Gene name - absent, small, or homeotic discs 2
Cytological map position - 96A13
Function - transcription factor
Keywords - trithorax family
Symbol - ash2
FlyBase ID: FBgn0000139
Genetic map position - 3-78.6
Classification - PHD finger, SPRY domain, nuclear localization signal
Cellular location - nuclear
The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. Ash2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. ASH2 binds directly to Skittles (Sktl), a predicted phosphatidylinositol 4-phosphate 5-kinase, and the association of these proteins is functionally significant. Histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that Ash2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation (Cheng, 2004).
ash2 was discovered in a screen for late larval/early pupal lethals with imaginal disc abnormalities (Shearn, 1971). Homozygous ash2 mutants exhibit homeotic transformations characteristic of loss-of-function mutants in homeotic genes (Shearn, 1987). For example, the haltere and third-leg imaginal discs of ash2 trans-heterozygotes show reduced accumulation of the homeotic gene product, Ultrabithorax, and the first leg discs show complete loss of Sex combs reduced (Lajeunesse, 1995). ash2 mutants display intergenic noncomplementation with other trxG gene mutants (Shearn 1989). In addition, ash2 mutants display pattern formation abnormalities of the legs and wings (Adamson, 1996). The predicted Ash2 protein contains a target sequence for early degradation (PEST sequence), a domain of unknown function (SPRY domain; Ponting, 1997), and a bipartite nuclear localization signal. The nuclear localization signal is functional; Ash2 is localized in nuclei of salivary gland cells (Adamson, 1996). Significantly, Ash2 contains a PHD finger, a putative double zinc finger involved in mediating protein-protein interactions and modifying chromatin structure and implicated in functioning as a nuclear phosphoinositide receptor (Gozani, 2003). Biochemical studies reveal that Ash2 is a subunit of a 500-kD multiprotein complex (Papoulas, 1998). Additional components of the complex have yet to be determined in Drosophila. However, biochemical purification of the SET1 protein complex in Saccharomyces cerevisiae reveals that one of the proposed subunits, Bre2p, contains a SPRY domain and another subunit, Saf41p, contains a PHD finger. It has been proposed that Bre2p and Saf41p together constitute a bipartite functional homolog of Ash2 (Nagy, 2002). The SET1 complex contains SET1, a SET domain-containing protein that has been shown to methylate lysine 4 of histone H3 (Miller, 2001; Roguev, 2001; Nagy, 2002). Human and Schizosaccharomyces pombe versions of this complex have Ash2 homologs that contain both a SPRY domain and a PHD finger; these complexes have also been shown to methylate lysine 4 of histone H3 (Roguev, 2003; Wysocka, 2003). The human Ash2 homolog (Ikegawa, 1999) is 47% identical to Drosophila Ash2 (Cheng, 2004).
Phosphoinositol lipids in the cytoplasm play important roles in growth, differentiation, and vesicular secretion. Phosphatidylinositol 4-phosphate [PtdIns(4)P or PIP] is phosphorylated by phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to become phosphatidylinositol 4,5-bisphosphate [Ptd(4,5)P2 or PIP2]. PIP2 is a second messenger thought to modulate the functions of cytoskeletal regulatory proteins such as profilin, coilin, fascin, and gelsolin. PIP2 also regulates vesicular trafficking and platelet activation. Phospholipase C hydrolyzes PIP2 to produce the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC), while IP3 releases calcium from intracellular stores. In addition, PIP2 is converted into phosphatidylinositol 3,4,5-trisphosphate, which activates some PKC isoforms. Phosphoinositide metabolism is also involved in signal transduction and cytoskeleton regulation via interaction with the Rho family of small G proteins. An interaction has also been implicated between phosphoinositides and receptor tyrosine kinases. There are two types of PIP5Ks, PIP5KI and PIP5KII, with distinct biochemical properties. PIPKIs prefer to phosphorylate PI-4-P to PI-4,5-P2, while the preferred substrate of PIPKII is PI-5-P rather than PI-4-P. PIP5KI has been shown to be required for vesicular secretion in PC12 cells, while PIP5KII may be involved in vesicular trafficking in budding yeast (Cheng, 2004 and references therein).
Phosphoinositides are also present in the nucleus. There is growing evidence that members of the phosphoinositide pathways are involved in post-transcriptional modification and chromatin-mediated gene regulation. Biochemical experiments have shown the association of phosphatidylinositol (PI), phosphatidylinositol phosphate kinase (PIPK), and DAG activities with the nuclear matrix. Multiple isoforms of PIPKs localize to the nucleoplasm and are concentrated at nuclear speckles containing mRNA-processing components. PIP2 was also detected at these speckles, consistent with its production by PIPKs localized to these sites. Genetic evidence has implicated nuclear phosphoinositides and their hydrolysis products in the export of mRNA via the nuclear pore complex. PIP2 is a necessary component of the pre-mRNA splicing machinery. A Dictyostelium nuclear phosphatidylinositol phosphate kinase is required for developmental gene expression. Nuclear inositol 1,4,5-trisphosphate kinase in yeast also has a role in transcriptional control. Phospholipids are able to bind histones and nonhistone chromosomal associated proteins. PtdInsP and PtdInsP were shown to bind to the PHD fingers of the chromatin-associated protein ING2 and several other proteins (Gozani, 2003). PIP2 stabilizes the association of the SWI/SNF-like BAF complex with chromatin and the nuclear matrix (Zhao, 1998), and it binds histone H1 (Yu, 1998), which leads to the inhibition of histone-H1-mediated repression on RNA polymerase II activity (Cheng, 2004 and references therein).
The Drosophila gene skittles encodes a putative PIP5KI, which is required for cell viability and germline and bristle development; sktl mutations affect the ovary, dorsal appendage, egg, and wing (Knirr, 1997; Hassan, 1998). Ash2 and Sktl bind directly to each other in vitro and in vivo and sktl mutations enhance the homeotic transformation phenotype of ash2 mutations. This study also shows that histone H1 hyperphosphorylation within euchromatin is dramatically increased on ash2 and sktl mutant polytene chromosomes. These results support a model in which PIP2 plays a role in maintaining transcriptionally active chromatin via histone H1 modification (Cheng, 2004).
Sktl was identified in a yeast two-hybrid screen as a protein that binds to Ash2. The direct physical association between Ash2 and Sktl, as indicated by the yeast two-hybrid assay, was confirmed in vitro by GST pull-down. Full-length Ash2 and Ash2.MC (which contains Ash2 amino acids 260-404 including the entire SPRY domain), were sufficient to pull down Sktl. The SPRY domain was named after the two proteins that possess this domain: yeast SPla and ryanodine receptor. Although the function of the SPRY domain is unknown, the GST pull-down results suggest that one of its possible functions is mediating protein-protein interactions (Cheng, 2004).
The physical association between Ash2 and Sktl, as indicated by the yeast two-hybrid assay and GST pull-down, also occurs in vivo in Drosophila embryos as shown by co-immunoprecipitation. Antibody against the FLAG epitope was able to immunoprecipitate Ash2 from embryos expressing FLAG-Sktl. This result shows that Ash2 and Sktl are physically associated in a complex. In embryos, the Ash2 antibody recognizes two proteins of different sizes, one ~48 kD and one ~94 kD. At other developmental stages (larval to adult), the 48-kD protein is also present but the larger Ash2 protein is ~65 kD. The 48- and 65-kD proteins are the sizes expected from the translation of two ash2 transcripts of 1.4 and 2 kb, respectively, which are detectable by RNA blotting (Beltran, 2003). The 94-kD protein in embryos may result from post-translational modification of the 65-kD protein. Despite the fact that the 94-kD protein is more abundant, only the 48-kD protein is immunoprecipitated by Sktl from embryonic nuclear extracts. Assuming that the 48-kD protein is translated from the 1.4-kb transcript, it would lack the PEST sequence and PHD finger of the full-length Ash2 protein, but would still contain the NLS-BP and SPRY domains. The SPRY domain was found in GST pull-down experiments to be sufficient to bind to Sktl in vitro. The 94-kD protein presumably would also contain the SPRY domain, yet it is not immunoprecipitated by Sktl. Perhaps its SPRY domain is modified in some way to prevent physical association with Sktl. Ash2 antibody was not able to immunoprecipitate FLAG-Sktl from embryos expressing FLAG-Sktl. This may be because the Ash2 antibody recognizes the same site on Ash2 that mediates binding with Sktl. The 94-kD Ash2 protein was found to associate with a 500-kD multimeric protein complex (Papoulas, 1998). Sktl could be a component of this complex. However, it is more likely that Sktl is in a distinct complex with the 48-kD Ash2 protein, because Sktl seems to immunoprecitate only with the 48-kD Ash2 and not with the 94-kD Ash2 found in the 500-kD complex. In S. pombe, the Ash2 homolog is present in two distinct complexes (Roguev, 2003). One complex contains Set1 and the other contains a homolog of Drosophila LID (Gildea, 2000) (Cheng, 2004).
Both Ash2 and Sktl accumulate on polytene chromosomes and in the nucleolus. These results suggest that their physical association and functions involve chromatin and, perhaps, ribosomal DNA transcription. Ash2 accumulates normally on sktl mutant chromosomes while Sktl does not accumulate on ash2 mutant chromosomes. There are two possible explanations for this result. Either Sktl is not made in ash2 mutants or Sktl requires Ash2 protein and/or function to bind to polytene chromosomes. Sktl has been shown to accumulate to normal levels in ash2 mutants that express FLAG-Sktl, so Sktl needs Ash2 protein and/or function to bind to chromosomes. The ash2 mutant combination used to represent the null mutant state is a trans-heterozygote of an inversion, ash2703, and a deletion, ash2X2; it does not accumulate either normal-sized ash2 transcript (Adamson, 1996) or Ash2 protein. Since this mutant lacks Ash2 protein, it necessarily lacks Ash2 function; it cannot be distinguished with certainty whether Sktl requires Ash2 protein or function to bind to chromosomes. However, since Sktl binds directly to Ash2, the idea is favored that Sktl requires the Ash2 protein to bind to chromosomes (Cheng, 2004).
It was also found that Ash2 does not accumulate on ash1 mutant chromosomes, but Ash1 still accumulates on ash2 mutant chromosomes. This suggests that Ash2 needs Ash1 to bind chromosomes. It is predicted that Sktl will not be able to bind to ash1 mutant chromosomes, since Sktl requires Ash2 and Ash2 requires Ash1 to bind to chromosomes. This could also explain why intergenic noncomplementation is seen between ash1 and sktl mutations (Cheng, 2004).
Since Sktl was the only Drosophila PIP5K found to have a NLS and has been shown to accumulate in nuclei, it is thought that Sktl serves a function distinct from those of other Drosophila PIP5Ks. The cytoplasmic PIP5Ks may be able to bind Ash2 in vitro, because they contain protein domains similar to Sktl that Ash2 recognizes. However, since Ash2 and the cytoplasmic PIP5Ks are not localized to the same cellular compartment, it is not expected that the other Drosophila PIP5Ks will have a functional physical association with Ash2. The PHD finger of ING2 was shown to function as a nuclear phosphoinositide receptor (Gozani, 2003), so it is attractive to speculate that the PHD finger of Ash2 can also bind phosphoinositides that can be processed into PIP2 via its interaction with Sktl. It is likely that other members of the nuclear phosphoinositide signaling pathway will have functional interactions with Ash2. As examples, if a sktl transcription factor were mutated, then less Sktl would be made or if genes that encoded enzymes that generated Sktl substrates such as PIP or metabolites such as IP3 were mutated, then fewer of these metabolites would be generated. ash2 mutants might be expected to display intergenic noncomplementation with these mutants as well (Cheng, 2004).
Several lines of evidence show that the physical association between Ash2 and Sktl is functionally significant. First of all, there is the complete loss of Sktl accumulation on ash2 mutant chromosomes, suggesting that Sktl requires Ash2 to bind to chromosomes. The intergenic noncomplementation between sktl and ash2 mutants also shows that the physical association between the two gene products has functional significance. Ash1 and Ash2, like other trxG proteins, play a role in maintaining transcription activation. Reduced Ubx accumulation in sktl mutants, as well as the intergenic noncomplementation seen with sktl mutants in combination with ash1 or ash2, suggests a similar role for Sktl in transcription regulation. The PIP2 generated in the nucleus by Sktl activity could be hydrolyzed to DAG and IP3. IP3 can be further phosphorylated to IP4 and IP5, both of which have been shown to activate transcription (Shen, 2003; Steger, 2003; Cheng, 2004).
Another result that shows functional significance of the physical association between Ash2 and Sktl is a similar dramatic increase in histone H1 hyperphosphorylation on both ash2 and sktl mutant chromosomes compared to wild-type chromosomes. Histone H1 is thought to be a general repressor of transcription by RNA polymerase II. The presence of histone H1 affects the ability of transcription factors to interact with DNA and is associated with transcription repression, while the removal of histone H1 is associated with transcriptional activation. Studies in mammals and Tetrahymena have found a correlation between transcriptional activation and increased histone H1 phosphorylation. Dephosphorylated histone H1 bound to chromatin over the mouse mammary tumor virus promoter is thought to restrict chromatin remodeling and transcription factor access. Phosphorylation of histone H1 has also been shown to regulate ATP-dependent chromatin-remodeling enzymes. The effect of phosphorylation is to create a region of negative charge, which may displace histone H1 from chromatin, allowing the binding of specific regulating factors. Alternatively, proteins that regulate transcription may recognize the phosphorylated residues (Cheng, 2004 and references therein).
However, histone H1 hyperphosphorylation has the opposite effect and is linked to high chromatin condensation, possibly by allowing the binding of accessory factors. During mitosis, histone H1 becomes hyperphosphorylated, which may facilitate the interaction with the DNA minor groove and factors involved in metaphase chromosome condensation. Therefore, increased histone H1 hyperphosphorylation as observed in ash2 and sktl mutants implies increased chromosome condensation and reduced transcription (Cheng, 2004).
Ash1 has been shown to be able to methylate K4 of histone H3 and ash1 mutant chromosomes show complete loss of histone H3 K4 methylation. This result suggests that Ash1 is required for all of the histone H3 K4 methylation that occurs in vivo. The S. cerevisiae SET1 complex, which contains two subunits that are thought to represent a bipartite functional homolog of Ash2, has also been shown to methylate K4 of histone H3 (Miller, 2001; Roguev, 2001; Nagy, 2002). In Drosophila, if Ash2 was also in a complex that could methylate histone H3 K4, then it would be predicted that ash2 mutant chromosomes would show a decrease in histone H3 K4 methylation. Indeed, a decrease is seen in histone H3 K4 methylation on ash2 mutant chromosomes (Cheng, 2004).
During the assembly of nucleosomes, histone acetylation regulates the binding of histone H1 and chromatin condensation. Displacement of histone H1 is required prior to acetylation of target genes and activation of transcription, because histone H1 inhibits histone H3 acetylation by hindering the access of histone acetyltransferases to the histone H3 tail. It has been predicted that chromatin-remodeling complexes would contain components that modify the interaction of histone H1 with chromatin. Ash2 and Sktl may represent such components. The results suggest that Ash2 and Sktl are direct binding partners that are associated in a complex. When the Ash2-Sktl complex binds to chromatin, a source of PIP2 (Sktl) is brought to the chromatin. PIP2 can bind to and displace histone H1 and/or be metabolized to IP3 and phosphorylated derivatives. The displacement of histone H1 would prevent its hyperphosphorylation and allow for chromatin decondensation, histone acetylation, and eventually, transcription activation. The presence of IP4 and IP5 would also stimulate transcription (Cheng, 2004).
The ash2 gene is a member of the trithorax group of genes whose products function to maintain active transcription of homeotic selector genes. Mutations in ash2 cause the homeotic transformations expected for a gene in this group but, in addition, cause a variety of pattern formation defects that are not necessarily expected. The ash2 gene is located in cytogenetic region 96A17-19 flanked by slowpoke and tolloid and is included in a cosmid that contains part of slowpoke. The ash2 transcript is 2.0 kb and is present throughout development. The Ash2 protein predicted from the nucleotide sequence of the open reading frame has a putative double zinc-finger domain, called a PHD finger, that is present not only in the products of other trithorax group genes such as Trx and Ash1, but also in the product of a Polycomb group gene, Pcl. Polyclonal antibodies directed against Ash2 detect the protein in imaginal discs and in the nuclei of salivary gland and fat body cells. On immunoblots these affinity-purified antibodies detect a 70-kDa protein in larvae and a 53-kDa protein in pupae (Adamson, 1996).
The ash2 gene product is a 2.0-kb transcript, The ash2 gene is one of four vital genes located in the interval defined proximally by slowpoke (96A17) and distally by tolloid (96A19). The ash218 mutation is a 20-kb deletion that removes all or part of three of these vital genes -- ash2 and two others. One of these three genes encodes a 2.0-kh transcript. Two lines of evidence indicate that this 2.0-kh transcript is the product of ash2. (1) The genomic region that encodes this transcript is disrupted by an insertion in the ash21 mutation and this transcript is absent in ash21/ash218 hemizygous mutant pupae. By itself this evidence shows that the insertion in the ash21 mutation is in the gene that encodes the 2.0-kh transcript, it does not prove that this transcript is the product of ash2. (2) Accumulation of the protein encoded by this 2.0-kh transcript is severely reduced not only in ash21/ash218 hemizygous mutant pupae as expected, but also in pupae hemizygous for other alleles. So this 2.0 transcript is referred to as the putative ash2 gene product (Adamson, 1996).
The ASH2 protein predicted from the open reading frame of ash2 contains 572 amino acids and has two regions of similarity to Pcl, the product of Drosophila Polycomblike, which is a Polycomb group gene. The most significant similarity is to one of the two PHD fingers of Pcl. The PHD finger is a putative double zinc finger that is found in many proteins that regulate transcription. In addition to Pcl, PHD fingers have been identified in the products of two other Drosophila genes, Trx, the product of trithorax, and ASHl, the product of ashl. The trithorax and ashl genes are both members of the trithorax group, as is ash2. Whereas, Pcl has two PHD fingers and Trx has four PHD fingers, Ash2 like Ash1, has a single PHD finger. Another domain, called the SET domain, has also been found in products of both trithorax and Polycomb group of genes. The SET domain occurs once in E(Z)/PCO, the product of Enhancer of zeste/polyhomeotic, a Polycomb group gene or perhaps both a Polycomb and a trithorax group gene, and once in Trx and Ashl both of which are trithorax group genes. Products of the trithorax and Polycomb groups of genes have antagonistic functions. Finding these domains shared among members of both groups may be hinting at some common mechanism of function. The Ash2 protein would be expected to have a molecular weight of ~65 kDa. On an immunoblot of extracts prepared from different developmental stages, affinity-purified antibodies raised against an Ash2-GST fusion protein recognize a single protein in larvae that is ~70 kDa and a single protein in pupae that is ~53 kDa. It is inferred that the 70-kDa larval protein is full length ASH2. The larger than expected size suggests that ASH2 in larvae is either posttranslationally modified or runs anomalously on SDSPAGE. Since the ash2 transcript in larvae and pupae is the same size, the smaller than expected size of ASH2 protein found in pupae suggests that a posttranslational modification such as cleavage occurs in pupae. There is a single PEST sequence in ASH2. PEST sequences are found in proteins that are rapidly degraded. The disappearance of the 70-kDa form during the third larval instar suggests that until this stage Ash2 protein is protected from degradation. Such protection could be afforded by posttranslational modification but could also be afforded by incorporation of Ash2 into a multimeric-protein complex. Regardless of the mechanism accounting for the difference in apparent sizes of Ash2 in larvae and pupae, the most significant point is that the transition from 70 to 53 kDa occurs after the middle of the third larval instar. This stage is significant because null mutant hemizygotes survive through the end of the third larval instar. Although no 2.0-kb ash2 transcript can be detected in ash2 mutant pupae, a normal sized transcript can be detected in mutant third instar larvae (Adamson, 1996).
date revised: 20 November 2004
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.