absent, small, or homeotic discs 2

The ash2 transcript detected in null mutant larvae is interpreted as a maternally derived product. This suggests that the zygotic requirement for ash2 begins during the third larval instar and is consistent with phenotypes of imaginal discs from mutant larvae. For example, in haltere discs from mid third instar mutant larvae there is no expression of an Antennapedia reporter gene and nearly uniform accumulation of Ultrabithorax protein as in normal haltere discs, whereas in haltere discs from late third instar mutant larvae there is ectopic expression of an Antennapedia reporter gene and patchy accumulation of Ultrabithorax protein. Finding the first detectable 53kDa Ash2 protein in late third instar larvae suggests that whatever activity is responsible for producing this smaller Ash2 product is not present before the end of the third larval instar. The fact that the final Ash2 protein is significantly different in larvae and pupae suggests that it has different functions in larvae and pupae. This may represent a novel mechanism for a single gene product to have multiple functions (Adamson, 1996).

Four lines of evidence place ash2 in the trithorax group of genes (Shearn, 1989; Lajeunesse, 1996). Nevertheless, in certain respects ash2 mutations behave differently from mutations in other trithorax group genes. Whereas amorphic and even hypomorphic alleles of the trithorax group genes, ashl, brahma or trithorax, give rise to high frequencies of homeotic transformations as double heterozygotes, amorphic mutations of ash2 as double heterozygotes with amorphic alleles of ashl, brahma or trithorax give rise to low frequencies of homeotic transformations. This differential behavior of ash2 mutations may be due entirely or in part to the extraordinary persistence of maternal transcript until the end of the larval period (Adamson, 1996).

One of the homeotic transformations caused by ash2 mutations is antenna to leg (Shearn, 1987). This transformation is also caused by gain of function mutations in the Antennapedia gene and is a consequence of ectopic accumulation of Antp. The ectopic accumulation of Antp is caused by juxtaposition of an antennal promoter and the Antp gene. Since no ash2 alleles affect the penetrance or expressivity of antennal transformations caused by heterozygosity for Antp73B, this suggests that ash2 does not affect transcription from this antennal promoter nor does it have any affect on translation of the Antp protein. The ectopic accumulation of Antp is caused by alteration of the Antp promoter itself. Since heterozygotes, amorphic alleles of ash2 increase the penetrance and expressivity of antennal transformations caused by heterozygosity for AntpNs. Other ash2 alleles that cause a similar enhancement are probably also amorphic. In contrast, two alleles suppress the penetrance and expressivity of antennal transformations caused by heterozygosity of AntpNs. These two alleles appear to be gain-of-function mutations. The levels of ectopic Antp in the eye-antenna discs of AntpNs+/+ ash2 double heterozygotes correlate well with the transformation frequencies observed. Enhancers of AntpNs, such as ash21 and ash218, increase the accumulation of ectopic Antp protein in antennal discs from doubly heterozygous larvae, while suppressors of AntpNs decrease the accumulation of ectopic Antp protein in antennal discs from doubly heterozygous larvae. The ectopic accumulation of Antp caused by AntpNs is primarily due to expression from the P2 promoter of Antennapedia. If the action of Ash2 on the Antennapedia gene were direct, the enhancement of AntpNs caused by amorphic alleles would imply that the normal function of Ash2 is to repress the P2 promoter of Antennapedia. However, it is more likely that action of Ash2 on the Antennapedia gene is not direct. In haltere discs from ash2 mutant larvae there is ectopic accumulation of Antp in cells that have lost Ubx accumulation (Lajeunesse, 1995). This ectopic Antp was interpreted as a consequence of derepression of the P1 promoter of Antennapedia due to the absence of Ubx. By analogy, it is possible that amorphic mutations of ash2 cause reduced accumulation in eye-antenna discs of a protein that represses transcription from the P2 promoter of Antennapedia. According to this hypothesis it is reduced accumulation of this other protein that leads to ectopic accumulation of Antp in the antenna (Adamson, 1996).

Many of the pattern formation defects found in ash mutants are not characteristic of mutations in the homeotic selector genes or trithorax group genes. These defects imply that Ash2 is required for normal expression of other genes in addition to homeotic selector genes. Ectopic expression of either wingless or decapentaplegic causes overgrowth of leg tissue. Ectopic expression of both wingless and decapentaplegic in the same cells leads to the formation of anadditional proximal-distal axis, and thus to the formation of a supernumerary leg. One possible explanation for the leg tissue overgrowth and supernumerary legs seen in ash2 mutants is that wild-type Ash2 negatively regulates wingless and decapentaplegic and that failure of this negative regulation in mutants leads to ectopic expression. However, altered expression of a wingless enhancer trap or decapentaplegic reporter gene was not seen in ash2 mutants. This failure to detect altered expression could have been because the regulatory elements reported by these constructs are different from those that are sensitive to Ash2. However, no altered expression of Distal-less was seen; this is invoked by wingless and decapentaplegic to form a proximaldistal axis. These data suggest that ash2 is functioning down-stream of wingless, decapentaplegic and Distal-less in this pattern formation signalling pathway (Adamson, 1996).

ash2 also appears to be playing a role in the determination of external sensory organs. Ectopic sense organs are found on pharate adult and/or adult ash2 transheterozygotes. This is correlated with an abnormal expression pattern of neuralized, which is expressed in all sense organ precursors. There are also transformations from one sensory organ type to another type. A similar transformation of campaniform sensilla to bristle is seen in embryos deficient for both BarHl and BarH2. BarHl and BarH2 are two closely related homeobox genes, whose expression is necessary for the subtype determination of external sensory organs. It seems reasonable to suppose that transcription of BarHl and/or BarH2 is reduced by loss of ash2 function (Adamson, 1996).

The phenotypic analysis of ash2 mutants makes it clear that ash2 is functioning as more than a regulator of homeotic selector genes. The neural and pattern formation phenotypes observed in ash2 mutants have not been found in other trithorax group gene mutants and suggest that ash2 activity is necessary in a variety of cell types to promote proper cell fate determination. It is possible that each of the two different forms of Ash2 that have been discovered has a different activity. For example, the PHD finger domain in the 70-kDa form may allow Ash2 to function as part of the trithorax group of genes. If the PHD finger were removed in a cleavage that leads to formation of a carboxy terminal 53-kDa form, then according to this speculation, Ash2 would no longer function as part of the trithorax group but could function in some other mechanism of gene regulation (Adamson, 1996).

Effects of Mutation or Deletion

Mutations in the ash-1 and ash-2 genes of Drosophila cause a wide variety of homeotic transformations that are similar to the transformations caused by mutations in the trithorax gene. Based on this similar variety of transformations, it was hypothesized that these genes are members of a functionally related set. Three genetic tests were employed here to evaluate that hypothesis. The first test was to examine interactions of ash-1, ash-2 and trithorax mutations with each other. Double and triple heterozygotes of recessive lethal alleles express characteristic homeotic transformations. For example, double heterozygotes of a null allele of ash-1 and a deletion of trithorax have partial transformations of their first and third legs to second legs and of their halteres to wings. The penetrance of these transformations is reduced by a duplication of the bithorax complex. The second test was to examine interactions with a mutation in the female sterile (1) homeotic gene. The penetrance of the homeotic phenotype in progeny from mutant mothers is increased by heterozygosis for alleles of ash-1 or ash-2 as well as for trithorax alleles. The third test was to examine the interaction with a mutation of the Polycomb gene. The extra sex combs phenotype caused by heterozygosis for a deletion of Polycomb is suppressed by heterozygosis for ash-1, ash-2 or trithorax alleles. The fact that mutations in each of the three genes give rise to similar results in all three tests represents substantial evidence that ash-1, ash-2 and trithorax are members of a functionally related set of genes (Shearn, 1989).

Genes of the trithorax group appear to be required for the maintenance of expression of the homeotic selector genes of the Antennapedia and bithorax complexes. According to genetic criteria, the Drosophila melanogaster genes ash1 and ash2 are members of the trithorax group. The consequences of ash1 and ash2 mutations on the expression of homeotic selector genes in imaginal discs have been examined. The results of these experiments demonstrate that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. Complete loss of ash2 activity causes the loss of expression of Sex combs reduced in first leg imaginal discs, loss of expression of Ultrabithorax in third leg discs, and a late-patterned loss of expression of Ultrabithorax within haltere discs, yet has no effect on engrailed or Antennapedia expression. These results suggest that the range and action of trithorax group genes is varied and complex and argue against any model in which all of the products of the trithorax group act together in a single mechanism or complex (LaJeunesse, 1995).

The ash2 gene is a member of the trithorax group of positive transcriptional regulators of the homeotic genes. Evidence that loss-of-function of ash2 results in patterning alterations in the developing wing. Homozygous adults of a weak allele of ash2 develop extra cross-veins. However, clonal analysis of a stronger allele, ash2, shows that this allele results in reduction of intervein tissue and increase of longitudinal veins and cross-vein tissue in the wing except the region between vein L3 and L4. These results suggest that ash2 function is required for both activation of intervein tissue and repression of vein tissue. Moreover, cross-vein development can be rescued in the absence of crossveinless-2 when the levels of ash2 were reduced (Amoros, 2002).


Adamson, A. L. and Shearn, A. (1996). Molecular genetic analysis of Drosophila ash2, a member of the trithorax group required for imaginal disc pattern formation. Genetics 144(2): 621-33. 8889525

Amoros, M., Corominas, M., Deak, P. and Serras, F. (2002). The ash2 gene is involved in Drosophila wing development. Int. J. Dev. Biol. 321-4. 12068954

Angulo, M., Corominas, M. and Serras, F. (2004). Activation and repression activities of ash2 in Drosophila wing imaginal discs. Development 131(20): 4943-53. 15371308

Beltran, S., et al. (2003). Transcriptional network controlled by the trithorax-group gene ash2 in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100(6): 3293-8. 12626737

Cheng, M. K. and Shearn, A. (2004). The direct interaction between Ash2, a Drosophila trithorax group protein, and Sktl, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for phosphatidylinositol 4,5-bisphosphate in maintaining transcriptionally active chromatin. Genetics 167(3): 1213-23. 15280236

Dou, Y. L., Milne, T. A., Ruthenburg, A. J., Lee, S., Lee, J. W., Verdine, G. L., Allis, C. D. and Roeder, R. G. (2006). Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 13: 713-719. Medline abstract: 16878130

Eissenberg, J. C. and Shilatifard, A. (2010). Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev. Biol. 339: 240-249. PubMed Citation: 19703438

Gildea, J., Lopez, R. T. and Shearn, A. (2000) A screen for new trithorax group genes identified little imaginal disks, the Drosophila melanogaster homolog of human retinoblastoma binding protein 2. Genetics 156: 645-663. 11014813

Goo, Y. H., et al. (2003). Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins. Mol. Cell. Biol. 23(1): 140-9. 12482968

Gozani, O., et al. (2003). The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 114: 99-111. 12859901

Guccione, E., et al. (2007). Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 449: 933-937. Medline abstract: 17898714

Hassan, B. A., et al. (1998). skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release. Genetics 150: 1527-1537. 9832529

Hughes, C. M., et al. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol. Cell 13: 587-597. PubMed Citation: 14992727

Ikegawa, S., Isomura, M., Koshizuka, Y. and Nakamura, Y. (1999). Cloning and characterization of Ash2L and Ash2l, human and mouse homologs of the Drosophila ash2 gene. Cytogenet. Cell Genet. 84(3-4): 167-72. 10393421

Knirr, S., Santel, A. and Renkawitz-Pohl, B. (1997). Expression of the PI4P 5-kinase Drosophila homologue skittles in the germline suggests a role in spermatogenesis and oogenesis. Dev. Genes Evol. 207: 127-130

Kraut, R., Menon, K. and Zinn, K. (2001). A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr. Biol. 11: 417-430. 11301252

LaJeunesse, D. and Shearn, A. (1995). Trans-regulation of thoracic homeotic selector genes of the Antennapedia and bithorax complexes by the trithorax group genes: absent, small, and homeotic discs 1 and 2. Mech. Dev. 53: 123-139. 8555105

Lee S., et al. (2008). Activating signal cointegrator-2 is an essential adaptor to recruit histone H3 lysine 4 methyltransferases MLL3 and MLL4 to the liver X receptors. Mol. Endocrinol. 22: 1312-1319. PubMed Citation: 18372346

Lloret-Llinares, M., Perez-Lluch, S., Rossell, D., Moran, T., Ponsa-Cobas, J., Auer, H., Corominas, M. and Azorin, F. (2012). dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes. Nucleic Acids Res 40: 9493-9505. Pubmed: 22904080

Miller, T., et al. (2001) COMPASS: a complex of proteins associated with a trithorax-related SET domain protein. Proc. Natl. Acad. Sci. 98: 12902-12907. 11687631

Miyazaki, H., Higashimoto, K., Yada, Y., Endo, T. A., Sharif, J., Komori, T., Matsuda, M., Koseki, Y., Nakayama, M., Soejima, H., Handa, H., Koseki, H., Hirose, S. and Nishioka, K. (2013). Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing. PLoS Genet 9: e1003897. PubMed ID: 24244179

Mohan, M., Lin C., Guest, E. and Shilatifard, A. (2010). Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nat. Rev. Cancer 10: 721-728. PubMed Citation: 20844554

Mohan M., et al. (2011). The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31: 4310-4318. PubMed Citation: 21875999

Nagy, P. L., Griesenbeck, J., Kornberg, R. D. and Cleary, M. L. (2002). A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3. Proc. Natl. Acad. Sci. 99(1): 90-4. 11752412

Nislow, C., Ray, E. and Pillus, L. (1997). SET1, a yeast member of the trithorax family, functions in transcriptional silencing and diverse cellular processes. Mol. Biol. Cell 8: 2421-2436. 9398665

Papoulas, O., et al. (1998). The Drosophila trithorax group proteins BRM, Ash1 and Ash2 are subunits of distinct protein complexes. Development 125(20): 3955-66. PubMed Citation: 9735357

P´rez-Lluch, S., et al. (2011). Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing. Nucleic Acids Res. 39(11): 4628-39. PubMed Citation: 21310711

Ponting, C., Schultz, J. and Bork, P. (1997). SPRY domains in ryanodine receptors (Ca2+-release channels). Trends Biochem. Sci. 22: 193-194. 9204703

Pullirsch, D., et al. (2010). The Trithorax group protein Ash2l and Saf-A are recruited to the inactive X chromosome at the onset of stable X inactivation. Development 137(6): 935-43. PubMed Citation: 20150277

Roguev, A., et al. (2001). The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4. EMBO J. 20(24): 7137-48. 11742990

Roguev, A., et al. (2003). High conservation of the Set1/Rad6 axis of histone 3 lysine 4 methylation in budding and fission yeasts. J. Biol. Chem. 278(10): 8487-93. 12488447

Shilatifard, A. (2008). Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. Curr. Opin. Cell Biol. 20:341-348. PubMed Citation: 18508253

Shearn, A., Rice, T., Garen, A. and Gehring, W. (1971). Imaginal disc abnormalities in lethal mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. 68: 2695-2698

Shearn, A., Hersperger, E. and Hersperger, G. (1987). Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations. Roux's Arch. Dev. Biol. 196: 231-242.

Shearn, A. (1989). The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related. Genetics 121(3): 517-25. 2497049

Shen, X., et al. (2003) Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 299: 112-114. 12434013

Smith E. and Shilatifard, A. (2010). The chromatin signaling pathway: diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes. Mol. Cell 40: 689-701. PubMed Citation: 21145479

Steger, D. J., et al. (2003). Regulation of chromatin remodeling by inositol polyphosphates. Science 299: 114-116. 12434012

Wang, G. G., Song, J., Wang, Z., Dormann, H. L., Casadio, F., Li, H., Luo, J. L., Patel, D. J. and Allis, C. D. (2009). Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger. Nature 459: 847-851. Pubmed: 19430464

Wang, J., et al. (2001). Ash2L: alternative splicing and downregulation during induced megakaryocytic differentiation of multipotential leukemia cell lines. J. Mol. Med. 79(7): 399-405. 11466562

Wysocka, J., et al. (2003). Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1. Genes Dev. 17(7): 896-911. 12670868

Yokoyama, A., et al. (2007). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell. Biol. 24(13): 5639-49. Medline abstract: 15199122

Yu, H., et al. (1998). Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. Eur. J. Biochem. 251: 281-287. 9492295

Zhao, K., et al. (1998). Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 95: 625-636. 9845365

absent, small, or homeotic discs 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 3 July 2014

Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.