Gene name - osa
Synonyms - eyelid
Cytological map position -
Function - presumptive transcription factor
Symbol - osa
Genetic map position -
Classification - Bright family
Cellular location - nuclear
osa, also know and referred to here as eyelid, antagonizes wingless signaling during Drosophila development and affects patterning of the eye imaginal disc. eyelid was originally isolated as a suppressor of a dominant mutation in the rough gene roughDOMINANT. Eye discs from roDOM third-instar larvae display a so-called "furrow-stop" phenotype, the hallmarks being the unusual presence of mature ommatidia that contain a full complement of photoreceptor cells in the anterior-most ommatidial row, and the loss of dpp expression in the furrow. Surprisingly, an analysis of eye discs from larvae heterozygous for both roDOM and eyelid reveals that the observed increase in eye size is not attributable to relief of the roDOM-induced block to furrow progression in the central region of the disc. Rather, photorecepter differentiation reinitiates at the dorsal and ventral edges of these discs. This produces two ommatidial fields, each preceded by decapentaplegic expressing furrows, which move anteriorly, presumably fusing later along the midline. The mechanism of roDOM suppression by eyelid suggests that eyelid is most critical at the lateral edges of the disc, regions in which wingless has been shown to inhibit precocious differentiation. A reduction of wingless has the opposite effect on roDOM, reducing the eye size and the extent of photoreceptor differentiation along the disc margins. Thus eyelid mutations show opposite effects to those of wingless mutations, suggesting that eyelid may normally function as an antagonist of wg signaling (Treisman, 1997).
eyelid is required for embryonic segmentation. Embryos zygotically mutant for eyelid appear to have normal cuticle patterning and normal expression of the segment polarity gene engrailed. However, because Eld protein is present at high levels in the early embryo and is presumably contributed maternally, these embryos would still contain Eld. Embryos deprived of maternally encoded Eyelid show severe defects in the cuticle pattern, with many denticle belts either missing, fused, or otherwise abnormal. The provision of a paternal wild-type copy of the eyelid gene fails to rescue the maternal mutants, suggesting either that early expression of eyelid is critical for its function or that the levels of expression of the paternal copy is insufficient. The eyelid mutants fail to extend their germ bands normally, arresting at a partially extended stage (Treisman, 1997).
To some degree, eyelid affects the expression of pair-rule genes. For example, slight defects are seen in the expression pattern of even-skipped stripes 3 and 4 often appear weaker than normal, stripe 2 wider, and stripes 5 and 6 closer together. This pattern is similar to that of eve during its early expression in wild-type embryos, suggesting a failure of refinement. Clearly eld functions in embryogenesis before wingless is expressed, suggesting that Eld function is not restricted to the Wingless signaling pathway (Treisman, 1997).
In maternally mutant eld embryos, although the initial engrailed expression is initiated in a relatively normal fashion, its later expression is abnormal: several stripes appear broadened, others are partially missing, and their spacing is disrupted. Nevertheless, wingless stripes are not expanded. These results are consistent with eld acting to counteract Wingless signaling in regions posterior to the engrailed stripes, in addition to responding to earlier patterning signals that affect the positioning of pair-rule gene stripes (Treisman, 1997).
It has been suggested that eld acts downstream rather than upstream of wingless for the following reasons:
The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. "Osa" means "fate" in Norwegian. The osa gene was first identified as a trxG gene in the same genetic screens that identified brm (Kennison, 1988). osa turns out to code for the same transcript as eyelid. Regulation of Antennapedia by Brm and Osa proteins requires sequences 5' to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The Osa protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila Dead ringer proteins. It is proposed that the Osa protein may target the BRM complex to Antennapedia and other regulated genes (Vázquez, 1999).
osa and brm were first identified as suppressors of both the antenna to leg transformation caused by the Nasobemia (Ns) allele of Antp and the extra sex combs phenotype caused by derepression of Sex combs reduced (Scr) in Polycomb (Pc) mutants (Kennison, 1988). While examining genetic interactions among trxG mutations, it was noted that flies heterozygous for both brm and osa mutations have a held-out phenotype rarely seen in flies heterozygous for either mutation alone. The expressivity of the held-out wings phenotype is more severe in combinations of brm with some point mutations in osa than it is with the osa deficiency, suggesting that the osa point mutations make altered proteins that still bind to something in competition with wild-type Osa proteins, but then fail to function. Increasing the dosage of wild-type brm reduces the held-out wings phenotype, as expected (Vázquez, 1999).
The held-out wings phenotype is not rare in Drosophila. It is caused by mutations in many other genes, including dpp. This phenotype was also observed in flies trans-heterozygous for partially complementing brm alleles. Nevertheless, the interaction between brm and osa alleles is unusual because it results from the failure of complementation between mutations in two different genes (non-allelic non-complementation). Although a few other trxG mutations have been shown to interact in double heterozygotes, the penetrance in every other case is far less than that observed for the brm/osa interactions. In fact, the majority of trxG mutations show little if any interaction in double heterozygotes. brm interacts with the trxG genes trx and ash1 to cause partial transformation of the fifth abdominal segment to fourth, and metathorax to mesothorax, but these flies do not hold their wings out at any significantly higher frequency. The basis for the held-out wings phenotype in the brm/osa transheterozygotes was investigated. The Antp gene has two alternative promoters, P1 and P2. Genetic studies have shown that the functions of both promoters are essential. Two mutations that inactivate only the P2 promoter have been described. Flies heterozygous for the P2-specific mutations and the chromosome aberrations that remove P1 function were examined. All combinations appear as wild type, except flies carrying either one of two very specific Antp mutations, which produce chromosome aberrations that remove P1 function in combination with the P2- specific mutations. Many of these flies have held-out wings phenotype indistinguishable from the held-out wings phenotype of the brm/osa transheterozygotes. It is suggested that disruption of P2 promoter activity can result in a held-out wings phenotype. Moreover, when a brm mutation is introduced, there is a significant increase in the penetrance of the held-out wings phenotype. These results strongly suggest that brm is one of the factors required for normal expression of the P2 promoter to prevent the held-out wings phenotype (Vázquez, 1999).
That both brm and osa are required for activation of the Antp P2 promoter is also suggested by their interaction with the Antp Ns mutation. The Antp Ns mutant chromosome has a large insertion (including a second copy of part of the P2 promoter) upstream of the P2 promoter. This insertion derepresses the P2 promoter and causes the antennae to differentiate leg structures. The first alleles of both brm and osa were isolated because they fail to derepress the P2 promoter in the Antp Ns mutant. As noted by Kennison and Tamkun (1988) the trxG genes identified in their screen, including the osa gene, might regulate HOM gene function at a variety of different levels. They might regulate transcription or translation of the HOM genes, or encode cofactors that interact with the HOM proteins in regulating target genes. Since brm has been shown to affect HOM gene transcription, the genetic interaction with brm suggests that osa may also act at the level of HOM gene transcription. Antp proteins are normally not expressed in the cells that form the adult antenna. Misexpression of Antp proteins during the larval stage in these cells causes them to differentiate leg structures instead of antennal structures. The Antp Ns allele derepresses the Antp P2 promoter in the eye-antennal disc, expressing wild-type Antp transcripts from the Antp promoter. The penetrance of the antenna-to-leg transformation of Antp Ns mutants is greatly reduced in osa heterozygotes. High levels of osa expression are required only for the Antp P2 promoter, and not for the function of Antp proteins expressed from other promoters (Vázquez, 1999).
osa is also required maternally for proper embryonic segmentation. Although osa function appears to be important for expression of some HOM and segmentation genes in imaginal tissues, homozygous osa mutants die late in embryogenesis with no clear defects in either segmentation or segment identity. To determine whether wild-type maternal osa gene products deposited in the egg might be sufficient for segmentation and segment identity, homozygous germ cells were generated for the osa alleles that are strong Antp Ns suppressors. Loss of maternal osa functions has dramatic effects on the segmentation of the embryo. When rescued by a wild-type allele inherited from the father, the embryos secrete cuticle but have severe defects in segmentation, resembling mutants for the early-acting gap segmentation genes. When both maternal and zygotic osa functions are lacking, the embryos fail to differentiate any cuticle at all. The failure to detect obvious changes in the homozygous osa mutants from heterozygous mothers is clearly a consequence of the maternally encoded osa gene products, which function early in embryogenesis to activate transcription of target genes. Because of the severe defects in embryos lacking maternal osa function and the cascade of regulatory interactions between the segmentation and HOM genes early in embryogenesis, no attempt was made to identify the earliest-acting genes affected by loss of osa function (Vázquez, 1999).
Two regions of Osa have homology to other genes: within region I (residues 854 to 1104) there is a 97 amino-acid sequence (residues 993 to 1087) that contains a putative ARID (AT-rich interaction domain) that is conserved in the Drosophila Dead ringer (Dri) and mouse Bright proteins and in at least 10 other proteins. Although the Dri protein was identified in a screen for proteins that bound a consensus sequence for the EN homeodomain (Kalionis, 1993), Dri lacks any homology to the homeodomain (Gregory, 1996). The BRIGHT (B cell regulator of IgH transcription) protein binds to the minor groove of a consensus MAR (matrix attachment region) sequence. MARs organize chromatin fibers into looped domains by attachment to the nuclear matrix and may function as boundary elements for transcriptional domains. They may also collaborate with enhancers to generate extended domains of accessible chromatin. Dri and Bright are sequence-specific DNA binding proteins and the ARID domain is essential, but not sufficient for this binding. The consensus target sequences for Dri, Bright and En binding are very similar. Dri binds the PuATTAA sequence (Gregory, 1996); Bright binds the PuATa/tAA sequence, and En binds GATCAATTAAAT. All of these contain the same ATTAA core sequence (Vázquez, 1999).
Of the other 10 proteins reported to have an ARID domain, particular interest is found in the SWI1 protein, given the fact that it is a member of the SWI/SNF complex. The possibility that Osa might be the putative Drosophila SWI1 homolog was investigated. SWI1 has long tracks of polyasparagine, polyglutamine, and a putative Cys4 zinc-finger motif. Osa is very rich in proline but no zinc-finger motif is detected. SWI1 has in common with Osa clusters of sequence made up principally of only two or three amino acids. Very recently, a protein called p270 has been described as a member of the human BRG1 complex and has been proposed as a human SWI1 homolog (Dallas, 1998). p270, like OSA and SWI1, has glutamine-rich regions, an ARID domain and several copies of the LXXLL motif (where L is leucine and X is any amino acid). This motif mediates binding to nuclear receptors. Interestingly, Osa has three copies of this motif. Although Drosophila ESTs corresponding to proteins related to several yeast SWI/SNF subunits (including SWI2/SNF2, SWI3, SNF5, and SWP73) have been recovered, it is interesting to note that no EST corresponding to SWI1 has yet been identified. It is possible that OSA, SWI1 and p270 ARID-domain-containing proteins play similar roles in their respective organisms (Vázquez, 1999).
Is OSA essential for the function of the BRM complex? If so, one might expect brm and osa mutants to have identical phenotypes, and the mutation with the strongest effects in one assay should be the mutation with the stronger effects in all other assays. This is not observed. For example, there are much greater effects on Scr, Ubx, and Abd-B in brm heterozygotes than in osa heterozygotes, but the reverse is observed for Antp. Another important difference is the germ line requirements for brm and osa, i. e., brm clones do not make eggs while osa clones make normal appearing eggs that are fertilized but fail during embryogenesis. Thus, brm is required under conditions that do not appear to require osa. If Osa is a subunit of the BRM complex, it is not essential for all of the complexs functions. Consistent with this possibility, the Osa protein was not identified as one of the major subunits of the BRM complex in the Drosophila embryo. However, it remains possible that Osa is a substoichiometric subunit of the BRM complex, or that it is associated with Brm at other stages of development. Another possibility is that the Osa protein targets the BRM complex to specific promoters (e.g., Antp P2). To date, no protein from the SWI/SNF complex (including SWI3 or the ARID-domain protein SWI1), has been shown to bind DNA in a sequence-specific manner (Vázquez, 1999 and references).
It is proposed that the Osa protein may be involved in the targeting of the BRM complex in Drosophila. Whether an intrinsic member of the BRM complex or merely an associated partner, the OSA protein may interact with specific target sequences in cis-regulatory elements to anchor or recruit the BRM complex. Given the patterns of expression driven by Antp cis-regulatory sequences in a reporter gene transposon, it is likely that there are En DNA-binding sites in the 10 kb region 5' to the Antp P2 promoter. Since the ARID domain found in the Osa protein may bind to En target sites, it is possible that Osa proteins will bind directly to these sequences. It is also possible that Osa may bind AT-rich regions of DNA with little specificity. The delineation of brm and osa response elements should allow a clarification of whether they act in concert or independently. It is also possible that the BRM complex alters chromatin structure in order to facilitate the binding of Osa to its target sites. Subsequent to this, Osa would act independently of the BRM complex to activate transcription (Vázquez, 1999 and references).
The protein contains a region of homology to the DNA-binding domains of the Drosophila protein Dead ringer (Gregory, 1996) and the mouse protein Bright (Herrscher, 1995). There is a second region of homology to a protein predicted by the C. elegans genome project. The C. elegans open reading frame is adjacent to another open reading frame containing a region homologous to the potential DNA-binding domain of Eld. The Eld sequence is extremely proline-rich: proline comprises 17% of its amino acids.
A new family of DNA-binding proteins is represented by the newly discovered dead ringer (dri) gene of Drosophila melanogaster. dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with Dri over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of Dri is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity between Dri and these other homeodomain proteins in in vitro DNA-binding specificity compounds the problem of understanding the in vivo specificity of action in these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others (Gregory, 1996).
B lymphocyte-restricted transcription of immunoglobulin heavy-chain (IgH) genes is specified by elements within the variable region (VH) promoter and the intronic enhancer (E mu). The gene encoding a protein that binds a VH promoter proximal site necessary for induced mu-heavy-chain transcription has been cloned. This B-cell specific protein, termed Bright (B cell regulator of IgH transcription), is found in both soluble and matrix insoluble nuclear fractions. Bright binds the minor groove of a restricted ATC sequence that is sufficient for nuclear matrix association. This sequence motif is present in previously described matrix-associating regions (MARs) proximal to the promoter and flanking E mu. Bright can activate E mu-driven transcription by binding these sites, but only when they occur in their natural context and in cell lines permissive for E mu activity. To bind DNA, Bright requires a novel tetramerization domain and a previously undescribed domain that shares identity with several proteins, including SWI1, a component of the SWI/SNF complex (Herrscher, 1995).
Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk: see Btk family kinase at 29A), and these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk fail to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription is enhanced when Bright and Btk are coexpressed. In contrast, neither Bright nor Btk alone leads to activation of heavy chain transcription. Furthermore, Bright function requires both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright is not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitates with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk (Rajaiya, 2005).
date revised: 28 August 97
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