Mapping sites of deposition-related acetylation in newly synthesized H3 and H4 during chromatin assembly and maturation in vitro and in vivo

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




C. David Allis


Drosophila melanogaster, Tetrahymena, Acetylation, Chromatin

Subject Categories

Biochemistry | Biology


The amino termini of core histones H2A, H2B, H3 and H4 contain conserved lysine residues which are transiently modified by the addition and removal of acetyl groups. Non-random acetylation of these lysines in histone H4 is associated with histone deposition (Chicoine, et al. 1986), dosage compensation in Drosophila (Turner, et al. 1992) and mating efficiency in Saccharomyces (Park and Szostak 1990, Megee, et al. 1990). In most eukaryotes, H4 is "blocked" at its amino terminus preventing direct analysis of acetylation sites by microsequencing. Application of a new deblocking procedure (Wellner, et al. 1990) allowed me to examine the deposition-related sites of acetylation of new H3 and H4 during chromatin assembly and maturation in a wide range of eukaryotes.

A crude cytosolic histone acetyltransferase (HAT-type B) from Drosophila embryos was used to in vitro label H4 purified from either Tetrahymena or Drosophila. Deblocking and microsequencing showed that acetylation occurred almost exclusively on lysine 11/12 of both substrates. Similar results have been obtained with homogeneous HATB from yeast (Parthun, et al. 1996). In agreement with results previously obtained in Tetrahymena H4 (Chicoine, et al. 1986), newly synthesized and deposited H4 from Drosophila and HeLa cells was diacetylated exclusively at lysines 5 and 12, demonstrating conservation of this modification. A novel, simple method to pulse-label, extract and purify newly synthesized yeast histones in vivo showed that in contrast to the organisms above, new yeast H4 was deposited primarily as a monoacetylated form.

New H3 from all four organisms failed to show conservation of deposition-related acetylation sites. Tetrahymena H3 was deposited as mono- and diacetylated forms acetylated predominantly at lysine 9 and lysines 9 and 14. New Drosophila H3 was mono- and diacetylated with lysine 23 and lysines 23 and 14 being the preferred sites, respectively. New yeast H3 was also deposited as acetylated forms with lysines 9 and 27 being the major sites of acetylation. However, in HeLa no acetylation of deposition-related H3 is observed.

Deacetylation of H4 during chromatin maturation was also examined.