Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo

Abstract

The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) Elp3 as a subunit. Here we show that, in contrast to the isolated Elp3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits--Elp4, Elp5, and Elp6--are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.

Figure 1

Holo-Elongator is a nucleosomal histone H3 and H4 acetyltransferase. (A Left) HAT activity by using core histones or intact nucleosomes as substrates measured by scintillation counting. One-microgram substrate was incubated in the absence (open bars) or presence (filled bars) of holo-Elongator. (Right) Silver stain of the holo-Elongator fraction used in all assays. (B Left) HAT reactions containing increasing amounts of holo-Elongator by using core histones (5 μg) as substrates. (Upper) Fluorogram; (Lower) Coomassie-stained protein gel. (Right) HAT reactions using reconstituted recombinant nucleosomes (1 μg) as substrate. (Upper) Fluorogram of SDS/PAGE analysis; (Lower) Coomassie-stained protein gel. Indicated are the individual core histones. Note that reactions in the presence of core histones or nucleosomes were carried out in independent experiments by using different amounts of substrate, and different exposure times. As indicated by Fig. 1A, core histones and nucleosomes seem to work equally well as substrates. (C) HAT activity using synthetic peptides mimicking the N-terminal histone tails as substrates as measured by scintillation counting. Approximately 5 μg peptide substrate was incubated in the absence (open bars) or presence (filled bars) of holo-Elongator.

Figure 2

Lysine-14 of histone H3 and lysine-8 of histone H4 are the predominant acetylation sites by holo-Elongator. Synthetic peptides corresponding to the N-terminal tail of histones H3 (A) and H4 (B), respectively, were acetylated by holo-Elongator in the presence of [³H]acetyl-CoA. The peptides were subsequently subjected to automated N-terminal cycle sequencing and the radioactivity (³H) released in each cycle was determined by scintillation counting (28).

Figure 3

Elp4, Elp5, and Elp6 are required for HAT activity. (A Upper) Silver stained protein gel of the MonoQ column fractions indicated (19). (Lower) Immunoblot analyses of MonoQ column fractions using anti-Elp1, anti-Elp3, and anti-Elp6 antibodies, respectively (19). (B) HAT activity of MonoQ column fractions by using core histones (5 μg) as substrates measured by scintillation counting. The MonoQ load (L, purified holo-Elongator), and the fractions used are indicated, alongside a control reaction (−) carried out in the absence of Elongator.

Figure 4

Holo-Elongator can interact with both naked and nucleosomal DNA. (A Left) Gel-shift experiment by using a naked DNA probe. (Right) Gel-shift by using a reconstituted nucleosomal probe. Increasing amounts of holo-Elongator were used for binding to identical amounts of labeled naked and nucleosomal DNA. Asterisks indicate mobility shifts of the nucleosomal probe. (B) Antibody supershift experiment by using naked (Left) and nucleosomal DNA (Right). Increasing amounts of anti-HA antibody or a control monoclonal antibody were present during binding. The positions of the free naked DNA probe and of the unbound, labeled nucleosome probe are indicated. An asterisk indicates mobility shift of the nucleosomal probe.

Figure 5

Histone H3 and H4 are hypoacetylated in elp3Δ cells. (A) Chromatin immunoprecipitation analyses of the relative level of multiply acetylated histone H3 in 22 different chromosomal regions using antibodies specific for diacetylated histone H3. (B) Chromatin immunoprecipitation analyses of the relative level of multiply acetylated histone H4 in 20 different chromosomal regions, using antibodies specific for tetraacetylated histone H4. One thirtieth of immunoprecipitated or 1/20,000 of input DNA was used for quantitative PCR. Ratios of immunoprecipitated/input DNA were calculated, sorted in groups according to their relative value, and presented together in histograms. The x axis represents the value groups and the y axis the number of genomic regions in each group. For example, only two genomic regions had a level of multiply acetylated histone H3 that had a relative value between 0.5 and 1 in wild-type cells. By comparison, 9 regions had this characteristic in elp3 cells, and 10 regions had this characteristic in gcn5 cells (compare second bar in each of the histograms of A).