Gcn5 ortholog

REGULATION

Gcn5 regulates hsp70

The presence of general transcription factors and other coactivators at the Drosophila hsp70 gene promoter in vivo has been examined by polytene chromosome immunofluorescence and chromatin immunoprecipitation at endogenous heat-shock loci or at a hsp70 promoter-containing transgene. These studies indicate that before heat shock the hsp70 promoter is already occupied by TATA-binding protein (TBP) and several TBP-associated factors (TAFs), TFIIB, TFIIF (RAP30), TFIIH (XPB), TBP-free/TAF-containing complex (GCN5 and TRRAP), and the Mediator complex subunit 13. After heat shock, there is a significant recruitment of the heat-shock transcription factor, RNA polymerase II, XPD, GCN5, TRRAP, or Mediator complex 13 to the hsp70 promoter. Surprisingly, upon heat shock, there is a marked diminution in the occupancy of TBP, six different TAFs, TFIIB, and TFIIF, whereas there is no change in the occupancy of these factors at ecdysone-induced loci under the same conditions. Hence, these findings reveal a distinct mechanism of transcriptional induction at the hsp70 promoters, and further indicate that the apparent promoter occupancy of the general transcriptional factors does not necessarily reflect the transcriptional state of a gene (Lebedeva, 2005).

Functional analysis of Gcn5/Pcaf

To investigate the contribution of Gcn5 to histone acetylation, polytene chromosomes from larval salivary glands were immunostained using antibodies specific for the acetylation of various lysine residues of histones H3 and H4. In order to compare their acetylation levels, polytene chromosomes from wild-type and mutant larvae were squashed and stained together on the same slide. The expression of a histone 2B yellow fluorescent fusion protein (H2b-YFP) in wild-type larvae allowed the distinguishing of their chromosomes from Gcn5 mutant chromosomes. The acetylation of the H3 K14 and K9 residues was detected in numerous loci of wild-type polytene chromosomes. In striking contrast, the acetylation of H3 K14 was undetectable and the acetylation of H3 K9 was barely detectable in Gcn5 mutant chromosomes. Antibodies against the acetylated H4 K8 and H4 K16 residues revealed unchanged acetylation of these residues in Gcn5 mutants compared to that in wild-type animals. Whether the loss of H3 K9 and K14 acetylation in Gcn5 mutants affects other modifications of histone H3 residues was analyzed. Like H3 K9 and H3 K14 acetylation, the phosphorylation of H3 S10 and di- and tri-methylation of H3 K4 have been associated with the transcriptional activation of target loci. It was found that global levels of these modifications are not affected in Gcn5 mutant polytene chromosomes. In contrast, methylation of the H3 K9 residue acts as a marker for the recruitment of the heterochromatin protein HP1 and transcriptional silencing. The loss of H3 K9 acetylation in Gcn5 mutants could have favored ectopic H3 K9 methylation and the subsequent delocalization of HP1. However, it was found that both H3 K9 methylation levels and HP1 localization in the pericentromeric regions of polytene chromosomes were unchanged in Gcn5 mutants (Carré, 2005).

The contribution of Gcn5 to histone acetylation in imaginal tissues was examined by immunostaining of imaginal discs in which Gcn5 was silenced in the P compartment by UAS-IR[gcn5] under the control of en-GAL4. The acetylation of H3 K9 and H3 K14 residues was strongly reduced in the nuclei of the Gcn5-depleted imaginal cells, while the acetylation of H4 K8 and H4 K16 residues was not affected. As seen with polytene chromosomes of Gcn5 mutants, Gcn5 depletion did not affect the level of H3 K4 or H3 K9 methylation in the nuclei of imaginal discs. When expression of the UAS-IR[Gcn5] transgene was driven by the ubiquitous da-GAL4 driver, animals arrested their development at the onset of metamorphosis. Western blot analysis of such late-third-instar larvae showed a strong depletion of histone H3 acetylated at the K9 and K14 residues, while the level of histone H4-AcK8 remained unchanged. Hence, the effect of the Gcn5 loss of function on polytene chromosomes, together with the results of Gcn5 RNAi studies with imaginal discs and larval tissues, points to dGcn5 as the major histone H3 K9 and H3 K14 acetyltransferase in Drosophila (Carré, 2005).

To analyze the functional requirement of the evolutionarily conserved domains of the Gcn5 protein, lines transgenic for Gcn5 variant genes were established under the control of the UAS promoter. The variant genes were designed in order to express Gcn5 with a deletion of the Pcaf homology domain (DeltaPcaf), the catalytic domain for acetylation (DeltaHAT), the conserved domain shown in yeast to be involved in interaction with the Ada2 protein (DeltaAda), or the bromodomain (DeltaBromo) were examined. It was verified by Western blot analysis that the variant transgenes are expressed in the presence of da-GAL4 at levels comparable to that of the UAS-Gcn5 wild-type transgene and then their ability to rescue the Gcn5 function was tested. In contrast to the UAS-Gcn5 wild-type construct, the UAS-Gcn5DeltaHAT construct did not rescue the lethal phenotype of the Gcn5E333st/sex204 heteroallelic combination. As expected from the disruption of the catalytic acetyltransferase domain, the acetylation of polytene chromosomes at the H3 K9 and K14 residues was not restored by the Gcn5DeltaHAT variant in the mutant animals compared to the rescue of acetylation by the wild-type Gcn5 protein. However, it should be noted that the Gcn5DeltaHAT variant still localized to the interbands of the polytene chromosomes in a manner similar to that of the wild-type dGcn5 protein expressed under the control of the da-GAL4 driver (Carré, 2005).

It was recently shown that the dAda2b component of the Drosophila SAGA-like complex directly interacts with Gcn5 and is required for the acetylation of histone H3. This result suggested that the interaction with dAda2b is essential either for the HAT activity of dGcn5 or for its targeting to chromatin (Qi, 2004). Surprising, the Gcn5DeltaAda protein appeared normally distributed on polytene chromosomes and restored histone H3 acetylation in Gcn5 mutants. However, Gcn5 mutants were not rescued by Gcn5DeltaAda and still arrested at puparium formation (Carré, 2005).

The Gcn5DeltaPcaf variant protein also appeared to be normally distributed at the interbands of polytene chromosomes and to restore the acetylation of histone H3. In addition, about half of the Gcn5 mutant animals expressing Gcn5DeltaPcaf formed their puparium and completed metamorphosis but died before eclosion as pharate adults, while the other half gave rise to adult flies. However, rescued adults displayed held-out, misshapen wings, legs with a severe femur kink, and rough eyes. They died a few hours after eclosion, indicating that the UAS-Gcn5DeltaPCAF construct only partially rescues Gcn5 mutants (Carré, 2005).

Strikingly, the Gcn5DeltaBromo variant protein not only restored histone H3 acetylation but also restored complete viability of the Gcn5 mutants. Adult flies were indistinguishable from Gcn5 mutant flies rescued by the full-length Gcn5 protein. The only observed defect was female sterility, a result expected from the absence of activity of the UAS promoter in the female germ line. It was not possible to examine the chromosomal localization of the Gcn5DeltaBromo protein because the Gcn5 antibody was prepared against the Gcn5 bromodomain. Nevertheless, the full phenotypic rescue by this variant strongly suggests that its localization to chromosomes was restored (Carré, 2005).

Protein Interactions

dTAF(II)24 is associated with the histone acetyltransferase dGCN5

TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF(II)s. Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAF(II)s, leaving a number of yeast and human TAF(II)s [e.g., hTAF(II)55, hTAF(II)30, and hTAF(II)18] without known Drosophila homologues. Drosophila has not one but two hTAF(II)30 homologues, dTAF(II)16 and dTAF(II)24 (Taf10), which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5' extremities overlap. These novel dTAF(II)s are expressed and they are both associated with TBP and other bona fide dTAF(II)s in dTFIID complexes. dTAF(II)24, but not dTAF(II)16, is also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAF(II)16 and dTAF(II)24 are functional homologues of hTAF(II)30, and this is the first demonstration that a TAF(II)-GCN5-HAT complex exists in Drosophila. The two dTAF(II)s are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAF(II)16 and dTAF(II)24 may have similar but not identical functions (Georgieva, 2000).

Two different Drosophila ADA2 homologues are present in distinct GCN5 histone acetyltransferase-containing complexes

A novel Drosophila gene, Rpb4, has been isolated coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, Ada2a, and a subunit of RNA polymerase II (Pol II), Rpb4. Moreover, another gene was identified in the Drosophila genome encoding a second ADA2 homologue (Ada2b). The two ADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The Ada2a/Rpb4 and Ada2b genes are differentially expressed at various stages of Drosophila development. Both Ada2a and Adab interact with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and Ada2b, but not Ada2a, also interact with Drosophila Ada3 (diskette). Both Drosophila ADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against Ada2a or Ada2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, Ada2a and GCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas Ada2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes (Muratoglu, 2003).

In Saccharomyces cerevisiae the coactivator-adaptor protein Gcn5 is part of large multisubunit complexes, the largest of which is the 1.8- to 2-MDa SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. Yeast SAGA comprises products of at least four distinct classes of genes: (1) the Ada proteins (yAda1, yAda2, yAda3, yGcn5 [yAda4], and yAda5 [ySpt20]), which have been isolated in a genetic screen as proteins interacting functionally with the yeast activator Gcn4 and the herpes simplex virus activation domain VP16; (2) the TBP-related set of Spt proteins (ySpt3, ySpt7, ySpt8, and ySpt 20), initially identified as suppressors of transcription initiation defects caused by promoter insertions of the Ty transposable element; (3) a subset of TBP-associated factors (TAFs) including scTAF5 (formerly TAFII90), scTAF6 (formerly TAFII60), scTAF9 (formerly TAFII17), scTAF10 (formerly TAFII25), and scTAF12 (formerly TAFII68/61), and (4) the product of the essential gene Tra1, which has been shown to be a component of SAGA (Muratoglu, 2003 and references therein).

Another type of GCN5-containing HAT complex identified in yeast is the 0.8-MDa ADA complex (for 'alteration/deficiency in activation'). The ADA complex differs from SAGA in many aspects. In contrast to the 1.8 to 2-MDa ySAGA complex, the only components of the 0.8-MDa yADA complex are the three adaptor proteins (Ada2, Ada3, and Gcn5) and Ahc1. The ADA complex does not contain yAda1, yAda5, or the other ySpt proteins found in SAGA. Furthermore, the structural integrity of the yADA complex, but not that of ySAGA, is dependent on the presence of the AHC1 gene product. The SAGA complex physically interacts with the acidic activators yGcn4 and VP16, whereas ADA fails to do so. Moreover, ADA and SAGA HAT complexes generate overlapping yet distinct patterns of lysine acetylation on histone H3. These results taken together, strongly suggest that in yeast two distinct ADA-Gcn5 HAT complexes exist (Muratoglu, 2003 and references therein).

A number of similar multiprotein complexes have been characterised in mammalian systems as well, such as the human TBP-free TAF-containing complex (TFTC), the PCAF-GCN5 complex (Ogryzko, 1998), and the SPT3-TAF9(TAFII31)-GCN5 acetyltransferase complex (STAGA) (Martinez, 2001), which all contain the GCN5 HAT, ADA proteins, SPTs, TAFs, and the human homologue of yTRA1, TRRAP (Muratoglu, 2003 and references therein).

TFTC is able to direct preinitiation complex assembly on both TATA-containing and TATA-less promoters in vitro. Similarly to other TBP-free TAFII complexes, TFTC contains the hGCN5 HAT and is able to acetylate histone H3 in both free and nucleosomal contexts (Brand, 1999). The fact that histone acetylation has been linked to the activation of transcription suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and potentiate transcription initiation. Additional recently identified TFTC subunits common to other human TAF-HAT complexes include TAF9, hADA3, hSPT3, hPAF65alpha, hPAF65ß, and TRRAP). Moreover, the Drosophila melanogaster dTAFII24 coimmunoprecipitates with dGCN5 (Georgieva, 2000), suggesting the existence of a TFTC-like HAT complex in Drosophila (Muratoglu, 2003 and references therein).

RPB4 is the fourth largest of the 12 subunits of yeast Pol II. Its unusual feature is that in optimally growing cells it is present only in a small fraction of Pol II complexes; however, it is required for efficient transcription during temperature extremes and certain other stress conditions like starvation. RPB4 and another subunit, RPB7, are thought to form a subcomplex which is incorporated into the Pol II enzyme under suboptimal growth conditions to play a stress-protective role by inducing a closed Pol II conformation. Interestingly, unlike the other subunits of Pol II, the level of RPB4 is posttranscriptionally regulated. In yeast, RPB4 is nonessential under normal growth conditions. Both its function and specific role in higher eukaryotes remain unclear. Nevertheless, the recent finding that its homologue is already present in archaea supports the assumption that it plays an important role (Muratoglu, 2003 and references therein).

This study reports the identification of two novel Drosophila homologues of the yeast Ada2 protein (ADA2a and ADA2b). Interestingly, two genes that encode ADA2 homologues were also found in the Arabidobsis thaliana and human genomes but not in the fully sequenced Caenorhabditis elegans genome. The analysis of the gene encoding the Drosophila ADA2a protein revealed that in addition to ADA2a, this gene encodes the Pol II subunit Rpb4 by alternative splicing. The N-terminal end of the two proteins is encoded by the same exon. Evolutionarily conserved protein-protein interactions were found among Ada2a and Ada2b proteins and their predicted partners of interaction. Several lines of evidence are presented that show that the two novel Drosophila ADA2 homologues are present in different GCN5 HAT-containing complexes (Muratoglu, 2003).

Two Drosophila Ada2 homologues function in different multiprotein complexes

The reversible acetylation of the N-terminal tails of histones is crucial for transcription, DNA repair, and replication. The enzymatic reaction is catalyzed by large multiprotein complexes, of which the best characterized are the Gcn5-containing N-acetyltransferase (GNAT) complexes. GNAT complexes from yeast to humans share several conserved subunits, such as Ada2, Ada3, Spt3, and Tra1/TRRAP. These factors have been characterized in Drosophila, and it was found that the flies have two distinct Ada2 variants (dAda2a and dAda2b). Using a combination of biochemical and cell biological approaches it was demonstrated that only one of the two Drosophila Ada2 homologues, Ada2b, is a component of Spt-Ada-Gcn5-acetyltransferase (SAGA) complexes. The other Ada2 variant, Ada2a, can associate with Gcn5 but is not incorporated into dSAGA-type complexes. This is the first example of a complex-specific association of the Ada-type transcriptional adapter proteins with GNATs. In addition, Ada2a is part of Gcn5-independent complexes, which are concentrated at transcriptionally active regions on polytene chromosomes. This implicates novel functions for Ada2a in transcription. Humans and mice also possess two Ada2 variants with high homology to Ada2a and Ada2b, respectively. This suggests that the mammalian and fly homologues of the transcriptional adapter Ada2 form two functionally distinct subgroups with unique characteristics (Kusch, 2003).

The Drosophila homologues of the Gcn5-associated factors Ada3, Ada2, Spt3, and Tra1/TRRAP have been characterized; these proteins are components of large multiprotein complexes and they associate with Gcn5 HAT activities. The human and yeast homologues of these factors were shown to be components of the SAGA-related GNAT complexes SAGA, PCAF, STAGA, and TFTC. SAGA-related complexes share a significant number of conserved subunits, and the SAGA, PCAF, and STAGA complexes have similar molecular masses of ~1.8 to 2 MDa. The TFTC complex is ~1.35 MDa, suggesting that higher eukaryotes might have more SAGA-related subcomplexes than yeast. Drosophila SAGA-related complexes elute from size exclusion columns in a comparable molecular-mass range. Moreover, size exclusion chromatography using a S400 column suggests that dGcn5, dAda3, dAda2b, and dSpt3 elute in two peaks of ~2 and 1.6 MDa. It therefore appears likely that Drosophila also might have different subtypes of SAGA-related complexes, as has been described for humans. The striking similarity in the sizes of Drosophila and human SAGA-type complexes suggests that their compositions might be very similar. This is supported by findings that Drosophila homologues of SAGA-specific human Tafs, such as dmTaf5, dmTaf9, and dmTaf10, associate with the dSAGA subunits dAda3, dAda2b, and dSpt3. In addition, dSAGA-type complexes interact with the acidic activator VP16 and the Drosophila p53 homolog. This suggests that these chromatin-remodeling complexes might have similar functions in the regulation of gene expression in flies and humans (Kusch, 2003).

The existence of a 440-kDa GNAT complex containing dAda2a, dGcn5, and dAda3 indicates that Drosophila has smaller GNAT complexes distinct from dSAGA. This novel finding suggests that higher eukaryotes also might possess non-SAGA-type GNAT complexes, as found in yeast. So far, only two yeast non-SAGA-type GNAT complexes, namely, the 800-kDa ADA and the 200- to 500-kDa HAT A2, have been identified. However, the functions of these complexes are not yet understood. Unfortunately, it was not possible to find specific subunits of non-SAGA complexes, such as a fly homologue of the yeast Ahc1 protein, but the possibility that a functionally equivalent protein exists in ADA-type complexes from higher eukaryotes cannot be excluded (Kusch, 2003).

In an attempt to address the functions of GNAT complexes in multicellular organisms, the genetically well-studied protostome model organisms Caenorhabditis elegans and D. melanogaster have been exploited. No sequences with homology to GNAT complex subunits have been detected in the genome of C. elegans, whereas D. melanogaster possesses homologues of Gcn5, Ada2, Ada3, and Spt3. The possibility that these factors are less conserved in nematodes cannot be excluded; however, the Tra1/TRRAP homologue from C. elegans is highly similar to Tra1/TRRAP from other organisms. Tra1/TRRAP is a component of the histone H4-specific NuA4 and Tip60 complexes (see Tip60), suggesting that C. elegans might have H4-specific HAT complexes. This is supported by the fact that C. elegans has homologues of Tip60 and Esa1, the catalytic subunit of NuA4. It remains unclear at this point why the protostome C. elegans may lack GNAT-type HAT complexes whereas the protostome Drosophila possesses GNAT complexes with significant homologies to their mammalian counterparts. Studies of mice indicate that GNAT complexes might play essential roles during development. The maternal load of GNAT components and their higher concentration in female flies suggest that they might play important roles during oogenesis and in the regulation of early embryonic gene regulation. The developmental functions of the distinct GNAT subunits in D. melanogaster are being investigated (Kusch, 2003).

It is interesting that both flies and mammals contain two subgroups of Ada2 proteins, namely, the Ada2a-like and Ada2b-like adapter proteins. Thus far, no sequence motif that is specific for either of the subgroups has been identified; however, phylogenetic comparisons strongly suggest that both dAda2a and dAda2b from flies and mammals form distinct subgroups. This is supported by findings that the Drosophila members of these subgroups are in distinct multiprotein complexes. It remains to be demonstrated that the human or mouse homologues have similar features and associate with distinct GNAT complexes. At present, only the human PCAF complex has been shown to contain a homologue of Ada2a. The PCAF complex contains subunits that are characteristic of SAGA-type complexes, suggesting that mammalian Ada2a homologues can associate with SAGA-type GNAT complexes. Nevertheless, phenotypic studies of mice have demonstrated that PCAF is dispensable for viability whereas the ubiquitously expressed Gcn5L protein is essential for embryogenesis. Thus, Gcn5L-containing complexes appear to be true functional homologues of SAGA-type complexes from flies and yeast. Intriguingly, the human Ada2a homologue was not detected in the Gcn5L-containing STAGA and TFTC complexes. Although an uncharacterized protein of the apparent molecular mass of the human Ada2b-like protein copurifies with STAGA, it remains to be demonstrated that these complexes contain Ada2b-like adapter proteins as do their fly homologues (Kusch, 2003).

All Ada2 proteins so far characterized show homologies within three conserved regions that mediate important functions of these proteins. The most N-terminal ZZ finger motif has been shown to be essential for the interactions of Ada2 and Gcn5 in vitro. The second conserved region is the Myb/SANT domain. Myb/SANT domains have been found in the Myb protein family as well as in several other chromatin-associated proteins, such as Swi3, TFIIIB, and the nuclear receptor corepressor N-CoR. Deletions of this domain cripple the ability of Ada2 to support transcriptional activation in vivo. Recent studies have suggested that this domain is important for the proper acetylation of nucleosomal histones. This is consistent with the finding that Ada2 potentiate the catalytic function of Gcn5. A third central region of high homology has been shown to mediate interaction with Ada3 in vitro. These three regions are conserved in virtually all Ada2 proteins so far characterized, supporting a tight functional link between Ada2- and Gcn5-containing complexes. The association of either of the two Drosophila Ada2 homologues is unexpectedly specific for distinct GNAT complexes. Only dAda2b associates with the dSAGA-specific subunits dmTaf5, dmTaf9, dmTaf10, dSpt3, and dTra1, and dAda2b-containing GNAT complexes have features that are typical of SAGA-related complexes from yeast and humans. dAda2a, however, exhibits an even more unexpected promiscuity and is incorporated into a large multiprotein complex lacking dGcn5. The possibility that a second copy of Gcn5 exists in flies as it does in mammals cannot be excluded and that this second Gcn5 homologue is a catalytic subunit of the larger dAda2a-containing complexes. However, several lines of evidence do not support this. Ada3 has been found in all GNAT complexes so far characterized and is essential for the acetyltransferase activities and specificities of these complexes. One would therefore expect dAda3 to be associated with the 2.5-MDa dAda2a-containing complexes. However, dAda3 did not coelute with these complexes in chromatography assays. Immunocolocalization studies of polytene chromosomes revealed that dAda2a is enriched at sites with high levels of RNA polymerase II-dependent transcription, but these puffed regions do not stain strongly for dAda3 or dGcn5. It therefore appears likely that dAda2a functions within large complexes independently of dAda3 and Gcn5, proteins with which Ada2 functions had previously been associated. Intriguingly, the large dAda2a-containing complex localizes to chromosomal regions with high RNA polymerase II activities, pointing to a novel transcription-linked function of this Ada2 variant (Kusch, 2003).

Although it will be necessary to identify subunits that are specific for these non-GNAT dAda2a complexes, several lines of evidence suggest that the ratio between distinct dAda2a-containing complexes might vary during the Drosophila life cycle. (1) Three different types of dAda2a-containing complexes have been found in nuclear extracts from 0- to 12-h-old embryos. In S2 cells, which derive from 16- to 24-h-old male embryos, one of these complexes was not detectable. (2) Antibodies against dAda2a immunoprecipitate relatively large amounts of dGcn5 from S2 nuclear extracts; however, only a little overlap between dAda2a and dGcn5 or dAda3 was observed on polytene chromosomes from third-instar larvae. Although the larger dAda2a complexes in general seem to be more abundant than the 440-kDa complex, these findings were rather unexpected. Nevertheless, developmental-expression studies indicate that dAda2a concentrations are significantly higher in 13- to 24-h-old embryos, from which S2 cells derive. In a third-instar larva, however, dAda2a levels are decreased relative to those in embryos or earlier larval stages. (3) While the concentrations of dGcn5 and other dSAGA-specific subunits, such as dSpt3 or dAda2b, are relatively low in adult male flies, the amounts of dAda2a are equally large in male and female flies. This indicates that the amount of dAda2a is not tightly linked to the levels of dGcn5 expression and suggests that the ratio between distinct dAda2a-containing complexes might be variable (Kusch, 2003).

Drosophila Ada2b is required for viability and normal histone H3 acetylation

Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interacts with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. Flies were generated that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein has been shown to interact with the SAGA-like complex in vitro, p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, it is speculated that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent (Qi, 2004).

The Drosophila Ada2b protein is present in a SAGA-like HAT complex. In accordance, dAda2b and dGcn5 directly interact and are expressed in similar patterns during embryogenesis. Ada2b is required for viability in Drosophila. Although dAda2b protein levels become greatly reduced in mutant embryos at stage 15, the animals survive until the pupal stage. Concomitant with reduced dAda2b protein amounts, a significant reduction is observed in acetylation of lysines 14 and 9 in histone H3. Lysine 14 acetylation is most severely affected, since a reduced staining intensity is observed even in dAda2b heterozygous embryos compared to the wild type. The preference for H3 K14, and the selective reduction in histone H3 over histone H4 acetylation, fits the substrate specificity of Gcn5. Furthermore, Gcn5's presence in the SAGA complex is dependent on Ada2 in yeast. It is therefore assumed that the loss of histone H3 acetylation in dAda2b mutants results from diminished dGcn5 activity, either as a consequence of reduced catalytic activity of the enzyme or due to a failure to target dGcn5 to chromatin (Qi, 2004).

The reduced histone H3 acetylation levels appear to persist during development; third-instar larval salivary gland chromosomes retained a similar reduction in H3 acetylation. Interestingly, chromatin acetylation is globally affected in dAda2b mutants, rather than affecting a subset of genes. Therefore, dAda2b is not likely to function solely as an adaptor that brings dGcn5 to only some promoter regions through associations with transcriptional activators. Instead, it is probable that dAda2b has a function similar to that of yeast Ada2, which is to retain Gcn5 within the SAGA complex and to stimulate its catalytic activity. This result also suggests that the SAGA complex, or at least a dAda2b-dGcn5 complex, has functions in addition to transcription-coupled gene-specific acetylation. Some heterochromatic regions with low gene density that stain with DAPI contain acetylated histone H3. These regions exhibit a decrease in H3 acetylation similar to that of other parts of the chromosomes (Qi, 2004).

Drosophila Gcn5 is present in complexes in addition to the dAda2b-containing SAGA-like complex. The fraction of dGcn5 that associates with dAda2b makes a significant contribution to the overall levels of histone H3 acetylation, and association with dAda2a cannot substitute for dGcn5's function within the SAGA complex. Interestingly, normal histone H3 acetylation appears to be dispensable for development from at least mid-embryogenesis until pupation. Furthermore, it is not known if the lethality observed at pupation is caused by reduced histone acetylation or by another function of dAda2b. In addition to its role in pupal development, dAda2b is also required for oogenesis. Females with dAda2b homozygous germ cells lack developed ovaries (Qi, 2004).

All Ada2 proteins share a ZZ zinc finger and a SANT domain in their N termini. The minimal Gcn5 interaction domain includes both the ZZ domain and the SANT domain, and the SANT domain additionally stimulates Gcn5's catalytic activity (Boyer, 2002; Sterner, 2002). The dAda2b gene is alternatively spliced, generating proteins with different C termini. Both protein isoforms contain the ZZ and SANT domains, but the longer isoform lacks another conserved element, Ada box 3. However, no evidence was found for differential usage of the two proteins during development. The Ada2-Gcn5 interaction, although conserved in evolution, is species specific. For example, human Ada2a cannot interact with yeast Gcn5, and both Drosophila Ada2a and Ada2b fail to interact with yeast Gcn5. As might be expected from these results, dAda2b fails to complement a yeast Ada2 mutant (Qi, 2004).

It is believed that the SAGA complex is recruited to target genes through interactions with sequence-specific transcription factors. At the promoter, the Gcn5 subunit acetylates histone H3 and perhaps other proteins, whereas the Spt 3 subunit facilitates preinitiation complex assembly by interacting with TATA binding protein. Several yeast activator proteins, including Gcn4, Pho4, and Gal4, require the SAGA complex for activity and recruit it to target genes in vivo. The activator proteins VP16 and Drosophila p53 can precipitate the SAGA-like complex from a Drosophila S2 cell nuclear extract (Kusch, 2003). Whether this interaction is of significance for the in vivo function of the Drosophila p53 protein was tested. p53-induced apoptosis and reaper gene expression in response to DNA damage are not impaired in dAda2b mutants. This result indicates that the Ada2/Gcn5 subcomplex of SAGA is not necessary for p53 function, but it does not exclude the possibility that p53 activity requires other SAGA components. In fact, many yeast genes show a differential requirement for the Ada2/Gcn5 and Spt3 subcomplexes of SAGA (Qi, 2004).

Unexpectedly, increased apoptosis in response to gamma radiation in dAda2b mutants was found. Irradiation causes DNA damage, which leads to activation of ATM/ATR kinases that phosphorylate a variety of substrates, including the Chk2 kinase, ultimately resulting in p53 activation. In Drosophila, p53 activation brings about apoptosis but does not lead to a G1 cell cycle arrest, which is another possible outcome in mammalian cells. It is possible that the reason for increased apoptosis in dAda2b mutants is that dAda2b normally has an inhibitory role in the generation of a DNA damage signal or in controlling p53 function. Another possibility is that DNA repair is not as efficient in dAda2b mutants as in the wild type, leading more cells to enter the apoptosis program. The role of dAda2b in DNA repair could be indirect. For example, dAda2b could be necessary for proper expression of a DNA repair factor. Alternatively, dAda2b might have a more direct function in DNA repair, presumably through chromatin modification. In dAda2b mutants, excessive apoptosis occurs after irradiation but not before irradiation or when apoptosis is induced by an alternative means. Furthermore, the extra apoptosis is p53 dependent. For this reason, it is unlikely that increased apoptosis in dAda2b mutants is the consequence of a higher sensitivity to stress in general. Instead, it is believed that dAda2b has a role in the p53-dependent pathway that is activated by DNA damage (Qi, 2004).

All eukaryotes respond to DNA lesions with activation of ATM/ATR kinases, although the downstream component p53 is absent from unicellular organisms such as the yeast S. cerevisiae. If the involvement of dAda2b in the DNA damage response is upstream of p53 activation, one would predict this function to be conserved in evolution. In fact, yeast strains lacking Ada2p, or Gcn5p or Ada3p, are sensitive to the genotoxic agent MMS. It is possible, therefore, that histone acetylation facilitates DNA repair or generates a DNA damage signal. Intriguingly, an increase in radiation-induced apoptosis of a magnitude similar to that observed in dAda2b mutants was observed in larvae in which the C terminus of a histone variant, H2Av, is missing. The H2Av C terminus contains a conserved serine that is phosphorylated in response to DNA double-strand breaks. Apparently, the absence of this phosphorylation event reduces DNA repair efficiency, which increases the amount of apoptosis twofold. Perhaps histone acetylation likewise contributes to the generation of a DNA damage signal, and maybe this is why chromatin is globally acetylated by Ada2/Gcn5. Future studies will be aimed at investigating this possibility (Qi, 2004).

The homologous Drosophila transcriptional adaptors ADA2a and ADA2b are both required for normal development but have different functions

In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. This study describes mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, allowing the functional study of individual ADA2s. Both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct (Pankotai, 2005).

Intimate relationship between the genes of two transcriptional coactivators, ADA2a and PIMT, of Drosophila

PIMT, a transcriptional coactivator which interacts with and enhances nuclear receptor coactivator PRIP function, was identified recently in mammalian cells and was suggested to function as a link between two major multiprotein complexes anchored by CBP/p300 and PBP. The Drosophila homologue of PIMT, designated as Dtl, is closely associated and has an overlapping promoter with a gene encoding another transcriptional coactivator, ADA2a, which in turn participates in GCN5 HAT-containing complexes. Ada2a also produces an RNA polII subunit, RPB4, via alternative splicing; consequently, an overlapping regulatory region serves for the production of three proteins, each involved in transcription. By studying expression of reporter gene fusions in tissue culture cells and transgenic animals it has been demonstrated that the regulatory regions of Ada2a/Rpb4 and Dtl overlap and the Dtl promoter is partly within the Ada2a/Rpb4 coding region. The shared regulatory region contains a DRE element, binding site of DREF, the protein factor involved in the regulation of a number of genes that play a role in DNA replication and cell proliferation. Despite the perfectly symmetrical DRE, DREF seems to have a more decisive role in Ada2a/Rpb4 transcription than in the transcription of Dtl (Papai, 2005).

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

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 have been identified and they have been shown to 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, novel components have been sought 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 (Guelman, 2006).

This work describes the affinity purification of Drosophila ATAC (Ada two A containing) HAT complex. dGcn5-containing complexes were purified and subjected to MudPIT analysis. MudPIT is an unbiased and sensitive mass spectrometry procedure that allows the identification of proteins in a complex mixture without prior electrophoretic separation Two new proteins were discovered that copurified with TAP-dGcn5: Atac1 (cg9200) and dHCF. Antibodies were generated against these two proteins and then utilized in a number of biochemical experiments that allowed demonstration that Atac1 and dHCF are stable components of the ATAC complex but not of dSAGA. To gain insight into the function of Atac1, homologues were sought in other species. Atac1 shares 35% identity with a human protein named zzz3 (NP_056349, 903 amino acids), which contains both a SANT and ZZ domain. However, no function has been assigned to this protein yet (Guelman, 2006).

Many chromatin remodelers and modifiers harbor a SANT domain in one of their subunits. Some examples include the yeast complexes SAGA, ADA, NuA4, RPD3, SWI/SNF, RSC, and ISW1/2. This 50-amino-acid motif was shown to be critical for acetylation by SAGA and remodeling by SWI/SNF. For example, SAGA complex purified from strains carrying a deletion in the SANT domain of Ada2 is intact but shows a drastic reduction in nucleosomal acetylation. There are a few cases where two SANT domains are present in the same complex. The nuclear receptor corepressor SMRT (silencing mediator of retinoid and thyroid receptors), which contains a pair of SANT domains, exists in a HDAC3-containing repressor complex. While the N-terminal SANT1 binds and activates HDAC3, its adjacent motif SANT2 is required for histone interaction. These two motifs are not functionally interchangeable. The fact that the ATAC HAT complex also contains two SANT domains is intriguing: one in dAda2A and the other one in Atac1. The possibility is considered that the former, like its yeast homologue, is involved in enhancing dGcn5 catalytic activity. Atac1, in contrast, could utilize its SANT domain for binding to nucleosomes or for other protein-protein interactions (Guelman, 2006).

Drosophila has only one orthologue that belongs to the HCF family of proteins, present in higher eukaryotes. This protein shares a high degree of conservation with human HCF-1. Analysis of the primary structure of dHCF does not reveal the presence of HCFpro repeats found in HCF-1. However, experiments done with S2 cells that express dHCF tagged at both ends demonstrated that this protein is cleaved after translation to generate two polypeptides that remain physically associated. By generating antibodies directed to the C terminus of dHCF, it was possible to confirm those results with the endogenous protein. No full-length protein was detected by Western blot analysis. Therefore, it is suggested that most of the dHCF precursor gets processed. Additionally, more than one form of dHCF was identified using the antiserum that recognizes the C terminus. It is believed that this reflects multiple processing sites in dHCF. This observation agrees with experiments done with HCF-1, which show that antibodies that recognize the N- or C-terminal subunits display several bands on Western blots (Guelman, 2006).

Peptide hits from the MudPIT analysis of affinity-purified ATAC complex have identified peptides corresponding to the N- and C-terminal subunits of dHCF. This finding indicates that both subunits are components of the ATAC HAT complex. Furthermore, antibodies against the dHCF C terminus coimmunoprecipitated the ATAC complex. Unfortunately, it was not possible to obtain antibodies against the N-terminal fragment to perform similar experiments (Guelman, 2006).

Anion-exchange fractionation revealed that dHCF is present in at least two other complexes that elute at different salt concentrations. The possibility of dHCF being present in other dGcn5-containing complexes was ruled out for two reasons: (1) immunoprecipitations using dSpt3 antibodies does not pull down dHCF; (2) S2 cell lines were establised expressing tagged dSAGA-specific subunits, complexes were affinity purified from these cells, and subjected to MudPIT. Peptides for dHCF were not identified in those complexes, leading to the conclusion that dHCF is uniquely associated with the ATAC HAT complex. Since dHCF is an abundant protein, it was not surprising to find it in column fractions where the ATAC complex was not present. It is likely that dHCF is a component of other chromatin-modifying complexes. In fact, the N-terminal subunit of human HCF-1 forms a supercomplex that contains subunits of the Sin3 histone deacetylase complex and the Set1/Ash2 histone methyltransferase. In addition, immunopurifications with antibodies directed to the proto-oncoprotein MLL reveal associations of HCF-1, HCF-2, menin (see Drosophila Menin-1), and components of the Set1/Ash2 complex (Guelman, 2006).

What could be the biological relevance of dHCF in a HAT complex? There is a strong link between HCF-1 and transcription. Experiments done with HCF-1 indicate that its N- and C-terminal subunits interact with cellular transcription activators (Luciano, 2000; Luciano, 2003). This could be a mechanism by which the ATAC complex is recruited to specific promoters. In addition, the possibility cannot be ruled out that the ATAC complex has a role during the cell cycle, since it is known that mammalian HCF-1N and HCF-1C are crucial for G1 phase progression and proper cytokinesis. Furthermore, yeast cells carrying deletions in Gcn5 and Sas3 genes arrest at G2/M, indicating that H3 HATs are essential for cell cycle progression. Mammalian cells lacking Gcn5 exhibit a delay in the progression from G1 to S phase and present abnormal levels of expression of cell cycle-related genes (Guelman, 2006).

It is anticipated that there are additional unidentified subunits in the ATAC complex. (1) The estimated molecular mass of the complex is 700 kDa. The sum of the molecular masses of the known subunits (dGcn5, dAda3, dAda2A, Atac1, and dHCF) is approximate 450 kDa, which suggests there could be few more subunits in this complex. (2) MudPIT analyses from TAP-dGcn5, dAda2A-TAP, and Atac1-FLAG purifications have revealed unconfirmed candidate proteins, one of which contains a putative HCF binding motif, identified in transcription factors that interact with the ß-propeller region of HCF-1. The identification of all the components of this novel HAT complex should provide additional evidence to help uncover its function in cell growth and Drosophila development (Guelman, 2006).

Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. This study analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5' end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression (Weake, 2011).

SAGA has been purified from Drosophila and mammalian cells and was found to contain homologs of most of the yeast SAGA subunits, including the Gcn5 (see Drosophila Pcaf) and Ubp8 catalytic subunits. In metazoans, SAGA may have roles in both normal development and cancer (Koutelou, 2010). Individual loss of the SAGA subunits Gcn5, Ada2b, Ada3, WDA, Sgf11, and SAF6 results in developmental defects and larval lethality in flies (Weake, 2009 and references therein). Similarly, Gcn5 deletion in mice leads to defects in mesoderm development and embryonic lethality (Xu, 2000). However, catalytic site mutations in Gcn5 survive longer but suffer neural tube closure defects and exencephaly (Bu, 2007). Furthermore, loss of the ubiquitin protease in Drosophila SAGA (Nonstop) leads to defects in photoreceptor axon targeting followed by lethality at late larval stages (Weake, 2011).

A system was designed in which SAGA could be isolated from different cell types in Drosophila embryos so that its composition and localization pattern could be determined in different tissues. To this end, the GAL4/UAS system was used to express a Flag-HA tagged version of the SAGA-specific protein Ada2b (Ada2bH1F2) in muscle or neuronal cells using the mef2-GAL4 and elav-GAL4 drivers, respectively. Expression of Ada2bH1F2 under the control of its genomic enhancer sequences rescues viability of the lethal ada2b1 allele. Whereas mef2 is expressed in committed mesoderm, the somatic and visceral musculature, and cardiac progenitors, elav is expressed prominently in neuronal cells and transiently in glial cells of the embryonic CNS. Ada2bH1F2 is expressed at levels similar to those of endogenous Ada2b using this system. To enrich for cell populations of interest that express tagged Ada2b, muscle and neuronal cells were labelled using GFP, and these cells were isolated using fluorescence-activated cell sorting (FACS). To determine whether the purified cells exhibit the characteristic gene expression profiles of each cell type, GFP-labeled neuronal and muscle cells were isolated from late stage embryos by FACS. RNA isolated from these tissues was compared with RNA extracted from whole embryos using cDNA microarrays, and genes were identified that are differentially expressed in muscle or neurons using significance analysis of microarrays. The differentially expressed genes identified using this approach were compared with ImaGO terms that describe the expression pattern of individual genes during Drosophila embryogenesis as determined by in situ hybridization. Genes identified as being expressed preferentially in muscle relative to neurons were enriched for ImaGO terms including embryonic/larval muscle system and dorsal prothoracic pharyngeal muscle. In contrast, genes identified as being expressed preferentially in neurons were enriched for ImaGO terms such as ventral nerve cord and dorsal/lateral sensory complexes. It is concluded that cells isolated using the FACS approach are enriched for the cell types of interest (Weake, 2011).

This study examined SAGA composition and localization in muscle and neuronal cells of late stage Drosophila embryos. Surprisingly, extensive colocalization of SAGA with Pol II was observed at both promoters and coding regions in muscle cells. Notably, genes at which SAGA was not detected in this assay have low levels of Pol II bound. It is suggested that SAGA might be important for recruitment and/or retention of high levels of Pol II at the promoter-proximal pause site in flies, and perhaps, therefore, more generally in higher eukaryotes. SAGA has been previously observed on the coding sequence of a small number of individual transcribed genes in yeast. Recently, low levels of Ada2b were detected on the 3' region of several different genes during larval development (Zsindely, 2009). It is noted that although some SAGA is present across the coding region of many genes, the peak of acetylated H3-Lys9 is restricted to the 5' region of the two genes that were examined: wupA and exba. A similar 5' bias of acetylated H3-Lys9 has been observed previously in genome-wide studies of histone modifications. It is speculated that the acetylation activity of SAGA in the 3' region of the gene is counteracted by histone deacetylases such as Rpd3S that have been shown to associate with the elongating form of Pol II (Weake, 2011).

SAGA localizes to different genes in muscle and neurons of late stage Drosophila embryos, and the number of genes bound by the complex in each tissue correlates with the number of transcription factors associated with the complex. These findings indicate that the differential localization of SAGA may be regulated by its association with different transcription factors in different cell types. A number of studies have found that transcription factor-binding sites tend to be clustered within the fly genome. This observed colocalization of transcription factors, together with the current data showing the association of SAGA with a large number of different transcription factors, indicates that multiple transcription factors might be involved in recruiting SAGA to its target genes (Weake, 2011).

SAGA is present at the promoter-proximal pause site together with Pol II at genes that are stalled or infrequently transcribed. The presence of SAGA together with paused Pol II is consistent with a role for SAGA in post-initiation deubiquitination of H2B, which has been shown in yeast to be important for phosphorylation of Ser-2 of the Pol II CTD and its subsequent transition into transcription elongation. In flies, phosphorylation of Ser-2 of the Pol II CTD by P-TEFb is also required for release of the paused polymerase into transcription elongation. Hence, the strong colocalization of SAGA with polymerase that has initiated transcription but is paused prior to elongation suggests a prominent function for SAGA in regulating tissue-specific gene expression at a step occurring post-initiation in metazoans. Consistent with the possibility, it was observe that the SAGA-bound genes that are most dependent on its ubiquitin protease activity for full expression are preferentially expressed in a specific tissue (Weake, 2011).


Gcn5 ortholog: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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