Gene name - Gcn5 ortholog
Synonyms - Pcaf
Cytological map position - 69C8--12
Symbol - Gcn5
FlyBase ID: FBgn0020388
Genetic map position - 3L
Classification - histone acetyltransferase, bromodomain, PCAF (N-terminal)
Cellular location - nuclear
|Ali, T., Krüger, M., Bhuju, S., Jarek, M.,
Bartkuhn, M. and Renkawitz, R. (2016). Chromatin
binding of Gcn5 in Drosophila is largely mediated by CP190.
Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27903907
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. This study searched for enzymes which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 colocalize with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency is further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. The dCas9 system was used to target CP190 or Gcn5 to a Polycomb repressed and H3K27me3 marked gene locus; both CP190 as well as Gcn5 activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.
|Liu, T., Wang, Q., Li, W., Mao, F., Yue, S.,
Liu, S., Liu, X., Xiao, S. and Xia, L. (2017). Gcn5
determines the fate of Drosophila germline stem cells through
degradation of Cyclin A. FASEB J [Epub ahead of print]. PubMed
The fluctuating CDK-CYCLIN complex plays a general role in cell-cycle control. Many types of stem cells use unique features of the cell cycle to facilitate asymmetric division. However, the manner in which these features are established remains poorly understood. The cell cycle of Drosophila female germline stem cells (GSCs) is characterized by short G1 and very long G2 phases, making it an excellent model for the study of cell cycle control in stem cell fate determination. Using a Drosophila female GSCs model, this study found Gcn5, the first discovered histone acetyltransferase, to maintain germline stem cells in Drosophila ovaries. Gcn5 is dispensable for the transcriptional silencing of bam, but interacts with Cyclin A to facilitate proper turnover in GSCs. Gcn5 promotes Cyclin A ubiquitination, which is dependent on its acetylating activity. Finally, knockdown of Cyclin A rescues the GSC-loss phenotype caused by lack of Gcn5. Collectively, these findings support the conclusion that Gcn5 acts through acetylation to facilitate Cyclin A ubiquitination and proper turnover, thereby determining the fate of GSCs.
|Torres-Zelada, E. F., Stephenson, R. E., Alpsoy, A., Anderson, B. D., Swanson, S. K., Florens, L., Dykhuizen, E. C., Washburn, M. P. and Weake, V. M. (2019). The Drosophila Dbf4 ortholog Chiffon forms a complex with Gcn5 that is necessary for histone acetylation and viability. J Cell Sci 132(2). PubMed ID: 30559249
Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes, respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. This study shows that only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon, forming the Chiffon histone acetyltransferase (CHAT) complex. Chiffon is the Drosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, this study finds that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, the CHAT-binding domain is essential for viability, but is not required for gene amplification, arguing against a role in DNA replication.
|Soffers, J. H. M., Li, X., Saraf, A., Seidel, C. W., Florens, L., Washburn, M. P., Abmayr, S. M. and Workman, J. L. (2019). Characterization of a metazoan ADA acetyltransferase complex. Nucleic Acids Res. PubMed ID: 30715476
The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been described in metazoans. Previous studies in Drosophila hinted at the existence of a small complex which contains Ada2b, a partner of Gcn5 in the SAGA complex. This study has purified and characterized the composition of this complex and shows that it is composed of Gcn5, Ada2b, Ada3 and Sgf29. Hence, it was named the metazoan 'ADA complex'. The fly ADA complex has histone acetylation activity on histones and nucleosome substrates. Moreover, ChIP-Sequencing experiments identified Ada2b peaks that overlap with another SAGA subunit, Spt3, as well as Ada2b peaks that do not overlap with Spt3 suggestingthat the ADA complex binds chromosomal sites independent of the larger SAGA complex.
|Wang, Y., Huang, Y., Liu, J., Zhang, J., Xu, M., You, Z., Peng, C., Gong, Z. and Liu, W. (2019). Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB. EMBO Rep: e48335. PubMed ID: 31750630
Accumulating evidence highlights the role of histone acetyltransferase GCN5 in the regulation of cell metabolism in metazoans. This study reports that GCN5 is a negative regulator of autophagy, a lysosome-dependent catabolic mechanism. In animal cells and Drosophila, GCN5 inhibits the biogenesis of autophagosomes and lysosomes by targeting TFEB, the master transcription factor for autophagy- and lysosome-related gene expression. GCN5 is a specific TFEB acetyltransferase, and acetylation by GCN5 results in the decrease in TFEB transcriptional activity. Induction of autophagy inactivates GCN5, accompanied by reduced TFEB acetylation and increased lysosome formation. It was further demonstrated that acetylation at K274 and K279 disrupts the dimerization of TFEB and the binding of TFEB to its target gene promoters. In a Tau-based neurodegenerative Drosophila model, deletion of dGcn5 improves the clearance of Tau protein aggregates and ameliorates the neurodegenerative phenotypes. Together, these results reveal GCN5 as a novel conserved TFEB regulator, and the regulatory mechanisms may be involved in autophagy- and lysosome-related physiological and pathological processes.
|Soffers, J. H. M., Alcantara, S. G., Li, X., Shao, W., Seidel, C. W., Li, H., Zeitlinger, J., Abmayr, S. M. and Workman, J. L. (2021). The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters. PLoS Genet 17(11): e1009668. PubMed ID: 34807910
The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. This study analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. Depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. These data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.
|George, S., Blum, H. R., Torres-Zelada, E. F., Estep, G. N., Hegazy, Y. A., Speer, G. M. and Weake, V. M. (2022). The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species. Insect Mol Biol 31(6): 734-746. PubMed ID: 35789507
Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, yeast two-hybrid assays were performed using Chiffon-B and Gcn5 from a variety of insect species; Chiffon-B and Gcn5 were found to interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. It is proposed propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.
|Farago, A., Zsindely, N., Farkas, A., Neller, A., Siagi, F., Szabo, M. R., Csont, T. and Bodai, L. (2022). Acetylation State of Lysine 14 of Histone H3.3 Affects Mutant Huntingtin Induced Pathogenesis. Int J Mol Sci 23(23). PubMed ID: 36499499
Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine-coding CAG repeat in the Huntingtin gene. One of the main causes of neurodegeneration in HD is transcriptional dysregulation that, in part, is caused by the inhibition of histone acetyltransferase (HAT) enzymes. HD pathology can be alleviated by increasing the activity of specific HATs or by inhibiting histone deacetylase (HDAC) enzymes. To determine which histone's post-translational modifications (PTMs) might play crucial roles in HD pathology, this study investigated the phenotype-modifying effects of PTM mimetic mutations of variant histone H3.3 in a Drosophila model of HD. Specifically, the mutations (K→Q: acetylated; K→R: non-modified; and K→M: methylated) of lysine residues K9, K14, and K27 of transgenic H3.3 was studied. In the case of H3.3K14Q modification, the amelioration was observed of all tested phenotypes (viability, longevity, neurodegeneration, motor activity, and circadian rhythm defects), while H3.3K14R had the opposite effect. H3.3K14Q expression prevented the negative effects of reduced Gcn5 (a HAT acting on H3K14) on HD pathology, while it only partially hindered the positive effects of heterozygous Sirt1 (an HDAC acting on H3K14). Thus, it is concluded that the Gcn5-dependent acetylation of H3.3K14 might be an important epigenetic contributor to HD pathology.
|Ciabrelli, F., Rabbani, L., Cardamone, F., Zenk, F., Loser, E., Schachtle, M. A., Mazina, M., Loubiere, V. and Iovino, N. (2023). CBP and Gcn5 drive zygotic genome activation independently of their catalytic activity. Sci Adv 9(16): eadf2687. PubMed ID: 37083536
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. This study used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. The screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. It was demonstrated that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, these data identify Nejire and Gcn5 as key regulators of ZGA.
|Li, J., Cao, Y., Yang, Y., Ma, H., Zhao, J., Zhang, Y. and Liu, N. (2023). Quantitative Acetylomics Reveals Substrates of Lysine Acetyltransferase GCN5 in Adult and Aging Drosophila. J Proteome Res. PubMed ID: 37545086
Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Useing Drosophila, this study performed label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. The dynamics were further characterize of protein acetylation with age, that exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, it was reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, this study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.
Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure and gene transcription, there is only little information on their developmental role in higher organisms. Pcaf, alternatively called Gcn5, was the first transcription factor with HAT activity identified in eukaryotes. Null alleles of Drosophila Gcn5 block the onset of both oogenesis and metamorphosis, while hypomorphic Gcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and K14 lysine residues of histone H3 in Gcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are observed. In vivo complementation experiments have revealed that Gcn5 integrates specific functions in addition to chromosome binding and acetylation. Defects displayed by Gcn5 mutant adults rescued by the Gcn5DeltaPcaf variant (deficient in the N-terminal Pcaf specific motif) suggest a role of Gcn5 in the ecdysone regulatory hierarchy during metamorphosis, possibly through interactions with nuclear receptors. Surprisingly, a Gcn5 variant protein with a deletion of the bromodomain, which has been shown to recognize acetylated histones, appears to be fully functional. These results establish Gcn5 as a major histone H3 acetylase in Drosophila that plays a key role in the control of specific morphogenetic cascades during developmental transitions (Carré, 2005).
Gene expression in eukaryotes has to accommodate the presence of nucleosomes and the packaging of DNA into higher-order chromatin structures. Nucleosomes are composed of octamers of histone proteins H2a, H2b, H3, and H4, whose N-terminal tails project outward from the nucleosomal core and are subjected to covalentmodifications such as acetylation, methylation, phosphorylation, and ubiquitination. The variety of these modifications and their association with distinct states of gene transcription suggested that they may act as a combinatorial code to specify downstream events such as the recruitment of transcription factors or modifications of the chromatin structure (Carré, 2005).
The acetylation of lysine residues is one of the most studied histone modifications and has long been linked to gene activation. For instance, a twofold up-regulation of transcription from the male X chromosome in Drosophila is correlated with histone hyperacetylation, while gene silencing in heterochromatin or X chromosome inactivation in mammals is correlated with histone hypoacetylation. Numerous sequence-specific activators, such as the nuclear receptors MyoD and CREB, have been shown to recruit coactivator complexes with histone acetyltransferase (HAT) activity, while transcriptional repressors have been found associated with corepressor complexes with histone deacetylase activity. HAT activity is also associated with more general transcription factors, such as TATA-binding protein-associated factor 1 (TAF1) and yeast elongation factor 3. Collectively, these data point to a causal role of histone acetylation in transcriptional activation. In support of this hypothesis, the acetylation of lysine 8 in histone H4 (H4-AcK8) and lysine 14 in histone H3 (H3-AcK14) has been implicated in the sequential recruitment of transcription factors leading to the activation of the human beta interferon gene in vitro, and distinct patterns of histone acetylation have been associated with groups of coexpressed genes in genome-wide studies (Carré, 2005).
The yeast adaptor Gcn5 was the first transcription factor identified as a bona fide HAT. It defines a family of evolutionarily conserved Gcn5-related N-acetyltransferases (GNATs) whose members were purified as essential subunits of ADA and SAGA (Spt-Ada-Gcn5 acetyltransferase) complexes in yeast and of PCAF, STAGA, and TFTC complexes in mammals. The GNAT complexes contain Ada transcriptional adapters, Spt proteins, and a set of TATA-binding protein-associated factors, and they are structurally related, suggesting that they perform similar functions in transcription. In vitro, Gcn5 acetylates lysine 14 of free, but not nucleosomal, histone H3. In contrast, Gcn5 acetylates an expanded set of lysines of nucleosomal histone H3 when copurified with native ADA or SAGA complexes (Grant, 1999), suggesting that one function of these complexes in vivo is to modulate the activity and specificity of Gcn5 (Carré, 2005).
Two distinct genes, hGCN5 and hPCAF, encode Gcn5 homologues in humans. Both hGCN5 isoforms, GCN5S and GCN5L, and hPCAF (P300/CBP-associated factor) share a close similarity with the complete yeast Gcn5 sequence, including more matches within the HAT catalytic domain as well as the bromodomain, which has been shown to bind acetylated histone lysines. PCAF and GCN5L share an additional N-terminal domain (Pcaf homology domain) of about 350 amino acids involved in binding to CBP/p300 and to several nuclear receptors. While the GCN5 gene knockout results in early embryonic lethality, the PCAF gene knockout has no detectable consequences on mouse development (Xu, 2000). However, GCN5-PCAF double mutants die earlier than single GCN5 mutants, indicating that PCAF and GCN5 functions are not completely redundant (Carré, 2005).
Although the Gcn5 HAT has been clearly involved in the control of Arabidopsis growth, development, and homeostasis (Bertrand, 2003; Vlachonasios, 2003), its contribution to the control of specific morphogenetic events during animal development remains poorly understood. There is only one Gcn5 homologue in Drosophila (Smith, 1998), which thus provides a simplified model for the study of the function of a GNAT in the context of a whole organism. Drosophila Gcn5 (dGcn5) has been isolated in at least two GNAT complexes that contain distinct Ada2 variants (Kusch, 2003; Muratoglu, 2003). A 1.8-MDa SAGA-like complex includes the Ada2b variant, the Ada3 and Spt3 homologues, and several TAFs. An Ada2b loss-of-function mutation is lethal and suppresses the histone H3 acetylation of polytene chromosomes (Qi, 2004), indicating that the SAGA-like complex plays an essential role in gene expression in Drosophila. In addition, Gcn5 associates with the Ada2a variant and with Ada3 in a 440-kDa non-SAGA-like complex. Interestingly, Ada2a and Ada2b mainly localize to different bands on polytene chromosomes, suggesting that GNAT complexes may play distinct functions depending on their composition (Carré, 2005).
In order to characterize the function of Drosophila Gcn5 and GNAT complexes during Drosophila development, two complementary approaches have been undertaken. Gcn5 loss-of-function mutants were isolated from a genetic screen, and in vivo targeting of Gcn5-specific RNA interference was performed, using inverted repeat transgenes. Gcn5 is shown to be the major HAT for the lysine residues K9 and K14 of histone H3, while the acetylation of histone H4 involves other HATs. These data indicate that Gcn5 is required for larva-to-adult metamorphosis and suggest an essential function of Gcn5 in the control of the cell cycle in imaginal tissues. An analysis of Gcn5 variant proteins revealed that the Pcaf homology domain, the domain of interaction with the Ada proteins, and the catalytic domain are all required for the function of the Gcn5 protein. In contrast, the bromodomain appears to be dispensable (Carré, 2005).
Gcn5 mutants have no discernible phenotype before the onset of metamorphosis. The considerable Gcn5 maternal stock may be sufficient to fulfill Gcn5 functions in mutant embryos. However, the Gcn5 protein is undetectable in Gcn5 mutant mid-third-instar larvae, and the proliferation of imaginal discs is already strongly impaired in such mutants at this stage. In addition, mutant larvae keep wandering when control animals have already formed a puparium, dramatically extending the duration of their third larval instar by 4 to 5 days before they die. Ada2b mutants die during pupal development (Qi, 2004), while Ada2a mutants fail to form a puparium in a manner very similar to that of Gcn5 mutant larvae (Pankotai, 2005). Together with these observations, the results strongly suggest that the function of the GNAT complexes is not required for Drosophila larval life (Carré, 2005).
The absence of normal induction of the three early puffs at 2B, 74EF, and 75B in Gcn5 mutants is indicative of a major failure in the regulatory hierarchy controlled by the steroid hormone ecdysone at the end of the third larval instar. The extended duration of this stage in Gcn5 null mutants is also characteristic of defects in pathways controlled by ecdysone. For instance, it is observed with EcR-B1-specific alleles of the ecdysone receptor gene and with mutant alleles of the broad gene. The Pcaf homology domain has been involved in interactions of human Pcaf with various nuclear receptors. Two Gcn5 hypomorphic alleles isolated in this work change amino acids in the Pcaf homology domain, resulting in a partial loss of function of the protein. Heteroallelic combination with the null Gcn5E333st allele led to impaired metamorphosis, with strong defects in adult appendage formation. Gcn5 adult mutant escapers rescued by the Gcn5DeltaPcaf variant consistently display very similar defects of leg elongation and blistered wings. These defects are also highly suggestive of an impaired ecdysone regulatory cascade during metamorphosis. From these observations, it is proposed that Gcn5 acts as a nuclear receptor coactivator at metamorphosis. The finding that the EcR protein is coimmunoprecipitated from embryonic nuclear extracts with Gcn5 in an ecdysone-dependent manner (A. Mazo, personal communication to Carré, 2005) points to the ecdysone receptor itself as a Gcn5 target. However, no interactions were detected between Gcn5 alleles and EcR mutant alleles in a trans-heterozygous genetic test. Although these results do not rule out a functional interaction between Gcn5 and the ecdysone receptor, they may also indicate that Gcn5 acts in the metamorphic ecdysone regulatory hierarchy through interactions with other Drosophila nuclear receptors (Carré, 2005).
This study shows that the lack of Gcn5 in the female germ line arrests oogenesis at an early stage. In addition, somatic Gcn5 mutant clones in ovarian follicles induce the formation of compound egg chambers indicative of an oocyte packaging defect (J. R. Huynh, personal communication to Carré, 2005). Together, these results indicate a strict requirement for Gcn5 during oogenesis, in both the germ line and the somatic line. In light of the role of Gcn5 during ecdysone-triggered metamorphosis, it is noteworthy that ecdysone regulatory hierarchies have also been shown to regulate Drosophila oogenesis (Carré, 2005).
Gcn5 mutant imaginal discs, as well as imaginal tissues silenced by Gcn5 RNAi, display slower cell proliferation. The role of CBP in the cell cycle has been documented for vertebrates, and a domain of interaction of CBP with the Gcn5 homologue Pcaf is targeted by viral and cellular factors (Schiltz, 2000) to regulate cell cycle progression (Carré, 2005).
The acetylation of E2F1 by Pcaf also results in cell cycle modulation (Martinez-Balbas, 2000). In yeast, Gcn5 regulates genes required for mitotic exit (Krebs, 2000). Together, these data suggest that functions of Gcn5 in cell cycle regulation have been conserved throughout evolution. Interestingly, a mouse knock-in mutant for Trrap, a conserved component of GNAT complexes, results in aberrant mitosis. Similarly, the higher mitotic index, together with the increased BrdU incorporation, of Gcn5-silenced imaginal cells suggests that they undergo an aberrant cell cycle. It has been shown that apoptosis induced in imaginal tissues by the loss of Gcn5 function may contribute to the net reduction in cell number. The Drosophila SAGA-like complex interacts in vitro with Dmp53 (Kusch, 2003), and several authors reported a role of dAda2b in DNA damage-induced p53-dependent apoptosis (Qi, 2003; Pankotai, 2005). However, this is the first time that apoptosis in a mutant with a deletion of a component of the Drosophila SAGA-like complex has been observed in the absence of X-ray-induced DNA damage. Strikingly, Gcn5 mouse mutants also display apoptosis in the absence of any exogenous inducers (Xu, 2000). Although further analysis is required to understand the complex roles of Gcn5 in cell proliferation, it is proposed that apoptosis in the Gcn5 mutant might be a consequence of cell cycle defects (Carré, 2005).
The data demonstrate that Gcn5 is the major acetylase for two distinct histone residues, H3 K9 and H3 K14, in Drosophila. In contrast, its contribution to histone H4 acetylation could not be detected in a global analysis. The loss of Ada2b results in a partial loss of H3 K9 and H3 K14 acetylation on polytene chromosomes, while the loss of Ada2a has no detectable effect on chromosome acetylation (Qi, 2004, Pankotai, 2005). In light of the results, these data suggest that the Drosophila GNAT complexes may retain partial Gcn5 HAT activity in the absence of the Ada2 components. Alternatively, Gcn5 could exert its HAT activity in other complexes which remain to be characterized. The substrate specificities of Drosophila and yeast Gcn5 proteins appear to be identical (Grant, 1999). However, it is interesting that in contrast to what was found for Drosophila, both the Gcn5 and Elp3 HATs must be invalidated in yeast to significantly impair histone H3 K9 and K14 acetylation (Wittschieben, 2000). Recent studies have extensively documented the relationship between histone H3 acetylation and gene transcription, but whether or not patterns of histone modification constitute a true code for the control of gene activity in eukaryotes is still a matter of debate. In either case, the results strongly suggest that specific histone acetylation profiles may be established in vivo through the activity of a very limited set of substrate-specific enzymes. The observation that Gcn5 mutant larvae survive for several days without detectable acetylation of H3 K9 and H3 K14 residues is striking and suggests that transcriptional regulation in larval versus embryonic or adult insect tissues involves distinct mechanisms (Carré, 2005).
Numerous data indicate that histone modifications can influence each other. The loss of H3 K9 and H3 K14 acetylation in imaginal discs or salivary glands has no detectable effect either on the levels of H3 S10 phosphorylation and H3 K4 methylation, both of which have been associated with transcriptional activation, or on the level of H3 K9 methylation, which marks HP1 recruitment and silencing. These results suggest that histone H3 acetylation is either a terminal or independent process in the cascade of histone H3 modifications (Carré, 2005).
Using transgenic Gcn5 variants, this study investigated the functions of the conserved regions in the Gcn5 protein. The deletion of the HAT domain completely abolishes the ability of Gcn5 to acetylate histones and to rescue Gcn5 mutant larvae. This result provides the first demonstration that Gcn5 in metazoans exerts its regulatory function during development through its histone acetylase activity. Defects displayed by Gcn5 mutant adults rescued by the Gcn5DeltaPcaf variant suggest a role of Gcn5 in the ecdysone regulatory hierarchy during metamorphosis, possibly through interactions with nuclear receptors. However, it is noteworthy that Gcn5DeltaPcaf retains important functions. The variant protein was distributed along polytene chromosomes similar to the dGcn5 wild-type protein and provided apparently normal H3 K9 and K14 acetylation. This suggests that the core functions of chromatin binding and substrate acetylation may be performed by the ancestral portion of Gcn5, which has been conserved throughout evolution from yeast to humans, and that the Pcaf homology domain has evolved to perform more specific functions in metazoans. The Ada2 interaction domain has been mapped in Gcn5 to an evolutionarily conserved region between the HAT domain and the bromodomain (Candau, 1996; Candau, 1997; Marcus, 1994). A direct interaction in vitro between dGcn5 and Ada2b was recently shown for Drosophila (Qi, 2004). The finding that the Gcn5DeltaAda variant was completely unable to rescue the lethality of the Gcn5 mutants at the time of puparium formation provides strong evidence that the interaction of dGcn5 with Ada2 proteins is crucial for the function of the Drosophila GNAT complexes. Strikingly, however, in Gcn5 mutants Gcn5DeltaAda could bind to polytene chromosomes and restore H3 K9 and H3 K14 acetylation patterns that were indistinguishable from those provided by the wild-type dGcn5 protein. This result is at odds with the finding that histone H3 acetylation is strongly reduced in Ada2b null mutant polytene chromosomes (Qi, 2004). It is possible that the loss of interaction between Ada2b and dGcn5 is deleterious for a specific function of the multiprotein SAGA-like complex but is not sufficient to disrupt the architecture of this complex as well as its ability to acetylate histones (Carré, 2005).
The bromodomain is conserved in a large number of transcription factors and has been shown to bind to acetylated lysines on histone tails. The yeast Gcn5 bromodomain has been shown to be involved in the stabilization of the SWI/SNF remodeling complex through interactions with acetylated nucleosomes at the PHO5 promoter (Syntichaki, 2000). The bromodomain-containing complexes SWI/SNF and TFIID have been shown to be targeted to acetylated histone H3 and H4 tails on the beta interferon promoter in vitro. Collectively, these findings suggest that the bromodomain is essential for targeting a large set of transcription factors to acetylated nucleosomes. In this context, the apparently normal chromosome acetylation by the Gcn5DeltaBromo variant, and most significantly, the complete rescue of Gcn5 mutant viability by this protein, raises a number of questions. The possibility cannot be excluded that the overexpression of the Gcn5DeltaBromo variant may overcome the lack of an essential Gcn5 function or that Gcn5DeltaBromo may be unable to supply an essential function under particular stress conditions. However, the observations clearly indicate that the Gcn5 bromodomain is not as strictly required as the other domains of the protein, since variants with truncations of these domains were expressed under the same conditions. Interestingly, others have reported a minor contribution of the bromodomain to the functions of various transcription factors. Notably, in yeast the deletion of the bromodomain of Drosophila Gcn5 has little consequence on its transcriptional activity (Candau, 1967; Sterner, 1999), while deletion of the bromodomain of Swi2/Snf2 or Spt7 has no phenotypic effect. Similarly, the Brahma protein with a deletion of its bromodomain binds normally to chromosomes in Drosophila and fully rescues brahma null mutants. The apparent lack of requirement for the bromodomain in Drosophila Gcn5 may be due to a functional redundancy of various components of Gcn5 complexes in targeting chromatin. This role has been proposed for the Spt7 factor and the large Tra1 protein, which are both components of the SAGA complex. The recent demonstration that a chromodomain of the yeast SAGA component Chd1 (see Drosophila Chd1) interacts with methylated lysine 4 of histone H3 also raises the interesting possibility that different factors in GNAT complexes interact with different histone modifications (Carré, 2005).
In summary, the deletion of the HAT domain in Gcn5 abolished both its HAT activity and its ability to rescue Gcn5 mutants. Gcn5 variants deleted in either the Pcaf homology domain or the Ada2 interaction domain acetylate chromosomes. However, Gcn5DeltaPcaf is able to partially rescue the Gcn5 mutants, while Gcn5DeltaAda is not. Finally, the deletion of the Gcn5 bromodomain has no noticeable consequences. This remarkable variety of effects revealed in complementation experiments strongly suggests that Gcn5 integrates multiple functions during development (Carré, 2005).
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).
PCAF and hGCN5 are distinct human genes that encode proteins related to the yeast histone acetyltransferase and transcriptional adapter GCN5. The PCAF protein shares extensive similarity with the 439 amino acids of yGCN5, but it has an approximately 350 amino acid N-terminal extension that interacts with the transcriptional co-activator p300/CBP. Adenoviral protein E1a can disrupt PCAF-CBP interactions and prevent PCAF-dependent cellular differentiation. This report describes the cloning and initial characterization of a Drosophila homolog of yGCN5. In addition to the homology to yGCN5, the Drosophila protein shares sequence similarity with the N-terminal portion of human PCAF that is involved in binding to CBP. In the course of characterizing dGCN5, it was discovered that hGCN5 also contains an N-terminal extension with significant similarity to PCAF. Interestingly, in the case of the h GCN5 gene, alternative splicing may regulate the production of full-length hGCN5. The presence of the N-terminal domain in a Drosophila GCN5 homolog and both human homologs suggests that it was part of the ancestral form of metazoan GCN5 (Smith, 1998).
To demonstrate that dGCN5 possesses HAT activity, both full-length dGCN5 and a fragment corresponding to the catalytic domain of dGCN5 (amino acids 469-634) were expressed as 6×His N-terminal tagged fusion proteins in E.coli and purified on Ni+-agarose. As expected and in excellent agreement with other GCN5 family members, each protein acetylates histone H3 when presented with a mixture of core histones. The similar substrate specificity between the full-length and catalytic fragment is consistent with evidence that a corresponding catalytic fragment from yGCN5 contains all of the sequences required for full HAT activity. An additional substrate utilized by hPCAF is nucleosomal histones; the ability to weakly acetylate nucleosomal substrates was attributed to the N-terminal domain. However, despite sequence similarities between the N-termini of hPCAF and dGCN5, no significant acetylation of nucleosomal histones by dGCN5 was detected (Smith, 1998).
date revised: 10 August 2023
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