA in the in vitro succinylation assays. Heat‐inactivated HAT1 was used as a negative control. Western blot analysis was performed with the indicated antibodies.
  4. HAT1‐catalyzed histone H3 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl‐CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.
  5. HAT1‐mediated histone H3 succinylation was assessed by mixing purified HAT1, histone H3, and succinyl‐CoA (2 μM) with or without acetyl‐CoA (2 μM). Western blot analysis was performed with the indicated antibodies.
  6. Diagram illustrating the catalytic pocket of HAT1 bound to succinyl‐CoA. Structures of HAT1 (PDB ID 2P0W) and succinyl‐CoA (PDB ID 5TRL) were used for the modeling by Discovery Studio.
  7. Histone H3 was immunoprecipitated from HAT1 KO HepG2 cells that were either reconstituted with wild‐type HAT1 or mutant HAT1 (T188A). The levels of succinylation, acetylation, histone H3, HAT1, and β‐actin were tested by Western blot analysis in these cells.