Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila

Abstract

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.

FIG. 1.

Affinity purification of dGcn5-containing complexes. (A) Thirty micrograms of nuclear extracts from WT cells (lane 1) or cells that stably express TAP-tagged dGcn5 (TAP-dGcn5, lane 2) were analyzed by Western blotting using an antibody that recognizes the protein A moiety of the TAP tag (α TAP). The migration of the molecular weight standards is shown to the left. (B) Silver stain gel showing the purified material from WT (lane 1) and TAP-dGcn5 extracts (lane 2). The migration of the tagged protein is shown with an arrow (CBP-dGcn5). (C) The affinity-purified material from the TAP-dGcn5 cells (lane 4) and the mock purification (WT, lane 3) were analyzed by Western blotting using antibodies against dGcn5 (α dGcn5) and dAda3 (α dAda3). The inputs correspond to 30 μg of nuclear extract from wild type (lane 1) or tagged cells (lane 2). The migration of TAP-dGcn5 and endogenous dGcn5 are indicated with arrows.

FIG. 2.

MudPIT analysis identifies peptides for dHCF and the uncharacterized protein cg9200 (Atac1). (A) Results from MudPIT, showing the number of nonredundant spectra for each protein (total peptides) and the amino acid sequence coverage (% coverage). (B) Primary structure of Atac1 gene product. The peptides identified for this protein are shown in boldface type. The sequence underlined indicates the SANT domain.

FIG. 3.

Tagging of Atac1, dHCF, and dAda2A confirms their association. (A) S2 cells were transfected with pRmHa3-dAda3-FL₂, pRmHa3-Atac1-HA₂FL₂, and pACXT-T7-dHCF-FLAG plasmids. After a 1-day induction, whole-cell extracts were prepared and incubated with M2-agarose beads. Untransfected S2 cells (WT) were used as a negative control. The immunoprecipitated material was analyzed by Western blotting using antibodies against dGcn5 (α dGcn5, rabbit), dAda2A (α dAda2A, rat), and dSpt3 (α dSpt3, rabbit). Lanes 1 to 4 correspond to 40 μg of whole-cell extract (2% input). Lanes 5 to 8 correspond to the immunoprecipitated material (IP). (B) Protein complexes from a stable line expressing Atac1-HA₂FL₂ were affinity purified using anti-FLAG-agarose beads. dAda2A-containing complexes were purified from dAda2A-TAP-expressing cells, according to the TAP protocol. These complexes were analyzed by MudPIT. The number of nonredundant spectra for each protein (total peptides) and the amino acid sequence coverage (% coverage) are shown.

FIG. 4.

Polyclonal antibodies against Atac1 and dHCF coimmunoprecipitate HAT activity and components of the ATAC complex. (A) Thirty micrograms of nuclear extracts from S2 cells were used in Western blots to determine the specificity of the antibodies against Atac1 (α Atac1) and dHCF (α dHCF). The primary antibody used is shown at the top of each blot. (B) One milligram of whole-cell extract from wild-type cells or cells expressing T7-dHCF-FLAG was immunoprecipitated with M2-agarose beads. The immunoprecipitated (IP) material was analyzed by Western blotting using antibodies against dHCF (α dHCF, rabbit and dHCF rat) and FLAG (α FLAG). Lanes 1, 3, and 5 correspond to 40, 40, and 20% FLAG beads from wild-type cells, respectively. Lanes 2, 4, and 6 correspond to 40, 40, and 20% FLAG beads from T7-dHCF-FLAG cells, respectively. (C and D) One milligram of nuclear extract was immunoprecipitated with rabbit antibodies against dAda3, Atac1, dHCF, or a preimmune bleed (pre). Immunoprecipitation reactions were subjected to HAT assays using radiolabeled acetyl coenzyme A and core histones (C) or nucleosomes (D) as substrates. The top panels show the protein gels stained with Coomassie blue to show total histone substrate. The bottom panels show fluorographies of the same gels to show the acetylated histones. The migrations of H3 and H4 are indicated. (E) Antibodies against dAda3, dHCF, Atac1, dSpt3, and a rabbit preimmune bleed (pre) were incubated with 1 mg of nuclear extract from S2 cells for immunoprecipitation reactions. The immunoprecipitated material was analyzed by Western blotting using antibodies against dGcn5 (α dGcn5, rabbit), dAda2A (α dAda2A, rat), dHCF (α dHCF, rat), Atac1 (α Atac1, rat), and dAda2B (α dAda2B, guinea pig). Lane 1 corresponds to 2% of the input.

FIG. 5.

dHCF, Atac1, and dAda2A are components of a 700-kDa complex. (A) Nuclear extract was incubated with Ni-NTA-agarose, eluted with imidazole, and applied to a Mono Q anion-exchange column. The bound proteins were resolved with a linear gradient of 100 to 500 mM NaCl, and odd fractions were analyzed by Western blotting, using antibodies against dAda2B (α dAda2B, rat), dGcn5 (α dGcn5, rabbit), dAda2A (α dAda2A, rabbit), dHCF (α dHCF, rabbit), and Atac1 (α Atac1, rabbit). I, 10 μl of imidazole eluate (input); FT, 10 μl of Mono Q flowthrough. (B) Fractions 29 to 33 were pooled, concentrated, and loaded onto a Superose 6 gel filtration column. Molecular mass standards were run under the same conditions, and their migration is indicated with arrows. I, 10 μl of pooled MonoQ fractions (input).

FIG. 6.

dHCF colocalizes with Atac1 and dAda3 on polytene chromosomes. (A) Polytene chromosomes were stained with antibodies against dAda3 (α dAda3, rabbit, green) and dHCF (α dHCF, rat, red). The green- and red-stained images were overlapped to generate the merge panel (yellow). (B) Magnification of the boxed region from panel A. Green and red arrowheads indicate chromosome sites that are bound exclusively by dAda3 or dHCF, respectively. Yellow arrowheads point to chromosomal loci occupied by dAda3 and dHCF (C) Polytene chromosomes were stained with antibodies against Atac1 (α Atac1, rabbit, green) and dHCF (α dHCF, rat, red). (D) Magnification of the boxed regions from panel C. The arrowheads point to chromosome sites that are bound by Atac1 (green), dHCF (red), or both (yellow).