Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

Abstract

Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gcn5 subcomplex of SAGA which acetylates histones H3 and H2B on nucleosomes, and the Piccolo NuA4 complex which acetylates histones H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify Escherichia coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes.

Figure 1

The Ada2/Ada3/Gcn5 and Piccolo NuA4 complexes are functionally sufficient for the nucleosomal HAT activity of the megadalton SAGA and NuA4 complexes. Gcn5 is the catalytic HAT subunit of SAGA, but acetylates only histone tails and weakly at that, whereas the Ada2/Ada3/Gcn5 is the sufficient subcomplex with similar robust HAT activity and histone H3 and H2B specificity for nucleosomal histones as the full megadalton SAGA complex. Similarly, Esa1 is the catalytic subunit of the NuA4 complex with weak activity on free and nucleosomal histones, but the Piccolo NuA4 subcomplex comprised of the Epl1, Yng2 and Esa1 subunits acetylates nucleosomal histones with the same preference for histones H4 and H2A as the full NuA4 complex.

Figure 2

Use of the BL21-CodonPlu host strain improves Talon metal affinity purification of the recombinant yeast Ada2/Ada3/Gcn5 complex compared to BL21 cells. BL21(DE3)pLysS and BL21-CodonPlus(DE3)-RIL cells containing the pST44-yAda3Δ2HIS-yAda2Δ1-Gcn5 expression plasmid were induced at 18°C overnight, harvested by centrifugation, resuspended in buffer, Lysol by sonication and purified using Talon metal affinity resin with imidazole elution. Lanes 1 and 2 represent equivalent volume samples eluted from the Talon resin for BL21(DE3)pLysS and BL21-CodonPlus(DE3)pLysS cells respectively. The contaminating bands in lane 1 do not appear to result from prematurely truncated Ada3, Ada2 or Gcn5 polypeptides since anti-Ada3, anti-Ada2 or anti-Gcn5 antibodies do not recognize these bands in Western blots (data not shown). The 43 kDa contaminating band just below the truncated Gcn5 band was identified by mass spectrometry as the E. coli EF-Tu polypeptide.

Figure 3

Purification of recombinant yeast Ada2/Ada3/Gcn5 complex expressed in E. coli. The whole cell extract of BL21-CodonPlus(DE3)-RIL cells with the pST44-yAda3Δ2HIS-yAda2-Gcn5 plasmid before induction with IPTG is shown in lane 1. Lane 2 shows the crude soluble extract of the same cells after IPTG induction while the pooled fractions after Talon metal affinity, Source Q anion-exchange and Source S cation-exchange chromatography are shown in lanes 3, 4 and 5 respectively. The major contaminant after Source Q and S chromatography is a 43 kDa contaminant identified by mass spectrometry as the E. coli translation elongation factor EF-Tu. Molecular weight markers are shown in lane 6.

Figure 4

Purification of the recombinant yeast Piccolo NuA4 complex of Epl1/Yng2/Esa1 expressed in E. coli. The whole cell extract of BL21(DE3)pLysS cells containing the pST44-HISyEsa1-yEpl1Δ3-Yng2Δ1 plasmid before IPTG induction is shown in lane 1, while the soluble extract after IPTG induction is shown in lane 2. The pooled fractions after Talon metal affinity, Source Q anion-exchange, Source S cation-exchange and Source ISO hydrophobic interaction chromatography are shown in lanes 3, 4, 5 and 6 respectively. The 43 kDa contaminant removed by hydrophobic interaction chromatography is presumed to be EF-Tu.