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Molecular and Cellular Biology, January 2008, p. 376-385, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01307-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcriptional Adaptor ADA3 of Drosophila melanogaster Is Required for Histone Modification, Position Effect Variegation, and Transcription{triangledown} ,{dagger}

Benjamin Grau,1,{ddagger} Cristina Popescu,2,{ddagger} Laura Torroja,1,# Daniel Ortuño-Sahagún,1,§ Imre Boros,2,3 and Alberto Ferrús1*

Department of Cellular, Molecular, and Developmental Neurobiology, Cajal Institute, C.S.I.C., Ave. Dr. Arce 37, Madrid 28002, Spain,1 Institute of Biochemistry, Biological Research Center, Temesvári krt. 62, H-6726 Szeged, Hungary,2 Department of Biochemistry HAS-USZ Chromatin Structure and Gene Expression Research Group, University of Szeged, Közép Fasor 52, H-6726 Szeged, Hungary3

Received 20 July 2007/ Returned for modification 25 September 2007/ Accepted 17 October 2007


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ABSTRACT
 
The Drosophila melanogaster gene diskette (also known as dik or dAda3) encodes a protein 29% identical to human ADA3, a subunit of GCN5-containing histone acetyltransferase (HAT) complexes. The fly dADA3 is a major contributor to oogenesis, and it is also required for somatic cell viability. dADA3 localizes to chromosomes, and it is significantly reduced in dGcn5 and dAda2a, but not in dAda2b, mutant backgrounds. In dAda3 mutants, acetylation at histone H3 K9 and K14, but not K18, and at histone H4 K12, but not K5, K8, and K16, is significantly reduced. Also, phosphorylation at H3 S10 is reduced in dAda3 and dGcn5 mutants. Variegation for white (wm4) and scute (Hwv) genes, caused by rearrangements of X chromosome heterochromatin, is modified in a dAda3+ gene-dosage-dependent manner. The effect is not observed with rearrangements involving Y heterochromatin (bwD), euchromatin (Scutoid), or transvection effects on chromosomal pairing (white and zeste interaction). Activity of scute gene enhancers, targets for Iroquoi transcription factors, is abolished in dAda3 mutants. Also, Iroquoi-associated phenotypes are sensitive to dAda3+ gene dosage. We conclude that dADA3 plays a role in HAT complexes which acetylate H3 and H4 at specific residues. In turn, this acetylation results in chromatin structure effects of certain rearrangements and transcription of specific genes.


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INTRODUCTION
 
Chromatin structure is modulated by proteins that alter the packaging of DNA, thus changing the accessibility of factors involved in transcription, replication, repair, and other DNA-dependent processes (1, 6, 33, 44, 60, 65). One of the most effective means of changing chromatin structure is the posttranslational modification of amino-terminal extensions of nucleosomal core histones, which are nearly invariant among eukaryotes. Histone N-terminal-tail lysine modifications neutralize positive charges and lower the affinity between histone octamers and DNA, thus relaxing chromatin. Furthermore, acetylation and other histone modifications serve as recognition signals for proteins acting on DNA, including transcription regulators. Indeed, many histone acetylase and deacetylase enzymes are transcriptional regulators (61). In the Saccharomyces cerevisiae (yeast) SAGA (SPT-ADA-GCN5-acetyltransferase) complex, the histone acetyltransferase (HAT) activity is carried out by GCN5, while the interaction with activators is mediated by different adaptors (ADA and SPT proteins) (12, 32), transcription-associated proteins (e.g., TRA1) (10, 35), and TATA-binding-protein-associated factors (TAFIIs) (62).

In yeast and higher eukaryotes, several GCN5-containing HAT complexes also share ADA2- and ADA3-type adaptors (1, 38, 45). Two Drosophila melanogaster HAT complexes, SAGA and ATAC, exhibit different specificities in the targeted histone lysines, and these complexes differ in the adaptor type, ADA2a or ADA2b, present (15, 30). A further ADA-type adaptor, ADA3, is present in both SAGA and ATAC complexes (29, 37); its role in vivo, however, so far has not been addressed. ADA3 was first isolated as a suppressor of GAL4-VP16-induced toxicity in yeast (53). Yeast ADA3 mutants grow slowly in minimal medium, reduce the efficiency of some transcriptional activation domains, and alter the selection of initiation sites in basal transcription (53). Based on these observations, ADA3 was proposed to act as a coactivator bridging acidic activation domains with the basic transcriptional machinery. A human ADA3-like protein was isolated from the PCAF complex, a HAT complex related to yeast SAGA (48), but little is known about its precise function in transcriptional regulation. In addition to being modified by acetylation, histones can be modified by phosphorylation. H3, in particular, exhibits acetylation of lysine 9 and phosphorylation of serine 10 (47). Several factors playing a role in H3 S10 phosphorylation have been identified, but the relationship between complexes depositing these two adjacent histone marks is not known (38).

In Drosophila melanogaster and other species, it is well documented that gene transcription can be affected by chromatin structure changes in the vicinity of the initiation site, a phenomenon referred to as position effect variegation (PEV). Defective histone deacetylation in RPD3 mutants enhances PEV (17), and a lack of phosphorylation by JIL-1 kinase on H3 affects PEV (34). SAGA and TFIID share TAFII components, illustrating the coupling between the alteration of chromatin structure and transcription initiation (28, 46). Selective gene expression, however, requires that, in addition to functional coupling, chromatin remodeling and transcription initiation complexes act on selective genome sites and tissues and during developmental times. Hence, there is a need to identify the elements involved in the site specificity of HAT complex activity and to demonstrate their role in vivo.


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MATERIALS AND METHODS
 
Genetic materials. Lethal mutations dAda31, dAda32, and dAda33 have been referred to previously as l(1)114, l(1)7688, and l(1)17053, respectively (20). These alleles were obtained from independent mutageneses and induced on y-, w-, or f5 os-marked parental chromosomes. The first two types of chromosomes were further recombined into a y w-marked chromosome. Additional mutant alleles {Delta}6 and {Delta}9 were kindly provided by Pilar Carrera (IGBMC, Strasbourg, France). These were generated by imprecise excision of P{Mae-UAS.6.11}CG7536GG01344, which is located 5' in dik (dAda3) coding sequence. Both deletions remove the 5' end of dik (dAda3) and parts of the second exon of the gene CG7536 within which dik (dAda3) is nested. The IRO-C rearrangements and the AS-C enhancer constructs are from J. Modolell (Center for Molecular Biology, Madrid, Spain). AS-C enhancers correspond to the following genomic fragments from the achaete-scute region. sc1.1 extends 1.1 kb and includes the scute start site (42); (1.2/0.0) 3.7 sc extends 3.7 kb from the same site near the scute promoter (11), and we refer to it as 3.7 sc for brevity; and, finally, AS 1.4DC18 corresponds to 1.4 kb from the scute start site, but it contains a heat shock basal promoter (21). All these constructs carried the LacZ gene as the expression reporter. Alleles dAda2a189 and dAda2b842 have been described previously (49). The upstream activation sequence-dAda3+ construct was cloned in the pUAST vector and injected into y w embryos to obtain transgenic lines. Primer sequences used for cDNA cloning are available upon request. For genomic rescue experiments, the EcoRI fragment 422E4 (3) was inserted into the germ line transformation vector pCasper (54). The proximal XbaI fragment of 422E4, which contains an additional transcription unit, HL-IV, was deleted by partial restriction digestion. The resulting construct, pCasper422E4RX, was used for P element-mediated germ line transformation. We refer to this construct as E4RX for brevity. Other deficiencies, mutants, and chromosomal rearrangements were from the Bloomington Stock Center (Fly Base [http://flybase.bio.indiana.edu]).

Mosaic and phenotype analyses. Twin clones were scored on the cuticle of adult y w dik*os/f5 os females X-ray irradiated at 0 to 48 h after egg laying. A twin clone is composed of two adjacent patches generated in the same event of recombination, one marked with forked and the other with yellow, wherein the yellow clone is homozygous for the lethal mutation. The generation of germ line clones was elicited by X-ray irradiation of first-instar ovoKS1237 v/y w dAda3* larvae as described previously (20). To determine the lethality phase, dAda3*/FM6 females were crossed with wild-type Canton S males. Fertilized females were allowed to lay eggs for a 20-h period at 25°C. Groups of 20 to 30 eggs were examined at 1-day intervals; the surviving individuals were counted. The imaginal disk size was evaluated on dissected material from late-third-instar larvae. The extent of variegation was evaluated by comparing sibling individuals from crosses of males with genotype y w dAda3*; E4RX/TM3 against females with genotype In(1)wm4/FM6 and females with genotype y w dAda3*/FM6; Ki Sb Dp(1;3)JC153/TM2 against males with genotype In(1)wm4. An additional control reference was obtained from the cross, In(1)wm4/FM6 females against y w; E4RX/TM3 males. Variegation for the hairy wing (Hwv) phenotype was studied in the offspring from the cross: In(1)sc8/In(1)sc8 females against y w dAda3*; E4RX/TM3 males. The number of pleural bristles was counted in In(1)sc8/y w dAda3*; TM3/+ females and the sibling controls with the genotype In(1)sc8/y w dAda3*; E4RX/+. These values were subjected to Student's t test for statistical significance. All variegation studies were done on cultures raised at 25°C.

Gene expression studies. For Northern blotting, poly(A)+ mRNA was isolated using a QuickPrep Micro mRNA purification kit (Pharmacia Biotech), and the probe was [{gamma}-32P]dATP radiolabeled by the random priming method (19). In situ expression was analyzed by hybridization of a cDNA probe. The corresponding cRNA was labeled with digoxigenin and hybridized to 12-µm paraffin-embedded sections by following standard procedures. In vivo experiments on directed gene expression were done using the upstream activation sequence-Gal4 system (50, 51). For a source of the Iroquois gene complex component araucan, we used the UASara29 construct (18) under Gal4-AC216 and Gal4-MD751 enhancer-trap drivers, which correspond to the spalt and u-shaped (ush) genes, respectively (M. Calleja, personal communication).

Anti-dADA3 antibodies, bacterial expression, and Western blots. The dADA3-specific polyclonal serum was generated against the peptide DSLDKDEKRQDRRK, which corresponds to amino acids 126 to 139 in the dADA3 sequence. Peptide synthesis and immunization of rabbits were performed by Sigma Co. The antibodies were purified by affinity chromatography on peptide-bound Sepharose 4 Fast Flow matrix (Amersham Bioscience) according to the manufacturer's instructions. For protein expression in bacteria, dAda3 cDNA was generated by reverse transcription-PCR using nucleotide primers based on expressed sequence tag sequences. The fragment was inserted into pGEX-4T-3 expression vector (GE Healthcare Life Sciences) for transformation. Protein expression was induced by IPTG (isopropyl-β-D-1-thiogalactopyranoside; 1 mM) and analyzed on polyacrylamide gel electrophoresis. Nuclear extracts of third-instar larvae were prepared as described elsewhere (25). For Western blotting, protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membrane, and incubated with affinity-purified primary antibodies. Peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) and rabbit anti-mouse (Sigma-Aldrich) specific antibody were used as secondary antibodies, and the blots were developed using Super Signal West Pico chemiluminescent substrate (Pierce).

Immunostaining of polytene chromosomes. Salivary gland polytene chromosomes were obtained from wandering third-instar larvae of dAda3, dAda2a, dAda2b, and dGcn5 mutants (52). Immunopurified anti-ADA3 polyclonal rabbit serum was used in 100-fold dilution. H3 acetyl (Ac)K14 (1:100)-, H3 AcK9 (1:100)-, H3 AcK18 (1:300)-, and H3 P-S10 (1:50)-specific antibodies (Upstate), H4 AcK5 (1:100) and H4 AcK12 (1:150) (ABCAM) antibodies, and H4 AcK16 (1:200) (Serotec) antibody were used in dilutions as indicated. Mouse anti-RNA polymerase II (Pol II; 7G5) antibody was a gift of L. Tora (IGBMC, Strasbourg, France). Secondary antibodies, AlexaFluor488-conjugated goat anti-mouse Ig, AlexaFluor555-conjugated goat anti-rabbit Ig, and AlexaFluor568-conjugated goat anti-rabbit Ig (Molecular Probes) were used in 500-fold dilutions. Images for the semiquantitative analysis of fluorescence signal were acquired under constant conditions by using a Leica TCS SP5 confocal microscope. Image analysis was performed using ImageJ (http://rsb.info.nih.gov/ij/). Mean fluorescence intensities of the different signals of the different chromosomes were calculated after thresholding the channels. Normalization of the data was avoided by applying ratios of the corresponding signal couples for the following statistics. Data were compared using Mann-Whitney U test.

Nucleotide sequence accession number. We reported the sequence of the dADA3-encoding gene to the NCBI database (GeneID accession number 32787).


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RESULTS
 
The gene dik encodes the fly homologue of ADA3. The presence of a putative homologue of the human and yeast ADA3 proteins, dADA3, was previously identified in Drosophila extracts (30, 37); the encoding gene, however, was not characterized. We found that this protein is the product of the gene diskette (dik) we identified previously (56) (see Fig. S1A and B and S2 in the supplemental material). For clarity, we refer to this gene as dAda3 and to its product as dADA3. Northern blot analysis indicates that dAda3 gives rise to two mRNAs of approximately 2.6 and 2.8 kb that fit well with the size of the corresponding cDNAs. Both mRNA forms are present at all developmental stages, although the smaller form appears in a smaller quantity, and the two forms have no significant differences throughout developmental stages (Fig. 1A). Maternal mRNA is deposited in the oocyte, and the corresponding signal is abundant in unfertilized eggs (Fig. 1A). In situ hybridization experiments show that the zygotic expression of the gene is ubiquitous. In third-instar larvae, the signal is most noticeable in imaginal disks, muscles, the gut, salivary glands, and the central nervous system. The nervous system exhibits a prominent mRNA signal (Fig. 1B). High levels of transcripts are also found in follicle and nurse cells of all cyst stages in adult ovaries (Fig. 1C). In adult testes, the expression of this gene is also abundant in spermatocytes (not shown). All these cell types are characterized by high transcriptional activity.


Figure 1
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  		FIG. 1. dAda3 gene expression. (A) Northern blots showing expression from unfertilized eggs (lane UE); 4- to 10-h embryos (lane E); first-, second-, and third-instar larvae (lanes LI-II and LIII); pupae (lane P); and adults (lane A). Two mRNAs of 2.8 and 2.6 kb are detected. Note the continuous expression throughout development and the intensity of the maternal component of the unfertilized eggs. (B) In situ hybridization of an antisense RNA probe to a sagittal section of a third-instar larva showing the brain (br) and ventral ganglion (vg). Note the hybridization signal (blue). Bar = 35 µm. D, dorsal; A, anterior. (C) Equivalent hybridization to a horizontal section of an adult female abdomen showing nurse cells (n) in cysts (cys) of various stages. This ovarian signal represents the maternal component to the unfertilized eggs. Bar = 80 µm.

The longest open reading frame of dAda3 encodes a 556-amino-acid protein with a predicted molecular mass of 60 kDa and sequence identities of 29% and 18% with the human and yeast ADA3 factors, respectively (see Fig. S2 in the supplemental material). Antibodies raised against a dADA3 peptide (see Materials and Methods) identified a protein band of the expected molecular mass in fly nuclear extracts (Fig. 2A). The specificity of the antibody was demonstrated by the identification of a green fluorescent protein (GFP)-tagged dADA3 expressed in bacteria (Fig. 2B). In both types of Western blots, the specificity of the identified band was demonstrated by the lack of signal when preimmune or peptide preadsorbed serum was used instead of the affinity-purified serum. Immunostaining with this antibody showed a specific signal associated with chromosomes (Fig. 2C). The localization of dADA3 was further determined in relation to that of RNA Pol II. The dADA3 signal appears in a banding pattern that coincides with RNA Pol II at most but not all loci. In relation to the DNA marker DAPI (4',6'-diamidino-2-phenylindole), the dADA3 signal seems to correspond to the chromosomal interbands (Fig. 2D; see Fig. S3 in the supplemental material).


Figure 2
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  		FIG. 2. Specificity of the anti-dADA3 antibody. (A) Western blot from nuclear extracts of wild-type adults stained with the affinity-purified serum (ap), the preimmune serum (pi), and the peptide-preabsorbed serum (pap). For a loading control, the Western blot was hybridized with an anti-actin antibody. (B) Western blot from bacterial (bact.) extracts expressing a GFP-tagged dADA3 construct coupled to glutathione S-transferase. The tagged product justifies the higher molecular mass (in kDa, listed to the left) of dADA3. Note that the anti-GFP and anti-dADA3 antibodies recognize the same protein. The Coomassie-stained gel is included below as a loading control. (C) The dADA3 immunosignal localizes on the polytene chromosomes, counterstained with DAPI to identify DNA, and provides the in situ evidence for the specificity of the antibody. (D) Polytene chromosomes hybridized with anti-dADA3 (red) and anti-RNA Pol II (green) antibodies and DAPI (blue) to mark DNA. The X chromosome is indicated. Note the colocalization of dADA3 and RNA Pol II at some sites (arrowheads) but not others (arrows) (see also Fig. S3 in the supplemental material).

dADA3 is required for cell viability and growth. A total of five alleles were used for the description of mutant phenotypes. The molecular nature of all alleles was determined, and it was found that they correspond to truncations or deletions (see Fig. S4 in the supplemental material). All alleles are lethal at the early pupal stage. The severities of phenotypes were similar in all allele combinations, indicating that all represent null conditions. The most noticeable mutant phenotype is the reduced imaginal disks in homozygous larvae, hence the name diskette (Fig. 3A and B). This phenotype could indicate a requirement for proliferation and/or cell survival. Staining of mutant wing disks with vital colorants failed to indicate increased rates of cell death (data not shown). To analyze the cellular effects of these mutations, we generated germ line and somatic mosaics. Table 1 shows that dAda3 mutant cells are not recovered in somatic mosaics, while twin control cells are recovered (Table 1; see also Materials and Methods). The requirement is also evident in the germ line, as no egg-producing ovarioles were recovered (Table 1), indicating that the protein is required in oogenesis. Taken together, these data indicate that depletion of dADA3 leads to severe proliferation defects that, in systems with a high mitotic rate, result in cell lethality. In homozygous-mutant individuals, the requirements during larval development can be covered by the maternal deposit, although the imaginal tissues begin to show abnormal phenotypes by the end of the third larval instar and organism lethality occurs at metamorphosis.


Figure 3
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  		FIG. 3. Mutant dAda3 phenotypes. (A) Normal wing disk from a third-instar male larva (genotype y w dAda31; E4RX/+). (B) Equivalent disk from a sibling y w dAda31 larva. Bar = 150 µm. Note the reduced size of the disk. The images in panels A and B were taken at the same magnification. Phase-contrast images of polytene chromosome from male wild-type (C) and dAda32 mutant (D) third-instar larvae. Note the aberrant banding pattern, particularly the X chromosome (arrowheads), in the mutant (see the text). Bar = 50 µm.


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  		TABLE 1. Mosaic analysis of dAda3 mutationsa

Another phenotypic trait consists in the aberrant structure of polytene chromosomes (Fig. 3C and D). The banding organization is distorted, particularly on the male X chromosome, where it has virtually disappeared (Fig. 3D). In order to determine whether dAda3 mutations affect the structure of the X chromosome specifically, we quantified the relative amounts of dADA3 in relation to RNA Pol II and DNA (DAPI) on the male and female polytene chromosomes (see Fig. S5 in the supplemental material). We found that the ratio of dADA3 to DNA on the single male chromosome is higher than those on the two female X chromosomes and also those on autosomes of both sexes (see Fig. S5A in the supplemental material). However, the ratio of dADA3 to RNA Pol II is not different among sexes or chromosomes (see Fig. S5B in the supplemental material). These data strongly suggest that the apparent specificity of the mutant male X chromosome is an indirect consequence of its higher level of transcriptional activity due to dosage compensation. Also, the greater amount of dADA3 on the male X chromosome is likely a function of its transcriptional activity.

dADA3 is involved in site-specific acetylation/phosphorylation of histones. Since dADA3 is a component of several HAT complexes (30, 37), we first searched for genetic interactions between dAda3 alleles and mutants affecting subunits of HAT complexes. dAda2a, dAda2b, and dGcn5 mutations impair the function of ATAC and SAGA complexes (38). Similar to dAda3 mutants, mutants of these HAT complex components show lethality phases around metamorphosis (see Fig. S6 in the supplemental material). dAda3 and dGcn5 null mutations are lethal at early pupal and late larval stages, respectively. In contrast, dAda3-dGcn5 double mutants die at L1 and L2 stages, earlier than either single mutant (see Fig. S6 in the supplemental material). For dAda2a-dAda3 and dAda2b-dAda3 double mutants, there was no evidence of genetic interaction because there was no shift of the lethal phase (not shown). Also, we noticed that the distorted polytene chromosome structure characteristic of dAda2a and dGcn5 mutants, but not of dAda2b mutants (49), is very similar to that observed here in dAda3 mutants (Fig. 3D). Furthermore, in dGcn5 and dAda2a mutants, the dADA3 immunosignal on chromosomes is greatly reduced (Fig. 4A, B, and D). In contrast, the signal in dAda2b mutants appears with intensity not different from that of the wild type (Fig. 4C). It should be noted that the dAda2b allele used is a transcriptional null (49), which rules out the possibility that the normal dADA3 signal on chromosomes could be due to residual dADA2b protein content. These qualitative data were confirmed with Western blots from larval nuclear extracts (Fig. 4E and F).


Figure 4
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  		FIG. 4. dADA3 is depleted in some HAT mutants. Polytene chromosomes stained with RNA Pol II (green) and dADA3 (red) antibodies in wild-type (A) and dAda2a (B), dAda2b (C), and Gcn5 (D) mutant backgrounds. Note the severe reduction of anti-dADA3 signal in Gcn5 and dAda2a mutants but not in dAda2b mutants. (E and F) Western blots from nuclear extracts of whole larvae from the same mutants stained with the anti-dADA3 antibody.

These observations prompted a further analysis of the possible effects of dADA3 in histone modifications. As ATAC and SAGA complexes target specific lysine residues at the N-terminal tails of nucleosomal histones H3 and H4, we asked if the acetylations elicited by these complexes are affected in dAda3 mutants. To that end, we used antibodies specific to individual acetylated lysines of H3 and H4 and Western blots. Acetylation of H3 K9, H3 K14, and H4 K12 residues was clearly affected, while acetylation of others was not. Specifically, acetylation of H3 K18, H4 K5, H4 K8, and H4 K16 was not affected in dAda3 mutants. For examples, see Fig. 5. Acetylation and phosphorylation of histone H3 have been shown to be functionally related to active transcription (47). Yeast GCN5-dependent acetylation occurs preferentially at a H3 tail that has been prephosphorylated at serine 10 (41) and forms of H3 with both modifications, acetylated K9 and phosphorylated S10, exist in vivo (14). In this context, we analyzed a possible role of dADA3 in H3 S10 phosphorylation. The corresponding qualitative and quantitative data demonstrate that dADA3 is also required for this type of H3 modification (Fig. 6). Since ADA3 is not known to have a kinase activity in any species, we reasoned that this activity should be carried out by a protein complex in which dADA3 is a component. We tested dGCN5 as a representative component of Drosophila SAGA and ATAC complexes, and the data show also a drastic reduction of the phosphorylated H3 S10 immunosignal on chromosomes and Western blots (Fig. 6). We concluded that dADA3 plays a key role in the modification of histones H3 and H4, including acetylation and phosphorylation of specific residues. Some, but not all, of these traits are in common with other members of HAT complexes. For instance, in dAda2a mutants, both H4 K5 acetylation and H4 K12 acetylation are reduced (15), while dAda2b is required for acetylation of K9 and K14 residues of histone H3 (49).


Figure 5
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  		FIG. 5. Histone acetylation phenotypes in dAda3 mutants. Polytene chromosomes immunostained with antibodies specific for individual acetylated lysine residues of H3 and H4 (red) as indicated on the left, and a RNA Pol II-specific antibody (green). Genotypes are indicated at the top. Each experiment is represented by four panels, including panels showing RNA Pol II staining as an internal control. The images from the wild type (+) and the dAda32 mutant were obtained with identical datum-recording settings. Note the severe reduction of H3 K9 acetylation (A and D) and the lack of effect on H4 K5 acetylation (B). (C and D) Also note the reduction of H4 K12 acetylation. (D) Western blots further demonstrate the histone acetylation reduction in dAda3 mutants.


Figure 6
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  		FIG. 6. H3 S10 phosphorylation in HAT mutant components. +, wild type. (A) Polytene chromosomes immunostained with antibodies specific for the phosphorylated form of H3 S10 (H3 P-S10; red) and the DNA marker DAPI (blue). The immunosignal is clearly reduced in dAda3 and dGcn5 mutants. (B) Western blot of dAda3 mutants confirming the previously described observation. All images were taken under the same laser and filter settings.

dADA3 is an enhancer of variegation of X chromosome rearrangements. The multiprotein complexes in which ADA3 is included are thought to mediate chromatin structure changes that, eventually, modify gene transcription. In this context, we first studied the in vivo effects of dAda3 mutant backgrounds on the gene inactivation caused by different chromosomal rearrangements, a phenomenon called PEV. In(1)wm4 causes the random inactivation of the gene white, due to the presence of X chromosome heterochromatin in the vicinity of the gene. We compared the extent of PEV in females heterozygous for this rearrangement and dAda3 alleles to that in sibling female controls. Adults from crosses kept at 25°C to prevent temperature effects on variegation were analyzed (22). For all dAda3 alleles tested, the mutant genotypes showed an increase of variegation in heterozygous In(1)wm4/dAda3* females (Fig. 7A and B). By contrast, variegation of this rearrangement is reduced in genotypes with additional copies of the normal dAda3+ gene (Fig. 7C). Another variegating rearrangement, In(1)sc8, yields a dominant hairy wing phenotype (Hwv) due to relocation of X heterochromatin adjacent to the scute locus (11). We measured this PEV effect in heterozygous females by counting the number of extra bristles in the pleura. As in the previous case, dAda3 alleles significantly increase Hw variegation (the mean ± standard deviation was 7.88 ± 0.6 bristles in In(1)sc8/dAda3* genotypes versus 5.08 ± 0.31 bristles in In(1)sc8/+ genotypes [n, 18 and 12, respectively; P < 0.001]) and dAda3+-containing duplications reduce or suppress it. Two other cases of chromatin-related effects on transcription, bwD (55, 58) and the interaction between wDZL and z1 (7, 8, 31), however, are not influenced by dAda3 mutant backgrounds (not shown). The PEV case of bwD originates from the localization of Y chromosome heterochromatin adjacent to the brown locus, while the white/zeste interaction is dependent on proper pairing of homologue chromatids. Finally, the euchromatic rearrangement that brings the B and C components of the no ocelli (noc) gene to the vicinity of the snail gene Tp(2,2) Scutoid (Sco) (43), also failed to modify its phenotype in dAda3 heterozygous backgrounds.


Figure 7
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  		FIG. 7. dAda3 mutant effects on PEV and gene expression. (A) Adult eye of a y w/In(1)wm4h female showing the characteristic white variegation. (B) Enhanced variegation in y w dAda31/In(1)wm4h females. (C) Reduced variegation in y w dAda31/In(1)wm4h; E4RX/E4RX females. The genomic E4RX fragment carries a copy of dAda3+. The same effect was detected with other alleles and dAda3+-containing duplications [i.e., Dp(1;3)JC153]. (D) Dorsal view of an adult thorax of the genotype Df(3L)iro-2/+ showing the normal pattern of bristles. Note the scutum (scu) and scutellum (sc) regions of the thorax. The anterior is at the top of the panel. (E) Similar view of a sibling dAda32/+; Df(3L)iro-2/+ adult. Note the absence of scutellum and several bristles from the remaining scutum in the thorax. (F) Normal wing disk from male third-instar larvae showing the expression of the LacZ reporter under the control of enhancer 1.1 from the AS-C gene complex. (G) Equivalent disk from a dAda32 AS-C1.1-LacZ male larva. Note the reduced disk size and the absence of LacZ expression. The same effect was detected with AS-C enhancers 1.2 and 3.8.

A further case of PEV is caused by the insertion of uncharacterized sequences within the promoter region of the yellow gene in the allele yC4 (24). This small rearrangement perturbs, but does not prevent, transcription. In heterozygotes over the nonfunctional allele, y1, the resulting phenotype is a yellow body color but normal-colored bristles. In double heterozygotes (yC4/y1 dAda3*), however, the majority of bristles become mutant and show a yellow color (not shown). By contrast, the insertion of the transposable element gypsy in the allele y2, which prevents the functional interaction between wing and body enhancers with the promoter (23), does not seem to be modified in y2/y1 dAda3* adults. Finally, no change of expression pattern was detected in dAda3 heterozygotes when four position specific enhancers that drive the white reporter expression to specific eye regions (dorsal half, posterior gradient, anterior gradient, and two eye poles) (64) were assayed (data not shown). All these observations indicate that dADA3 participates, in a dosage-dependent manner, in the functional conformation of chromatin in some, but not all, chromosomal rearrangements.

dADA3 regulates the expression of specific genes. While testing chromosomal deficiencies for possible phenotype interactions with dAda3 alleles, we detected a significant interaction with the Df(3L)iro2 (see Table S1 in the supplemental material). This rearrangement deletes the Iroquoi gene complex (IRO-C), composed by the homeoprotein-encoding genes araucan, caupolican, and mirror (27). Double heterozygotes for dAda3 and IRO-C [the genotype for females was dAda3/+; Df(3L)iro2/+ and that for males was dAda3; Df(3L)iro2/E4RX] exhibit, with differing penetrances and expressivities, IRO-C phenotypes (Fig. 7D and E). In a given cross, two-thirds of the adult offspring (n = 54) showed a recognizable IRO phenotype, and about half of these showed the phenotype illustrated in Fig. 7E. In a stock established for more than two years [genotype y w dAda32/FM6; Df(3L)iro2/TM3] the penetrance of IRO-C phenotypes seems stabilized at around 80% in heterozygous females. Penetrance and expressivity of this effect is temperature sensitive, since cultures raised at 27°C exhibit stronger phenotypes than those raised at 17°C. The effect was reproduced, although with somewhat lower expressivity, when the mutants with partial deletions of the IRO-C, Df(3L)iroDFM1 (with araucan and caupolican deleted) and Df(3L)iroDFM3 (with araucan, caupolican, and the promoter of mirror deleted) mutants, were assayed (n, 47 and 62, respectively). As reference controls, the described phenotypes were never observed among heterozygote deficiencies for either dAda3 or IRO-C. Since Df(3L)iro2 includes also the Gcn5 locus (59), it may be argued that this component of the SAGA and ATAC complexes may contribute, in addition to dADA3, to the observed enhancing of IRO-C phenotypes.

The IRO-C-encoded functions seem to be partially redundant because deletions, but not single gene mutations, yield mutant phenotypes (27). We took advantage of this redundancy to test if the observed phenomenon in dAda3/+; Df(3L)iro2/+ adults was due to a depletion of IRO-C expression or to some other function included in this deficiency (e.g., GCN5). To that end, we generated adults of genotype dAda3/+; Gal4-AC216/+; Df(3L)iro2/UAS-ara29 in which the expression of the araucan component of IRO-C is driven to the wing disk by Gal4-AC216, an insertion under the control of enhancers from the spalt gene. Here, IRO-C phenotypes were never observed (n = 235), demonstrating that the phenomenon results from the combined reduced expression of dAda3 and IRO-C. In the corresponding crosses, the sibling genotype dAda3/+; Df(3L)iro2/UAS-ara29 exhibit the regular IRO-C phenotypes (Fig. 7E). To further document the functional relationship between dAda3 and IRO-C genes, we assayed one of the IRO-C targets, the regulatory regions of the achaete-scute gene complex, AS-C (27). We tested the effects of dAda32 on the expression of constructs in which the LacZ reporter is under the control of several AS-C enhancers. Mutant wing disks (n = 36) were tested for the expression of enhancers sc 1.1, AS 1.4DC, and 3.7 sc. In all cases, the expression was abolished in the mutant disks, while it was maintained in their sibling controls (Fig. 7F,G). Only a small number of mutant disks (n = 6) showed a weak LacZ expression in some, but not all, of the normal wing disk expression domains. This observation is consistent with a role of dADA3 in regulating the expression of AS-C, either directly or in conjunction with members of IRO-C. As an additional demonstration of these gene transcriptional effects of dADA3, we checked the transcriptome of dAda3 mutant larvae. The microarray data indicate the expected down regulation of scute, araucan, and caupolican genes (not shown).

Following the rationale inspired by the results with IRO-C, we analyzed other genes involved in bristle patterning in the dorsal notum. The mutant amosTft produces an excess of macrobristles in the same area of the thorax where most dominant IRO-C phenotypes are found in dAda3 heterozygotes, the scutellum. We found that the amosTft phenotype, like that of IRO-C, is enhanced in a dAda3/+ background (the mean ± standard deviation of thorax macrobristles was 22.5 ± 2.1 in the mutants, compared to 18.1 ± 3.1 in controls [16 and 8 adults, respectively]). A similar enhancement effect is detected with AntennapediaHu with respect to the number of extrabristles in the humerus (not shown). It should be noted that the humerus and the scutellum are equivalent metameric structures. The observed effects seem specific, since equivalent tests with other deletions [one of the deletions, Df(2L)TW201.6, included a cluster of histone-encoding genes], genetic constructs, or mutants failed to show evidences of functional interaction (see Table S1 in the supplemental material).


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DISCUSSION
 
We report an in vivo study of the functional properties of the Drosophila orthologue of human ADA3; these Drosophila and human proteins have amino acid identity of 29%. This low sequence conservation is also a feature between yeast and humans, for which the amino acid identity of the proteins is only 14%. Thus, it seems that ADA3 sequences are not well conserved across species. ADA3 was first identified in Saccharomyces cerevisiae as being involved in the response to acidic activators (53). Subsequently, yeast ADA3 was identified in several GCN5-containing HAT complexes, and its role in maintaining complex integrity and providing interaction surfaces with the transcription machinery was demonstrated. Mouse and human ADA3 proteins have been identified in a TATA-binding-protein-free TAF-containing complex which interacts with the estrogen receptor ER{alpha} (4) and also functions as a coactivator for p53-dependent (36) and retinoic acid X receptor alpha-mediated transactivation of target genes (66). More recently, the presence of an ADA3-like protein in several Drosophila HAT complexes has been demonstrated by biochemical dissociation of SAGA and ATAC subunits (37). In all these multiprotein complexes, ADA3 is thought to bind ADA2 and a transcriptional activator through its carboxy and amino termini, respectively (13, 32). In turn, ADA2 associates with GCN5, the acetylase component, sustaining the activity of the whole complex. Under the assumption that these interactions occur also in Drosophila, the mutant versions analyzed here would be unable to interact with dADA2 because of their truncation towards the C terminus.

The selective loss of histone H3 and H4 acetylation at specific lysine residues in dAda3 mutants provides an in vivo demonstration that dADA3 plays an essential role in HAT complexes. In Drosophila, dADA3 is present in the SAGA and ATAC complexes, both of which have a preference for H3 and H4 acetylation. Thus, the loss of selected acetylation activities in both histones indicates the functional deficiency of both complexes in dAda3 mutants. We have previously reported the involvement of fly ADA2b-containing SAGA complex in histone H3 K9 and K14 acetylation and the involvement of the dADA2a-containing ATAC complex in histone H4 K5 and K12 acetylation (15). Neither complex, however, seems involved in the acetylation of H3 K4 or K18 or H4 K8 or K16. Here, in dAda3 mutants, the acetylations of H3 K9 and K14 and H4 K12 are reduced, offering repertoires that are similar, but not identical, to those of dAda2b and dAda2a mutants. Surprisingly, we did not observe acetylation defects in H4 K5 in dAda3 mutants, while in dAda2a and dGcn5 mutants, this site is affected. This discrepancy probably reflects the relative contribution that each ADA component has to the selectivity of the HAT complex to which it belongs. Perhaps, in the absence of dADA3, a site specificity shift of the malfunctioning ATAC from H4 K5 to K12, as described for yeast, could be considered (2). The alternative possibility of additional, yet unidentified, dADA3-containing HAT complexes cannot be ruled out.

The phosphorylation effect on H3 S10 is a novel feature for an ADA3 component. It should be emphasized, however, that acetylation and phosphorylation of H3 appear as two closely related processes, since an adaptor and an acetylating enzyme affect both of them, suggesting that the two forms of histone modification are a property of HAT complexes. The TRA1 component of yeast SAGA exhibits kinase activity, but the H3 S10 phosphorylation is carried out by the SNF1 kinase (40). In Drosophila, however, the precise identity of the H3-phosphorylating complex remains to be determined. The H3-targeted JIL-1 kinase activity is known to associate with the MSL complex involved in the transcriptional modulation of male X chromosome genes (34). In addition, Jil-1 mutants show chromosome structure defects akin to those of dAda3, dGcn5, and dAda2a mutants (34, 49). However, it is still unknown if JIL-1 is a component of a HAT complex. Consistent with its role in histone modification, dADA3 is found on transcriptionally active regions of polytene chromosomes. The quantitative analysis of in situ data showed colocalization of dADA3 with RNA Pol II, albeit with variable stoichiometry according to chromosomal site. This feature is compatible with the proposal that dADA3-containing complexes could have different, probably sequential, effects on nucleosomes, for instance, phosphorylation (e.g., H3 S10 phosphorylation) followed by acetylation and finalizing with initiation of gene transcription by RNA Pol II. In the first event, dADA3 should not be expected to colocalize with RNA Pol II, while it should during initiation of gene transcription. Perhaps it is worth not considering this sequence of events the result of a single type of HAT complex activity or the ordered parade of different complexes on the same chromosomal site. It may be more appropriate to envision the whole sequence as a gradual transition of chemical interactions elicited by a complex in which its components are substituted at different rates. Under this speculative view, assigning a component to a complex becomes less relevant unless the functional context of the chromosomal site is specified. Thus, as an example, a HAT complex would become gradually transformed into a TFIID complex. A case reflecting this conceptual view is already documented for the yeast INO1 promoter (40).

The modification of histones is expected to result in structural changes of the chromatin. In this context, it is important to note that dAda3 mutant chromosomes appear abnormally structured in their polyteny. This is direct evidence that dADA3-containing complexes play a role in the maintenance of chromatin integrity, a process in which dADA2a and dGCN5 also participate. dADA2b, however, seems not to be involved in this structural role. In turn, chromatin structure is expected to result in gene expression effects. Gene variegation is a form of gene silencing elicited by changes in chromatin structure that can yield several types of position effects. Relocation of centromeric heterochromatin to the vicinity of transcription units is among the most widely studied effects (16); modification of pairing between homologous chromosome regions is another widely studied effect (26). Different types of heterochromatin, most notably that from the X chromosome in comparison to that from the Y or autosome chromosomes, exhibit differential variegation properties (39). The data reported here show that dADA3 is a modifier of variegation in a gene-dosage-dependent manner. Loss of function enhances the phenomenon, while additional gene copies reduce it. The effect, however, is dependent on the variegating rearrangement. The three X heterochromatin rearrangements tested showed the dAda3 background effect, while Y heterochromatin (bwD), euchromatic [Tp(2;2)Sco], and homologue-pairing (white and zeste) rearrangements have not. It appears, thus, that the mutant effect is selective of loci and/or heterochromatin type. This feature is compatible with targeting of the dADA3-containing complexes to selective chromatin regions. The site specificity of chromatin-binding complexes and the intervening mechanisms are still poorly defined (5, 9, 63), although some genetic functions have been described as being specific for certain types of variegation (57). Transcription factors such as GAGA and proliferation disrupter (Prod) are known to bind heterochromatin at specific times during the cell cycle (55). GAGA, in particular, binds AC-rich sequences in the heterochromatin associated with bwD. Consistent with the lack of dAda3 mutant background effect on bwD variegation, we tested the GAGA-encoding gene trithorax-like (Trl) for potential effects in double heterozygotes with dAda3 mutants, yielding negative results (data not shown). The loss of dADA3 causes a reduced transcriptional activity of specific genes, as demonstrated for the IRO-C and their targets on scute enhancers. This is direct evidence that dADA3 plays a role in gene expression and that this role is locus specific. In this context, unraveling the specificity of adaptors and other HAT components opens new avenues to target gene expression in vivo in a tissue- and time-controlled manner.


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ACKNOWLEDGMENTS
 
We acknowledge the ASC gene constructs from J. Modolell and his colleagues. The help from lab members O. Komonyi, A. Ciurciu, K. Ökrös, M. C. Álvarez, and C. Hernández-Capitán is deeply appreciated. We are grateful to L. Tora for kindly providing RNA Pol II antibody.

Research was funded by grants from the Spanish Ministry of Science (BFU2006-10180), the Hungarian Science Fund (OTKA T046414), and the European Network TAF-CHROMATIN (MRTN-CT-2004-504288).


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FOOTNOTES
 
* Corresponding author. Mailing address: Cajal Institute, CSIC, Ave. Dr. Arce 37, 28002 Madrid, Spain. Phone: 34 91 5854739. Fax: 34 91 5854754. E-mail: aferrus{at}cajal.csic.es Back

{triangledown} Published ahead of print on 29 October 2007. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back

{ddagger} These authors have contributed equally to this report. Back

# Present address: Departamento de Biología, Facultad de Ciencias Biológicas, Universidad Autónoma de Madrid, Madrid, Spain. Back

§ Present address: Department of Cellular and Molecular Biology, Instituto de Neurobiología, Universidad de Guadalajara, Guadalajara, México. Back


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Molecular and Cellular Biology, January 2008, p. 376-385, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01307-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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