INTRODUCTION

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For proper cellular function and the elaboration of developmental programs, gene expression must be regulated tightly. There is increasing evidence that large transcription complexes, composed of unique combinations of sequence-specific activators and repressors, coactivators, and corepressors, play an important role in determining the temporal and spatial patterns of gene expression (for review, see reference 39).

The CREB binding protein (CBP) is one of most extensively characterized coactivator proteins. CBP was first identified through its ability to link the cyclic AMP protein kinase (PKA)-phosphorylated form of CREB to components of the basal transcriptional machinery, including TFIIB (14, 34), TATA-binding protein (65), and the RNA polymerase II holoenzyme complex (28, 44). CBP is highly related to the adenovirus E1A binding protein p300 (17), and CBP and p300 are considered to be functional homologues (4, 38), although a few differences in their activities have been reported (27). CBP and p300 associate with a wide variety of transcriptional activators in addition to CREB, suggesting that each may serve as a transcriptional integrator of different signaling cascades (reviewed in references 20 and 60). Thus, one model for the function of CBP and p300 is bridging DNA binding transcription factors to components of the basal transcriptional machinery.

Another function of coactivators appears to be the modification of chromatin structure. In this regard, CBP and p300 have also been proposed to mediate transcriptional activation via intrinsic (6, 46) and associated (9, 63, 81) histone acetyltransferase (HAT) activity. Targeted HAT activity is thought to facilitate the access of nuclear factors to their target sites by relaxing the interaction between histones and the DNA (for a review, see reference 77). Moreover, recent studies suggest that transcriptional activation mediated through CBP or p300 occurs only in the context of chromatin (31, 32). Therefore, CBP and p300 may regulate gene expression by interacting with components of the transcriptional machinery as well as by augmenting the access of factors to DNA through their HAT activities. Acetylation of basal and sequence-specific transcriptional regulators may also contribute to CBP function (22, 24).

Genetic studies indicate an essential role for CBP in cellular function and development (reviewed in reference 21). In humans, CBP loss of function is associated with Rubinstein-Taybi syndrome, a haploinsufficiency disorder characterized by mental retardation, developmental defects, and an increased predisposition to cancer (42, 48). Chromosomal translocations that fuse CBP with MOZ (monocytic zinc finger protein) or MLL (mixed-lineage leukemia protein, a trithorax group-like protein) are associated with various types of myeloid leukemia (7, 62). In addition, somatic mutations of the p300 gene have been detected in colorectal and gastric carcinomas (43). Gene knockouts in mice indicated that CBP and p300 are required for normal embryonic development and viability (69, 82). Finally, mutations in the Caenorhabditis elegans homologue of CBP (CBP-1) affect the differentiation of several embryonic tissues (59).

In Drosophila melanogaster, Drosophila CBP (dCBP) loss-of-function mutations cause embryonic lethality. Specifically, dCBP serves as a coactivator for transcription factor Cubitus interruptus (CI) and mediates its activity in the hedgehog pathway (2, 12). dCBP is also a coactivator of the dorsal protein (D1) and Mad, mediating dl-dependent twist expression and dpp-induced transcriptional stimulation, respectively (3, 79). However, dCBP does not always function as a coactivator. Recent studies have shown that dCBP binds to the Drosophila homologue of the T-cell factor (dTCF) and facilitates dTCF-mediated repression in the Wnt/Wingless signaling (78). Therefore, dCBP can function as both a coactivator and a corepressor during embryogenesis.

To further define the developmental processes and the signaling pathways that require dCBP, we have taken advantage of the yeast GAL4 enhancer trap (8) system to generate transgenic flies that overexpress dCBP in a variety of cell types. The dominant overexpression adult phenotypes generated with this system were used to screen for suppressors of dCBP overactivity in specific tissues. In this report, we describe a functional and specific interaction between transcriptional coactivator dCBP and ASH1, a member of the trithorax group (trxG) of chromatin modifiers.

The trxG proteins are required to maintain the continued and efficient expression of homeotic and other genes throughout development. Loss-of-function mutations in the trithorax group genes cause homeotic transformations because they fail to maintain the expression pattern of homeotic selector genes. While the trxG proteins function as transcriptional activators, members of Polycomb group (PcG) genes form stable complexes that maintain a repressed pattern of homeotic gene function (for reviews, see references 49, 50, and 61). Current models envision that trxG and PcG proteins lock in the active or inactive state, respectively, by creating a stable chromatin organization. trxG represents a heterogeneous family of proteins with diverse functions. Some of these proteins, such as Trithorax (TRX), ASH1, ASH2, GAGA, and ZESTE, are associated with particular sites on polytene chromosomes (1, 13, 33, 51, 71, 74), while others, such as Brahma (BRM) and SNR1, are found in chromatin-remodeling complexes that may not be associated with specific chromosomal regions (16, 68). There is some evidence that one of the functions of the trxG proteins may be to recruit chromatin-remodeling complexes to DNA. GAGA is required for the function of one chromatin-remodeling complex, the Drosophila NURF complex (74), and TRX was shown to physically interact with SNR1, a component of the Drosophila SWI/SNF complex (54). These studies strongly support the model that trxG proteins are important regulators of higher-order chromatin structure. However, the precise role of each of the diverse trxG members and the functional relationships that might exist among them and with other transcriptional regulatory factors are still poorly understood.

Our studies show that mutations in the ash1 gene suppress a wing phenotype caused by the overexpression of dCBP in specific central nervous system (CNS) cells. This suppression is specific for ash1 because other members of the trithorax family do not have the same effect. At the cellular level, ASH1 expression coincides with the overexpression pattern of dCBP and, in wild-type flies, ASH1 and dCBP colocalize in the nucleus of the ASH1-expressing neurons. Finally, at the molecular level, we show that dCBP interacts strongly with ASH1 and that the two proteins colocalize to specific sites on polytene chromosomes. Our results strongly suggest that coactivator dCBP and trithorax factor ASH1 are part of a functional complex in vivo. These findings implicate a new type of chromatin-associated proteins in mediating dCBP function and imply that, in addition to its HAT activity, dCBP may participate in the regulation of higher-order chromatin structure.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Drosophila strains. All the ash1 alleles and the ash2 allele were kindly provided by Allen Shearn (Johns Hopkins University). They are described by Tripoulas et al. (70, 71) and Adamson and Shearn (1). The PKA-C1/DCO null allele, G9, (36) and the UAS-R* line containing the upstream activation sequence (UAS) dominant-negative catalytic subunit of PKA (37) were kindly provided by Dan Kalderon (Columbia University). The GMR-GAL4 line was provided G. Rubin, (University of California, Berkeley). Other mutant alleles and most of the deletions used in this study came from the Bloomington Stock Center. UAS-dCBP transformants (Tr21 and Tr36) were established by standard methods. The NotI dCBP fragment that includes the entire dCBP cDNA was cloned into the NotI site of pUAST (8). yw embryos were injected with this DNA and the p2-3 helper as described previously, and the transformants were mapped and put into stock (64). The UAS-dCBP Tr21 insert is on the fourth chromosome, and the Tr36 insert is on the X. The UAS-386 and UAS-363 lines were kindly provided by C. O'Kane (Cambridge University). The balancer chromosomes MKRS, TM3,Stubble (Sb), and TM6B,Tubby (Tb), which are used in these studies, have been described by Lindsley and Zimm (37a). The balancer chromosome strain pk-sple³³ pr cn/T(2;3)SM6.TM6B,Tb was kindly provided by J. Roote and M. Ashburner (Cambridge University). The wild-type strain used in these studies is Canton-S. All crosses were reared at 25°C on standard cornmeal yeast extract source media.

Detection of -galactosidase (-GAL) activity in imaginal discs and CNS. Third-instar larvae were dissected into saline, fixed for 1 h in phosphate-buffered saline (PBS) and 4% formaldehyde, and rinsed several times with PBST (PBS with 0.3% Triton X-100). Fixed samples were then placed in a 0.02% X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) solution (53) at 30°C until the appearance of the blue color (about 1 h for the GAL4-386 CNS, no staining after 24 h for the GAL4-386 imaginal discs). After staining, samples were washed several times in PBST. Larval tissues were mounted in PBS-50% glycerol and photographed with Echtachrome 64 film using Nomarski optics.

Screen to isolate dominant suppressors of the dCBP overexpression wing phenotype. The screen was performed at 25°C where the uninflated-wing phenotype has a 100% penetrance. GAL4-386/MKRS; Tr21/+ females were collected and crossed to males from the Bloomington Stock Center deficiency kit [Df(2)/Balancer or Df(3)/Balancer] (see Fig. 2). Approximately 150 different deletions, which cover more than 80% of the second and third chromosomes, were tested. From these crosses, one-fourth of the progeny were expected to be GAL4-386/+; Tr21/+ and have uninflated wings. In fact, this ratio is less and approximates one-eighth of the total population because this genotype is weak. Although Tr21 is inserted on the fourth chromosome, it is linked to a miniwhite transgene and produces a characteristic pale-orange eye color that can be monitored in a w¹ background. All of the second and third chromosome balancers used in this study were tested and had no effect on the uninflated-wing phenotype (data not shown). When the balancer chromosome of a deficiency stock had a dominant wing marker, for example, Curly or Serrate, the balancer was exchanged with one carrying a more convenient marker, i.e., SM6.TM6B,Tb or TM3,Sb. In this screen, a deletion was considered to have no effect on the uninflated-wing phenotype when the population of GAL4-386 heterozygotes had approximately the same number of flies with uninflated wings regardless of whether they carried the deletion chromosome or the balancer. When the number of uninflated-wing GAL4-386/+ flies with the balancer chromosome was greater than the number of uninflated-wing GAL4-386/+ flies with the deletion chromosome by at least a factor of two, then putative suppressed males (partially inflated or wild-type wings) were crossed to w' females to determine the presence of the Tr21 transgene in the next generation. Approximately 20 to 30 putative suppressed males were tested for the presence of Tr21. When Df/+; GAL4-386/+; Tr21/+ males were obtained, the deletion was considered a potential suppressor. These deletions were rescreened in the same way to confirm the suppression. Only the deletions for which the number of Df/+; GAL4-386/+; Tr21/+ flies with partially inflated or wild-type wings (suppressed phenotype) was at least 50% greater than the number of flies with the same genotype and uninflated wings were considered suppressors (deletions in Table 2). The same procedure was used to determine suppression by mutations in single genes (alleles in Table 3). Each experiment was repeated at least twice.

-Gal expression tests. This assay allows us to measure the possible effect of a deletion on the expression of UAS-transgene and distinguish between suppressors that repress the expression of the transgene and those that represent dCBP-interacting genes (see Table 2). Deficiency stocks were put over a balancer chromosome with a dominant pupal marker: SM6.TM6B,Tb or TM6b,Tb. Df(2)/SM6.TM6B,Tb or Df(3)/TM6b,Tb males were crossed to UAS-LacZ; GAL4-386 homozygous females. Three-day-old pupae were collected and assayed for -Gal activity using o-nitrophenyl--D-galactopyranoside (ONPG) as the substrate. Briefly, a single pupa was squashed and permeabilized in 800 µl of Z buffer (Na₂HPO₄ · 7H₂O [16.1 g/liter], NaH₂PO₄ · H₂O [5.5 g/liter], KCl [0.75 g/liter], MgSO₄ · 7H₂O [0.246 g/liter], pH 7.0) containing -mercaptoethanol (2.7 µl/ml) plus 50 µl of 0.1% sodium dodecyl sulfate (SDS) and 50 µl of chloroform. ONPG was used as the substrate (160 µl of a solution of 4 mg/ml in 0.1 M phosphate buffer, pH 7.0), and assays were performed at 30°C. After 30 min, the reaction was stopped with 0.4 ml of 1 M Na₂CO₃ and the optical density at 420 nm was determined. For each test, at least five pupae were analyzed separately. -Gal activity in Tb flies was compared to -Gal activity in the non-Tb flies that contained the deletion. If the activities were similar, then the deletion has no effect and we considered the test positive. If the activity was largely reduced in the non-Tb flies, then the deletion represses the transgene either by affecting GAL4, the UAS, or the stability of the gene product and we considered the test negative. Using the same strategy (Tb versus non-Tb larvae), we were also able to compare qualitatively the expression of -Gal in fixed larval brains stained with X-Gal (53). In all cases, the qualitative results in whole tissues were consistent with the quantitative values obtained for the pupal squashes (data not shown).

Confocal analysis of larval and pupal CNS. Third-instar larvae or pharate adults were dissected into saline, and the CNSs were processed. They were fixed for 1 h in PBS-4% paraformaldehyde, rinsed several times with PBST, and blocked at least for 1 h in PBST-10% HS. The CNSs were incubated overnight at 4°C with the primary antibody, washed several times in PBST-10% HS (at least three times for 20 min each over 1 h), and then incubated for 1 h at room temperature with the secondary antibody, washed several times in PBST, and mounted in a "slow-fade" buffer specific for immunofluorescence (Molecular Probes). ASH1 was detected by a rabbit polyclonal antibody (71) (affinity purified; kindly provided by Allen Shearn, Johns Hopkins University) at a dilution of 1:40, and dCBP was detected by a chicken polyclonal antibody raised against the CREB binding domain (CBD) of dCBP at a dilution of 1:800. FMRFamide (PT-2) and peptidylglycine--hydroxylating mono-oxygenase (PHM) rabbit polyclonal antibodies (30, 67) (kindly provided by Paul Taghert) were used at dilutions of 1:2,000 and 1:500, respectively. Fluorescein anti-rabbit (Vector) and rhodamine anti-chicken (Jackson) secondary antibodies were used at a dilution of 1/200. CNSs were examined with a confocal laser scan microscope (Bio-Rad 1024ES laser and Nikon Eclipse TE300 microscope).

Plasmid constructions. Various dCBP fragments (from pBSK-dCBP [2]) were inserted by PCR cloning into the pGEX-KG vector (Pharmacia) to generate pGST-dCBP-825-1043, pGST-dCBP-1699-1997, and pGST-dCBP 2274-2508, corresponding, respectively, to the CBD, BrZn, and C/H3 domains. The BglII restriction fragment of pBKS-ASH1 (kindly provided by Allen Shearn, Johns Hopkins University) was inserted into a pCITE vector (Novagen) for in vitro translation of the nearly full-length ASH1 (amino acids [aa] 49 to 2011). Other ASH1 fragments were inserted by PCR cloning into the pCITE vector for in vitro translation of ASH1-47-456, ASH1-458-853, ASH1-855-1255, ASH1-1639-2011, and the ASH1-SET domain (aa 1245 to 1525). Two N-terminal fragments of ASH1 were inserted by PCR cloning into the pGEX-KG vector to generate pGST-ASH1-47-456 and pGST-ASH1-458-853.

In vitro binding assays. GST fusion proteins were produced in Escherichia coli BL21 and purified by affinity on GST-agarose beads according to the Pharmacia protocol. In vitro-translated [³⁵S]methionine-labeled proteins were incubated with immobilized GST fusion proteins for 1.5 h at room temperature in Harlow buffer (50 mM HEPES [pH 7.5], 100 mM NaCl, 0.2 mM EDTA, 0.01 mM NaF, 1 mM dithiothreitol, 0.5% NP-40) with 5 mg of bovine serum albumin (BSA)/ml. After four washes in high-stringency Harlow buffer (containing 300 mM NaCl), bound proteins were eluted by boiling in SDS loading buffer, resolved by SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography. Proteins from Kc cell nuclear extracts (65 µg of total protein) were incubated with immobilized GST fusion proteins using the procedure described above. In this case, the incubation was performed in the presence of 2.5 mg of BSA/ml instead of 5 mg of BSA/ml. For the E1A competition assays, proteins from Kc cell nuclear extracts were incubated with immobilized GST-ASH1-47-456 in the presence of 1, 4, or 12 µg of purified E1A or E1A-RG2 (kindly provided by J. Lundblad, Oregon Health Sciences University). This corresponds to approximately 50, 200, and 500 nmol of the E1A proteins, respectively. Incubations were done in the absence of E1A and E1A.RG2 as controls. The presence of bound dCBP from nuclear extracts was examined by Western blotting using the dCBP chicken polyclonal antibody at a dilution of 1/1,000. The Kc cell nuclear extracts were prepared as described previously (23).

Confocal analysis of polytene chromosomes. Polytene chromosomes were fixed and squashed for immunohistochemistry as described previously (83). ASH1 was detected by the ASH1 rabbit polyclonal antibody at a dilution of 1/40, and dCBP was detected by the chicken antiserum at a dilution of 1/800. Fluorescein anti-rabbit (Vector) and rhodamine anti-chicken (Jackson) secondary antibodies were used at a dilution of 1/200. Polytene chromosomes were examined by confocal laser scanning microscopy.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Dominant adult phenotypes obtained by overexpressing dCBP in specific cell types. Our goal was to obtain dominant adult phenotypes by overexpressing dCBP in specific cell types during development. To generate transgenic flies that overexpress dCBP in various tissues, we used the yeast GAL4 enhancer trap system (8). We generated two independent transgenic lines, Tr21 and Tr36, that carry the UAS-dCBP construct. These two lines are viable and show no visible phenotypes. We crossed the Tr21 and Tr36 transgenic lines to approximately 50 different enhancer trap GAL4-expressing lines and characterized the progeny in which the UAS-dCBP transgene is transcribed in a specific GAL4-dependent pattern. In most cases, overexpression of dCBP resulted in lethality at different stages of the development. A few GAL4 strains generated visible adult phenotypes with the Tr21-UAS-dCBP line (Table 1). Two of the dominant phenotypes had 100% penetrance: a smooth-eye phenotype using the GMR-GAL4 driver (this phenotype will be described in a separate paper) and an uninflated-wing phenotype using the GAL4-386 line (Fig. 1A).

View larger version (71K): [in this window] [in a new window]   FIG. 1.   Uninflated-wing phenotype obtained by overexpressing dCBP in specific cells of the CNS using the GAL4-386 driver. (A) The uninflated-wing phenotype. (B to D) Expression of a LacZ reporter gene driven by GAL4-386 in larval tissues. -Gal staining was detected in specific cells of the CNS (brain lobes and ventral ganglion) (B) but not in wing discs (C) or haltere/leg imaginal discs (D). (E to J) Analysis by laser confocal microscopy of dCBP wild-type expression and dCBP overexpression in the CNSs of third-instar larvae and pharate adult pupae. (E) dCBP ubiquitous expression in wild-type larval brain; (F) dCBP overexpression in specific cells in larval brain lobes and larval ventral ganglion; (G) overexpression in larval ventral ganglion at higher magnification; (H) overexpression in pupal brain including the optic lobe (OL); (I) overexpression in pupa thoracic ganglion with the prothorax (PT), the mesothorax (MS), the metathorax (MT), and the abdominal ganglion (AB) indicated; (J) overexpression in PT and MS at higher magnification. Magnifications, ×20 (E, F, H, and I) and ×40 (G and J).

View larger version (72K): [in this window] [in a new window]   FIG. 2.   dCBP overexpression with the GAL4-386 driver coincides with the expression of the neuropeptides FMRFamide and PHM. Arrows, colocalization of dCBP (rhodamine) and FMRFamide (fluorescein isothiocyanate [FITC]) in the larval (A) and pupal (C) ventral ganglion and colocalization of dCBP (rhodamine) and PHM (FITC) in the larval (B) and pupal (D) ventral ganglion.

Screen for genes that can suppress the uninflated-wing phenotype caused by the overexpression of dCBP in specific CNS cells. The premise of the screen was that a twofold reduction in the dose of a gene that is involved in dCBP signaling may suppress the uninflated-wing phenotype that results from dCBP overexpression. Using the Bloomington Stock Center deficiency kit that covers at least 70% of the genome, we screened for deletions on the second and third chromosomes that are dominant suppressors of the uninflated-wing phenotype. The strategy of the screen is represented in Fig. 3 and is described in Materials and Methods. A suppression was considered positive when at least 50% of the flies had a suppressed phenotype (partially or completely inflated wings). Deletions identified as potential suppressors were rescreened at least twice to confirm the interactions. We obtained 10 different deletions in various portions of the genome (six on the second chromosome and four on the third chromosome) that suppress the uninflated-wing phenotype (Table 2). We expected two types of suppressors from our screen: first, deletions that reduce the level of dCBP production, either by reducing GAL4 expression or by affecting the expression of the transgene, and second, deletions of genes that affect dCBP function, either directly or indirectly and in a dosage-sensitive manner.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Screen for deletions that can dominantly suppress the dCBP overexpression wing phenotype. Males carrying deficiencies for the second [Df(2)] and the third [Df(3)] chromosomes over a Balancer (B) were crossed to GAL4-386/MKRS; Tr21 (UAS-dCBP)/+ females with the uninflated-wing phenotype. Suppressed flies with the nonbalanced phenotype were analyzed. The Tr21 transgene on the fourth chromosome was not marked. To determine its presence in the suppressed flies, totally or partially suppressed males were crossed to w' females to monitor the pale-orange eye color characteristic of flies with the Tr21 transgene. Examples of the mutant and suppressed-wing phenotypes are illustrated adjacent to the genotypes.

Specific expression of ASH1 protein in the same CNS cells that overexpress dCBP. To understand more precisely the relationship between dCBP and ASH1, we analyzed their localization at the cellular level. Using confocal laser microscopy, we first localized the expression of ASH1 protein in the CNS when dCBP is expressed under the control of GAL4-386. For this study, we used an ASH1 rabbit polyclonal antibody and the dCBP chicken polyclonal antibody produced in our laboratory. ASH1 was expressed in specific neurons in the larval ventral ganglion (Fig. 4A) and the pupal and adult thoracic ganglion (Fig. 4D, G, and J). No expression of ASH1 was detected in the brain, either in third-instar larvae, late pupae, or adults (data not shown). dCBP, which is normally expressed in every neuron of the CNS, was overexpressed in specific neurons when placed under the control of the GAL4-386 driver (Fig. 4B, E, H, and K). The overexpression of dCBP is seen both in the brain and the ventral ganglia at larval, pupal, and adult stages. We then determined whether dCBP and ASH1 were colocalized. In the ventral ganglia of third-instar larvae, approximately two-thirds of the overexpressing dCBP neurons were ASH1 positive (Fig. 4C). The same high level of coexpression was observed in the thoracic CNSs of pharate adults before eclosion and of adults 24 h posteclosion (Fig. 4F, I, and L). In the prothorax and mesothorax, 12 neurons that overexpressed dCBP expressed ASH1 as well (Fig. 4F). In the metathorax and the abdominal ganglion, about half of the dCBP-overexpressing cells were ASH1 positive (Fig. 4I), representing also about 12 neurons. Colocalization of ASH1 and dCBP overexpression was still observed in the 12 ASH1-positive cells that persisted 24 h after eclosion (Fig. 4L).

View larger version (64K): [in this window] [in a new window]   FIG. 4.   ASH1 expression coincides with dCBP overexpression in the CNS of GAL4-386/+; Tr21/+ larvae and pupae. An analysis by laser confocal microscopy was performed. (A to C) Colocalization in the thoracic region of a third-instar larval ventral ganglion. (A) ASH1 immunostaining with fluorescein isothiocyanate (FITC); (B) dCBP immunostaining with rhodamine; (C) merged image from panels A and B. (D to F) Colocalization in the thoracic CNSs, prothoraxes (PT), and mesothoraxes (MS) of pharate adult pupae. (D) ASH1-FITC immunostaining; (E) dCBP-rhodamine immunostaining; (F) merged image from panels D and E. (G to I) Colocalization in the thoracic CNSs metathoraxes (MT), and abdominal ganglia (AB) of pharate adult pupae. (G) ASH1-FITC immunostaining; (H) dCBP-rhodamine immunostaining; (I) merged image from panels G and H. Magnification, ×40.

View larger version (88K): [in this window] [in a new window]   FIG. 5.   CNS expression of ASH1 in dCBP-overexpressing or wild-type third-instar larvae, as analyzed by laser confocal microscopy. (A to C) Expression of ASH1 and dCBP in the larval ventral ganglion by dCBP-overexpressing cells. (A) ASH1 immunostaining with fluorescein isothiocyanate (FITC); (B) dCBP immunostaining with rhodamine; (C) merged image from panels A and B. (D to F) Expression of ASH1 and dCBP in the larva ventral ganglia of wild-type flies. (D) ASH1-FITC immunostaining; (E) dCBP-rhodamine immunostaining; (F) merged image from panels D and E. (G to I) Colocalization of ASH1 and dCBP in the nuclei of wild-type larval ganglion neurons. (G) ASH1-FITC immunostaining; (H) dCBP-rhodamine immunostaining; (I) merged image from panels G and H (arrowheads, fine haze of yellow colocalization). Magnifications, ×40 (A to F) and ×60 (G to I).

View larger version (50K): [in this window] [in a new window]   FIG. 6.   dCBP overexpression in a wild-type or ash122 heterozygous genetic background. Shown is an analysis by laser confocal microscopy of dCBP normal expression and overexpression in the ventral ganglia of third-instar larvae. (A) dCBP ubiquitous expression in wild-type larvae; (B) dCBP overexpression in GAL4-386/+; Tr21/+ larvae; (C) dCBP overexpression in GAL4-386/ash122; Tr21/+ larvae.

Direct association between dCBP and ASH1. Because both dCBP and ASH1 are involved in transcription and appear to interact at both the genetic and cellular levels, we asked whether dCBP and ASH1 could interact biochemically.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   dCBP interacts with ASH1. (A) Equimolar amounts of immobilized GST and GST-dCBP fusion proteins were incubated with in vitro-translated 35S-labeled ASH1 protein (nearly full-length ASH1 protein; aa 49 to 2011). (B) Equimolar amounts of immobilized GST fusion proteins were incubated with various in vitro-translated 35S-labeled ASH1 fragments. The most C-terminal ASH1 fragment (aa 1639 to 2011) contains the PHD domain, but none of these fragments contain the SET domain. (C) Equimolar amounts of immobilized GST fusion proteins were incubated with the in vitro-translated 35S-labeled SET domain. (D) Equimolar amounts of immobilized GST and GST-ASH1 fusion proteins were incubated with Kc cell nuclear extracts. dCBP was detected by Western blotting using the dCBP chicken polyclonal antibody. (E) GST-ASH1-47-456 fusion protein was incubated with Kc cell nuclear extracts in the presence of 1, 4, or 12 µg of E1A or E1A-RG2. Similar results were obtained for GST-ASH1-458-853 (data not shown).

View larger version (31K): [in this window] [in a new window]   FIG. 8.   Endogenous dCBP and ASH1 proteins colocalize on wild-type polytene chromosomes. Shown is an analysis by laser confocal microscopy. (A) Chromosome arm showing localization of ASH1. (B) Chromosome arm from panel A showing localization of dCBP. (C) Merged image from panels A and B. Arrowheads, loci that colocalize both dCBP and ASH1. Magnification, ×60.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies have shown that CBP affects transcription through interactions with components of the basal transcriptional machinery and through its intrinsic and associated acetyltransferase activities. In this report, we used a genetic approach in Drosophila to further examine the in vivo function of dCBP. Overactivity of dCBP in particular cell types causes several distinct adult phenotypes. By screening for deletions that could suppress one dCBP overexpression phenotype, we identified ASH1, a member of the trithorax group of chromatin modifiers, as a potential interacting partner of dCBP. ASH1 and dCBP colocalize to a subset of CNS neurons and to specific bands in polytene chromosomes. Furthermore, dCBP and ASH1 interact specifically at the molecular level. Our genetic and biochemical analyses link dCBP to a second class of proteins involved in epigenetic gene regulation.

Screen for suppressor genes of the uninflated-wing phenotype. Despite the fact that the dCBP-overexpressing flies with an uninflated-wing phenotype were weak and difficult to culture, they were fertile and could be used for our screen. The use of the deficiency kit allowed us to rapidly define regions of the genome that contain genes capable of influencing the effect of dCBP overexpression. In five deletions, the genetic elements that cause the suppressions remain unknown and must be characterized. We anticipate that the sequencing of the entire Drosophila genome and the generation of more P-element mutations in these regions will help us to identify novel dCBP interactors.

Suppressing effect of ash1 on the overactivity of dCBP. Lethal mutations of ash1 cause homeotic transformations of imaginal disc-derived structures (58). Gene ash1 (absent, small, or homeotic discs 1) is a member of the trxG genes that encode a variety of proteins with different biochemical properties that are thought to play an important role in modulating chromatin structure during development. Therefore, the identification of ash1 as a possible effector of dCBP function suggested a novel role for dCBP in regulating gene expression. Amorphic or strong hypomorphic alleles of ash1 that are thought to retain very little function (70, 71), suppress the dCBP overexpression phenotype. However, two other alleles, ash1²⁹ and ash1¹⁴, had no effect on the dCBP uninflated-wing phenotype. ash1²⁹ is a weak hypomorph that retains some degree of function, while ash1¹⁴ is a temperature-sensitive allele (70) that certainly retains most of its function at the temperature of our assays (25°C). These results indicate that the dCBP-ASH1 interaction, whether direct or indirect, is dosage sensitive. One of the deletions isolated as a suppressor contains gene ash2, another member of the trx family. The ash1 and ash2 genes are functionally related (57), but their gene products are structurally divergent (1, 71). Despite the fact that they belong to the same family, a strong mutation in ash2 does not suppress the dCBP overexpression phenotype. It is interesting that none of the other trxG genes tested in this study (brahma, trithorax, and trithorax-like) were capable of suppressing the wing phenotype. The specificity of the interaction could be explained by the restricted range and action of trxG genes in different cell types. Alternatively, it could be that the interaction of dCBP with other members of the trxG genes is not dosage sensitive in these neurons.

ASH1 expression coincides with the overexpression pattern of dCBP. The set of specific cells in the larval ventral ganglion and pupal thoracic ganglion that express ASH1 is a subset of the cells that overexpress dCBP in the GAL4-386 line. In the thoracic region of the larval ventral ganglion as well as in the prothorax and mesothorax of the pupal thoracic CNS, all of the ASH1-positive cells colocalized with dCBP-overexpressing cells. Some of the dCBP-overexpressing cells express the neuropeptide markers FMRFamide and PHM (30, 45, 56) and from their positions are likely to express ASH1 as well. This result suggests that dCBP and ASH1 could have common functions in peptidergic neurons.

Overexpression of dCBP in specific CNS cells causes wing inflation defects. In many tissues, overexpression of dCBP causes lethality, suggesting that the dose of this effector is important for its function. The overproduction of dCBP in specific cells of the CNS with two different GAL4 lines produced defects in wing inflation with various degrees of penetrance. However, overexpression of dCBP in wing tissues throughout development does not interfere with wing inflation.

Specific interaction between ASH1 and dCBP. Our biochemical experiments show that coactivator dCBP binds strongly to trxG protein ASH1. This observation supports the idea that ASH1 and dCBP interact in vivo and implicates a novel class of chromatin binding proteins in mediating dCBP function.