osa/eyelid: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - osa

Synonyms - eyelid

Cytological map position -

Function - presumptive transcription factor

Keywords - segmentation, segment polarity, eye, wing

Symbol - osa

FlyBase ID:FBgn0261885

Genetic map position -

Classification - Bright family

Cellular location - nuclear



NCBI links: | Entrez Gene
Recent literature
Valanne, S., Jarvela-Stolting, M., Harjula, S. E., Myllymaki, H., Salminen, T. S. and Ramet, M. (2020). Osa-Containing Brahma Complex Regulates Innate Immunity and the Expression of Metabolic Genes in Drosophila. J Immunol. PubMed ID: 32198143
Summary:
Negative regulation of innate immunity is essential to avoid autoinflammation. In Drosophila melanogaster, NF-kappaB signaling-mediated immune responses are negatively regulated at multiple levels. Using a Drosophila RNA interference in vitro screen, this study identified a set of genes inhibiting immune activation. Four of these genes encode members of the chromatin remodeling Osa-containing Brahma (BAP) complex. Silencing additional two genes of the BAP complex was shown to have the same phenotype, confirming its role in immune regulation in vitro. In vivo, the knockdown of osa and brahma was shown to enhance the expression of the Toll pathway-mediated antimicrobial peptides when the flies were challenged with Gram-positive bacteria Micrococcus luteus. In this setting, osa knockdown had a particularly strong effect on immune effectors that are predominantly activated by the Imd pathway. Accordingly, Drosophila NF-kappaB Relish expression was increased by osa silencing. These transcriptional changes were associated with enhanced survival from M. luteus + E. faecalis infection. Besides regulating the expression of immune effector genes, osa RNA interference decreased the expression of a large group of genes involved in metabolism, particularly proteolysis. Of note, the expression of the recently characterized, immune-inducible gene Induced by Infection (IBIN) was diminished in osa knockdown flies. Although IBIN has been shown to modulate metabolism upon infection, the expression of selected Osa-regulated metabolism genes was not rescued by overexpressing IBIN. It is concluded that the BAP complex regulates expression of genes involved in metabolism at least partially independent or downstream of IBIN. Moreover, Osa affects the NF-kappaB-mediated immune response by regulating Drosophila NF-kappaB factor Relish expression.
BIOLOGICAL OVERVIEW

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:

  1. wingless expression is present in stripes of approximately normal width in embryos containing no Eld protein.
  2. Ectopic wingless expression is not found in clones of eld mutant cells, indicating that eld does not repress wingless expression.
  3. En stripes are present in eld, wg double mutants, suggesting that eld is not upstream of wingless.
Eld meets the criteria for a direct nuclear effector: its expression is ubiquitous in the early embryo and imaginal discs, and therefore cannot be dependent on localized Wingless signaling. Eld appears to function as a repressor of engrailed expression although this effect need not be direct. Intriguingly, a homolog of Eld, Drosophila Dead ringer, binds to target sequences of Engrailed protein, which are derived from possible autoregulatory sites within the engrailed genomic region (Gregory, 1996). If Eld also binds to these sites, it might compete with En to prevent the establishment of autoregulation. This would imply that Eld acts as a repressor; several active repression domains have been shown to have a similarly high proline content (Treisman, 1997).

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 complex’s 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 transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop

Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. In this study a method was developed to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. This analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. It is proposed that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities (Abdusselamoglu, 2019).

Temporal patterning is a phenomenon where NSCs alter the fate of their progeny chronologically. Understanding how temporal patterning is regulated is crucial to understanding how the cellular complexity of the brain develops. This study presents a novel, FACS-based approach that enabled isolation of distinct temporal states of neural progenitors with very high purity from Drosophila larvae. This allowed a study the transitions between different temporal identity states. odd-paired (opa), a transcription factor that is required for INP temporal patterning, was identified. By studying the role of this factor in temporal patterning, a novel model is proposed for the regulation of temporal patterning in Drosophila neural stem cells (Abdusselamoglu, 2019).

Two different roles are established of the SWI/SNF complex subunit, Osa, in regulating INP temporal patterning. Initially, Osa initiates temporal patterning by activating the transcription factor D. Subsequently, Osa regulates the progression of temporal patterning by activating opa and ham, which in turn downregulate D and Grh, respectively. The concerted, but complementary action of opa and ham ensures temporal identity progression by promoting the transition between temporal stages. For instance, opa regulates the transition from D to Grh, while ham regulates the transition from Grh to Ey. It is proposed that opa achieves this by repressing D and activating grh, as indicated by the lack of temporal patterning in D and opa-depleted INPs. Loss of opa or ham causes INPs to lose their temporal identity and overproliferate. Moreover, it is proposed that D and opa activate Grh expression against the presence of ham, which represses Grh expression. As D and opa levels decrease as INPs age and become Grh positive, ham is capable of repressing Grh later on in temporal patterning. This explains how opa and ham act only during specific stages even though they are expressed throughout the entire lineage (Abdusselamoglu, 2019).

An open question pertains to the fact that the double knock-down of opa and ham, as well as that of D and opa, failed to recapitulate the Osa phenotype. Even though opa and ham RNAi caused massive overproliferation in type II lineages, no Dpn+ Ase- ectopic NB-like cells (as occurs in Osa mutant clones) were detected. It is proposed that this is caused by D expression, which is still induced even upon opa/ham double knockdown, but not upon Osa knock-down, where D expression fails to be initiated. Thus, the initiation of the first temporal identity state may block the reversion of INPs to a NB-state. In the future, it will be important to understand the exact mechanisms of how opa regulates temporal patterning (Abdusselamoglu, 2019).

This study further demonstrates that Osa initiates D expression earlier than opa expression. Osa is a subunit of SWI/SNF chromatin remodeling complex, and it guides the complex to specific loci throughout the genome, such as the TSS of both D and opa. The differences in timing of D and opa expression may be explained by separate factors involved in their activation. Previous work suggests that the transcription factor earmuff may activate . However, it remains unknown which factor activates opa expression. One possibility is that the cell cycle activates opa, since its expression begins in mINPs, a dividing cell unlike imINPs, which are in cell cycle arrest (Abdusselamoglu, 2019).

It is proposed that balanced expression levels of D and opa regulate the timing of transitions between temporal identity states. Indeed, Osa initiates D and opa, the repressor of D, at slightly different times, which could allow a time window for D to be expressed, perform its function, then become repressed again by opa. Deregulating this pattern, for example by overexpressing opa in the earliest INP stage, results in a false start of temporal patterning and premature differentiation. This elegant set of genetic interactions resembles that of an incoherent feedforward loop (FFL). In such a network, pathways have opposing roles. For instance, Osa promotes both the expression and repression of D. Similar examples can be observed in other organisms, such as in the galactose network of E. coli, where the transcriptional activator CRP activates galS and galE, while galS also represses galE. In Drosophila SOP determination, miR-7, together with Atonal also forms an incoherent FFL. Furthermore, mammals apply a similar mechanism in the c-Myc/E2F1 regulatory system (Abdusselamoglu, 2019).

The vertebrate homologues of opa consist of the Zinc-finger protein of the cerebellum (ZIC) family, which are suggested to regulate the transcriptional activity of target genes, and to have a role in CNS development. In mice, during embryonic cortical development, ZIC family proteins regulate the proliferation of meningeal cells, which are required for normal cortical development. In addition, another member of the ZIC family, Zic1, is a Brn2 target, which itself controls the transition from early-to-mid neurogenesis in the mouse cortex. Along with these lines, it has been shown that ZIC family is important in brain development in zebrafish. Furthermore, the role of ZIC has been implicated in variety of brain malformations and/or diseases. These data provide mere glimpses into the roles of ZIC family proteins in neuronal fate decisions in mammals, and this study offers an important entry point to start understanding these remarkable proteins (Abdusselamoglu, 2019).

These findings provide a novel regulatory network model controlling temporal patterning, which may occur in all metazoans, including humans. In contrast to existing cascade models, this study instead shows that temporal patterning is a highly coordinated ensemble that allows regulation on additional levels than was previously appreciated to ensure a perfectly balanced generation of different neuron/glial cell types. Together, these results demonstrate that Drosophila is a powerful system to dissect the genetic mechanisms underlying the temporal patterning of neural stem cells and how the disruption of such mechanisms impacts brain development and behavior (Abdusselamoglu, 2019).

SWI/SNF complex prevents lineage reversion and induces temporal patterning in neural stem cells

Members of the SWI/SNF chromatin-remodeling complex are among the most frequently mutated genes in human cancer, but how they suppress tumorigenesis is currently unclear. This study used Drosophila neuroblasts to demonstrate that the SWI/SNF component Osa (ARID1) prevents tumorigenesis by ensuring correct lineage progression in stem cell lineages. Osa induces a transcriptional program in the transit-amplifying population that initiates temporal patterning, limits self-renewal, and prevents dedifferentiation. The Prdm protein Hamlet was identified as a key component of this program. Hamlet is directly induced by Osa and regulates the progression of progenitors through distinct transcriptional states to limit the number of transit-amplifying divisions. These data provide a mechanistic explanation for the widespread tumor suppressor activity of SWI/SNF. Because the Hamlet homologs Evi1 and Prdm16 are frequently mutated in cancer, this mechanism could well be conserved in human stem cell lineages (Eroglu, 2014).

The data reveal an essential function for the chromatin-remodeling SWI/SNF complex in ensuring lineage progression in stem cell lineages. When neural stem cells/NBs progress toward the transit-amplifying intermediate neural progenitor (TA/INP) fate, the SWI/SNF complex activates a transcriptional program that limits self-renewal and initiates a temporal TF cascade to confer temporal identity. Failure to do so results in lineage reversion and tumor formation. The temporal TF Dichaete (D) and the Prdm protein Ham as direct SWI/SNF targets and show that induction of Ham limits the number of TA divisions by ensuring the progression of temporal patterning. Members of the SWI/SNF complex, particularly the Osa homologs ARID1A and ARID1B, are among the most frequently mutated genes in human cancer, and the findings provide a potential mechanism for their tumor-suppressing activity (Eroglu, 2014).

A model is proposed where two distinct transcriptional programs act in concert to ensure directionality in Drosophila neural stem cell lineages. In type II NBs, a 'self-renewal' program comprising the TFs Dpn, Klu, and HLHmγ allows long-term self-renewal. Upon asymmetric division, Numb and Brat terminate this program in one of the two daughter cells, which therefore progresses toward the imINP stage. As INPs undergo maturation, Brat and Numb disappear, allowing the program to reinitiate and self-renewal to resume. The data indicate that Osa activates a second 'self-renewal restriction' program before this reinitiation occurs to ensure that INPs, unlike NBs, differentiate after around five rounds of asymmetric cell division. In osa mutants, the restriction program is not activated. The self-renewal program, however, is unaffected, and therefore, INPs regain NB-like properties resulting in unlimited self-renewal and brain tumor formation (Eroglu, 2014).

Why does Osa not activate the self-renewal restriction program in NBs? In mammalian neural stem cells, a subunit switch in the SWI/SNF complex is thought to trigger the switch from self-renewal to differentiation, but this study failed to detect a similar switch in the Drosophila larval brain. More likely, Dpn, Klu, and HLHmγ prevent Osa binding in NBs, for example by competing with SWI/SNF for binding sites. In fact, all three factors can act as transcriptional repressors, and opa (one of the SWI/SNF targets identified in this study) is actually also a direct Dpn target in the embryonic CNS (Eroglu, 2014).

The results suggest a tight functional connection between the SWI/SNF complex and the temporal TF cascade that confers temporal identity to INPs. SWI/SNF directly induces transcription of D, the first member of this cascade. In addition, it induces Ham, a chromatin regulator that can limit self-renewal capacity in INPs but also when ectopically expressed in NBs. In INPs, Ham is specifically required for the transition from Grh+, Ey+ middle-aged INPs to Grh-, Ey+ old INPs. Because transition to the terminal transcriptional state is important for timely cell-cycle exit in mINPs (Bayraktar and Doe, 2013), this explains the overproliferation phenotype observed in ham mutants (Eroglu, 2014).

How could Ham mediate temporal progression of INPs? It has been previously shown that recruitment of the earliest component of the NB 'transcriptional clock' to the nuclear periphery permanently silences its expression and limits NB competence. Evi1 and Prdm16, the mammalian homologs of Ham, have been postulated to initiate heterochromatin formation by methylating H3K9. Because H3K9 methylation is crucial for recruiting gene loci to the nuclear periphery, it is interesting to speculate that Ham acts in INPs by driving the transition to the next transcriptional state and, ultimately, to differentiation (Eroglu, 2014).

Mutations in the mammalian SWI/SNF complex subunits are potential drivers of tumorigenesis in a wide variety of tissues including the brain. The Brm homolog SMARCA4 and the Osa homologs ARID1A and ARID1B are among the chromatin modifiers that are recurrently mutated in medulloblastoma, the most common malignant childhood brain tumor. Identifying the cell of origin in brain tumors is crucial in designing effective therapeutic strategies. Stem cells could acquire oncogenic mutations that initiate tumor formation. On the other hand, tumors could also originate from more restricted progenitors that inherit these mutations and become malignant. This study offers an alternative explanation: provided that the function of SWI/SNF is conserved in humans, mutations occurring in restricted progenitors could affect lineage progression causing progenitors to revert into stem cells. In this case, the cell of origin would be a progenitor despite the fact that tumors are made up of stem cells. In fact, this possibility has been proposed for other tumor suppressors and could be tested rigorously for SWI/SNF mutations given the recent significant advances in cell lineage tracing in tumors (Eroglu, 2014).


PROTEIN STRUCTURE

Amino Acids - 2713

Structural Domains

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.


EVOLUTIONARY HOMOLOGS

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).


osa/eyelid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 28 August 97  

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