brinker: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - brinker

Synonyms -

Cytological map position - 7B

Function - transcription factor

Keywords - Dpp pathway, D/V patterning, wing

Symbol - brk

FlyBase ID: FBgn0024250

Genetic map position -

Classification - novel

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene

Recent literature
Deignan, L., Pinheiro, M.T., Sutcliffe, C., Saunders, A., Wilcockson, S.G., Zeef, L.A., Donaldson, I.J. and Ashe, H.L. (2016). Regulation of the BMP signaling-responsive transcriptional network in the Drosophila embryo. PLoS Genet 12: e1006164. PubMed ID: 27379389
Summary:
The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. Using the Drosophila embryo as a model, this study combined RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. Multiple new BMP target genes were identified, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain were identified, and widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions was observed. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. 



BIOLOGICAL OVERVIEW

brinker is a potential transcription factor that negatively regulates decapentaplegic target genes (Bray 1999). brinker was independently characterized as a modifier of dpp signaling in three laboratories (Campbell, 1999; Jazwinska, 1999a, and Minami, 1999). In an enhancer trap screen designed to identify genes whose expression may be regulated by Dpp in the imaginal discs (Campbell, 1999), one potential line, X47, was selected that appeared to exhibit marker gene expression at the anterior and posterior extremes of the wing disc but not in the central region. Dpp is expressed along the compartment border within this central region, suggesting that the gene marked in the X47 line may be negatively regulated by Dpp signaling. In Jazwinska (1999a), a collection of 3200 X-linked lethal mutations was screened. One mutant was identified that showed a strong expansion of the dorsal epidermis and concomitant reduction of the ventral epidermis, indicating a defect in Dpp signaling. This indicated that brk negatively affects either the distribution of Dpp, the reception of the Dpp signal, or the activation of Dpp target genes. Minami (1999) isolated brinker by characterizing two strains with P-element insertions that generate expression patterns complementary to those of Daughters against dpp in the wing disc. The two strains have P-element insertions in polytene cytological location 7B, a region to which brinker had been genetically mapped by Roth and Wieschaus laboratories in the first two studies.

An abundance of attention has been paid to proteins which, together with the SMADs, act as activators of transcription downstream of Dpp. However, one case is known from C. elegans where activated SMADs counteract the specific repression exerted by a DNA-binding inhibitory SMAD (Patterson, 1997 and Thatcher, 1999). In addition, analysis of Drosophila Brinker suggests that one purpose of SMAD signaling is to antagonize transcriptional repression of Dpp target genes (Campbell, 1999; Jazwinska, 1999a and b, and Minami, 1999). In the wing, the Brinker protein acts as a repressor of low and intermediate level Dpp target genes. These genes are expressed upon loss of brk even in the absence of SMAD signaling, suggesting that Brk acts as a direct transcriptional repressor. Since brk expression itself is negatively regulated by Dpp, target gene activation, at least in part, results from downregulation of brk.

This overview will examine the role of Brinker in regulating Dpp target genes in the early zygote. Consistent with brk’s role in the wing disc, embryonic target gene activation in the absence of brk is independent of SMAD activity. Thus, in both contexts, brk acts either parallel to or downstream of SMADs as a specific repressor of low and intermediate level Dpp target genes. brk is expressed like another dpp antagonist, short gastrulation (sog), in ventrolateral regions of the embryo abutting the dorsal dpp domain, and in brk mutants dpp expression expands to cover the entire ectoderm. In this situation sog is largely responsible for Dpp gradient formation, since brk;sog double mutant embryos have almost no polarity information in the ectoderm. The double mutants consist mainly of mesoderm and unstructured dorsal epidermis. Thus, brk and sog together specify the neuroectoderm of Drosophila embryos (Jazwinska, 1999b).

The Dpp activity gradient that is established in the dorsal half of cellular blastoderm embryos subdivides the ectoderm into three major territories that can be identified by the cell types they will comprise and/or by the cuticular structures they secrete. High levels of Dpp activity specify the dorsalmost region, which differentiates into large amnioserosa cells that express Krüppel (Kr) protein. These cells form extraembryonic tissue that does not produce cuticle. Lower Dpp levels in the dorsolateral region specify the non-neurogenic ectoderm, harboring peripheral nervous system precursors that can be identified as small segmentally repeated snail (sna)-expressing cells. This region, the dorsal epidermis, gives rise to cuticle with weakly pigmented dorsal hairs. Finally, the ventral (neurogenic) ectoderm requires very low levels or the absence of Dpp to form the neuroblasts of the central nervous system, which also expresses sna. This region, the ventral epidermis, secretes cuticle with heavily pigmented ventral denticles. The cuticle of brk mutant embryos has an enlarged region carrying dorsal hairs and a smaller region carrying ventral denticles. The number of sna-expressing neuroblasts in the ventral neurogenic region is reduced. This indicates that brk mutations lead to an expansion of dorsolateral fates and a reduction of ventrolateral fates. However, despite these lateral fate shifts, the number of Kr-expressing amnioserosa cells is not different from wild type. Thus, brk specifically affects cell fates depending on low or intermediate levels of Dpp signaling, while those that require peak levels are not altered (Jazwinska, 1999b).

To identify the underlying causes of the visible changes in cell fate, the effect of brk was examined on the expression of two groups of dorsal/ventral (D/V) patterning genes. The first group consists of dpp, zerknullt (zen) and tolloid (tld), whose expression is initiated very early in syncytial blastoderm stages. Since they are ventrally repressed by Dorsal (Dl) protein, their expression domains are confined to the dorsal 40% of the egg's circumference. In brk mutant embryos dpp, zen and tld expression is initiated normally. However, in contrast to wild type their expression domains expand ventrally during mid-cellularization. These data demonstrate that brk is not required for the early ventrolateral repression of these genes, but is essential to prevent their lateral expansion during cellularization. Since at these stages Dl protein is still present in the lateral nuclei, brk might function to augment the Dl-mediated repression of dpp, zen and tld (Jazwinska, 1999b).

The second group of DV patterning genes includes rhomboid (rho), u-shaped (ush) and pannier (pnr), which are not direct targets of repression by maternal Dl. The initiation of their expression during cellularization requires prior formation of the Dpp activity gradient. Therefore, they are candidates for being direct targets of Dpp signaling in the embryo. They are expressed in domains straddling the dorsal midline that are 12 (rho), 14 (ush) and 32 (pnr) cells wide at cellular blastoderm (cell counts at approx. 50% egg length). The two narrowly expressed genes rho and ush are not changed in brk mutant embryos. This is also true for late zen expression, which in brk mutant embryos, as in wild type, refines to a narrow 5- to 6- cell-wide stripe along the dorsal midline despite the prior expansion. However, pnr expression expands in brk mutant embryos and low ectopic pnr levels can be seen in a broad lateral domain that stops about five cells short of mesodermal sna expression. Thus, brk does not affect the Dpp target genes that are expressed in dorsalmost regions and supposedly depend on highest Dpp levels. However, a target gene that is expressed in a wider domain, and is therefore presumably activated by intermediate levels of Dpp, is expanded (Jazwinska, 1999b).

The described alterations in DV patterning appear to result from complete loss of brk function, and deficiencies uncovering brk produce phenotypes identical to the original point mutation brk M68. This mutation contains a stop codon causing an early truncation of the protein (Jazwinska, 1999a). The phenotypes are also not enhanced in embryos derived from females in which germline clones homozygous for brk M68 have been induced, indicating that the zygotic phenotype is not ameliorated by maternal expression. In summary, brk mutations affect the Dpp activity gradient in the embryo by expanding the domains of expression of dpp and one of its activators (tld) into ventrolateral regions. Despite the uniform expression of dpp in the entire ectoderm, Dpp activity levels appear to be only mildly increased in the ventrolateral region since only low-level (zen) or intermediate-level (pnr) target genes are ectopically expressed, causing a reduction in the size of the nervous system and ventral epidermis accompanied by an expansion of dorsal epidermis. Peak levels of Dpp in dorsalmost positions appear to be normal, judging from both target gene expression and cell type differentiation (Jazwinska, 1999b).

The question arises as to why uniform expression of dpp in the ectoderm is compatible with the substantial degree of DV polarity exhibited by brk mutant embryos. In wild type, Dpp activity is polarized by sog expression in the ventrolateral region of the embryo, such that ventrolateral Dpp activities are reduced and a peak of activity is established centered on the dorsal midline. Embryos mutant for sog show a reduction of ventrolateral fates, albeit to a weaker degree than brk embryos. In contrast to brk embryos, they differentiate only a small number of scattered amnioserosa cells. The lateral fate shift is not accompanied by strong expansion of dpp transcription as seen in brk embryos, and pnr expression is not as greatly expanded as in brk embryos. In brk embryos, sog is still expressed in the ventrolateral domain of the ectoderm. To test whether this expression accounts for the DV polarity in the ectoderm of brk embryos, brk embryos were constructed that were also mutant for sog. In contrast to either single mutant, the ectoderm of brk sog embryos forms only dorsal-type cuticle hairs and completely lacks ventral denticles. During germ band extension, neuroblast expression of sna cannot be detected; instead, sna expression in the peripheral nervous system (PNS) precursors, normally restricted to dorsolateral regions, expands into the ventral region of the embryo. This suggests a complete deletion of the ventral neurogenic region (Jazwinska, 1999b).

Taken together, brk and sog have both overlapping and distinct roles in shaping the Dpp activity gradient of the Drosophila embryo. While sog has an important function in providing peak levels of the gradient necessary for amnioserosa development, brk and sog together are essential to limit the ventral extension of the anti-neurogenic activity of dpp. Interestingly, brk;sog double mutants do not completely eliminate all polarity of the ectoderm. This can be seen from the expression of pnr, which in the double mutant has uniformly high ectodermal levels except in a 5-cell wide stripe bordering the mesoderm. Thus, in addition to brk and sog, other ventrally localized factors provide patterning information for the ectoderm. Candidates for this function are members of the spitz group of genes (Jazwinska, 1999b).

In the wing disc, brk mutant clones have completely cell autonomous effects that lead to cell fate transformations corresponding to low or intermediate levels of Dpp signaling (Campbell, 1999; Jazwinska, 1999a, and Minami, 1999). Mutant cells autonomously express the Dpp target genes optomotor blind (omb) and spalt (sal) without expressing dpp. omb and sal each represent a target gene of a different class with regard to brk regulation. While omb shows normal levels of expression in brk clones, sal, the target gene that depends on higher Dpp levels, is only weakly expressed. This indicates that sal integrates brk-dependent and brk-independent mechanisms for its activation. High-level target genes that are not affected by brk have not been found in wing discs so far; however, the cell fate transformation caused by ectopic expression of constitutively active Tkv (Tkv*) indicate that such target genes exist (Jazwinska et al., 1999). Since in the complete absence of Mad, which is essential for all aspects of Dpp signaling, removal of brk leads to the ectopic expression of omb and sal, it is concluded that Dpp signaling acts to relieve brk repression. brk is expressed in lateral regions of the wing disc in a pattern which might largely result from transcriptional repression by Dpp. This might also be the predominant mechanism by which Dpp signaling relieves Brk’s repression of target genes (Jazwinska, 1999b).

The relationship between dpp and brk is more complex in the embryo than in the wing imaginal disc for several reasons: (1) brk influences dpp expression in the embryo and thus changes the Dpp activity gradient. Therefore, it is more difficult to judge whether target gene mis-expression in brk mutants is a direct result of removal of brk or of the expansion of Dpp. (2) Three of the genes, dpp, zen and tld, whose expression is expanded in brk, are also targets of the maternal Dl gradient, and so are subject to both Dl-mediated and Brk-mediated repression in early embryos. (3) brk itself is clearly an activated target of Dl when it is first required for repression of dpp, tld and zen; only later during gastrulation does Dpp negatively regulate brk. (4) In the embryo but not in the disc, brk acts with sog in an intricate way to shape the Dpp gradient (Jazwinska, 1999b).

Despite these complexities brk’s relationship to the Dpp activity gradient in early embryos and to the Dpp gradient in wing discs is very similar. In both contexts low and intermediate-level targets are misexpressed in Brk mutants independent of Dpp signaling. Using brk;dpp double mutant embryos it has been demonstrated that the ectopic expression of zen and pnr in brk embryos is not a secondary consequence of the activation of dpp, but occurs even in the complete absence of Dpp signaling. pnr activation in brk;dpp double mutant embryos furthermore confirms that repression by brk is not dependent on prior repression by maternal Dl since pnr is not a direct target gene of Dl repression. pnr’s regulation by brk is strikingly similar to brk’s regulation of sal in the wing disc. pnr and sal both are intermediate-level Dpp target genes. In brk mutant cells, they both show weak levels of expression as compared with the expression in their normal domains. Thus, in the embryo and in the disc three types of Dpp target genes can be distinguished with regard to regulation by brk: (1) those that are fully activated upon loss of brk; (2) those that are weakly activated, and (3) those that are not affected. The regulation of the first group might occur entirely by antagonizing brk’s repression; the second group requires both relief of repression by brk and positive activation by Dpp; and the third group might only be subject to direct activation by Dpp (Jazwinska, 1999b).

brk-mediated target gene regulation might be important for morphogen function of Dpp. The strongest phenotypic effects of brk mutations seen so far are in places where Dpp acts as a morphogen. In both the wing disc and embryo brk regulates Dpp target genes that are activated at the lower end of the gradient. In these regions special mechanisms of gradient interpretation might be required that are sensitive to small changes in Dpp activity. Target gene regulation by brk could provide such a mechanism. The full activation of low-level target genes requires only transcriptional repression of brk by Dpp. However, a more non-linear mechanism suited to establish sharp threshold responses would result if target gene promoters had both repressive brk and activating SMAD binding sites. Then, Dpp would simultaneously downregulate brk transcription and antagonize its function at the target gene promoters. Such a dual mechanism is the more likely because the data demonstrate that it operates for the intermediate targets sal and pnr. However, experimental support for such a model can only come from a detailed analysis of how brk interacts with the target gene promoters (Jazwinska, 1999b).

Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types

Transcriptional repressors function primarily by recruiting co-repressors, which are accessory proteins that antagonize transcription by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many can recruit more than one, with Drosophila Brinker (Brk) recruiting the co-repressors CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress targets in the wing, questioning why it should need to recruit CtBP, a short-range co-repressor, when Gro is known to be able to function over longer distances. To resolve this, genomic engineering was used to generate a series of brk mutants that are unable to recruit Gro, CtBP and/or have 3R deleted. These reveal that although the recruitment of Gro is necessary and can be sufficient for Brk to make an almost morphologically wild-type fly, it is insufficient during oogenesis, where Brk must utilize CtBP and 3R to pattern the egg shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling (Upadhyai, 2013).

A structure/function analysis of the transcriptional repressor Brk has been performed by replacing the endogenous brk gene with a ΦC31 bacteriophage attP site into which mutant forms of brk were introduced by integrase-mediated transgenesis. The goal was to generate mutations that disrupted the ability of Brk to recruit the CoRs Gro and CtBP and/or that deleted the less well characterized 3R repression domain and to test their activity in different tissues at different times of development to determine if and why they are required by Brk to repress transcription. Previous studies with Brk and other TFs that can recruit both CoRs indicated that Gro recruitment is essential for at least some of the activities of these TFs, but the reason for recruiting CtBP has proven more elusive. This study has confirmed that Gro recruitment is essential for Brk activity, but have also showed that Brk needs to recruit CtBP and to possess the 3R domain for full activity in some tissues, in particular during oogenesis (Upadhyai, 2013).

Lethality of the brkGM mutant reveals Gro recruitment is necessary for Brk activity. The brkΔ3RCM mutant, which utilizes Gro as its sole repressive activity, can progress from fertilization to an almost morphologically wild-type adult, indicating that Gro is close to sufficiency in this regard. However, brkΔ3RCM mutants often die as embryos and show defective oogenesis, with eggs having aberrant egg shell pattering, a characteristic of brk null mutants. The single mutants, brkΔ3R and brkCM, show less severe egg shell defects and reduced fertility, the latter probably relating to a defective micropyle, the structure through which sperm normally enter. The apparent inactivity of BrkΔ3RCM protein in follicle cells appears to be explained by active, unphosphorylated Gro being reduced there. The egg shell is patterned by the surrounding follicle cells, where Brk is expressed at high levels in the dorsal anterior. This coincides with high levels of EGFR signaling and previous studies have shown that Gro activity is attenuated following phosphorylation by MAPK downstream of EGFR signaling. As expected, lower levels of unphosphorylated or active Gro were found in the dorsal-anterior follicle cells. Consistent with the activity of BrkΔ3RCM being compromised by EGFR-dependent downregulation of Gro activity, upregulation of EGFR signaling in the wing disc of brkCM mutants results in derepression of the targets salE1 and ombZ (Upadhyai, 2013).

EGFR signaling also probably reduces the levels of active Gro available for Brk in other tissues, including the ventral ectoderm where Brk activity is required to ensure proper patterning of the denticle belts and where EGFR signaling is known play a key role. Many brkΔ3RCM mutants do not survive embryogenesis and demonstrate defects in denticle patterning similar to, but weaker than, those of null mutants. In addition, the VDB phenotype of brkGM mutants is less severe than in brkKO or brk3M mutants. Thus, CtBP and 3R appear to provide repressive activity in the ventral ectoderm (Upadhyai, 2013).

No Brk targets have been characterized in the follicle cells, but these would be expected to be partially derepressed in both brkCM and brkΔ3R mutants and possibly completely derepressed in brkΔ3RCM mutants based on the egg shell phenotypes, although there might be some differences between brkCM and brkΔ3R given the differences between CtBP and 3R just discussed. However, again, this would not imply that these targets are CtBP/3R specific, because the inability of Gro to participate in their repression is presumed to be due to its unavailability. Thus, although studies have indicated that TFs that have the ability to recruit both Gro and CtBP may only recruit one or other at specific targets, this might not reflect a CoR specificity for individual targets, but rather a cell-specific availability of CoRs (Upadhyai, 2013).

It is possible that if Gro were available in all cells then the CiM and 3R domain would be dispensable and so, at least for Brk, downregulation of Gro by MAPK phosphorylation could be considered inconvenient. This might be true for other TFs, including Hairy, Hairless and Knirps, which also function in multiple tissues, many of which are exposed to RTK signaling, and might explain why these TFs need to resort to recruiting CtBP as well as Gro. It should also be noted that Gro activity can be downregulated in other ways, including phosphorylation by Homeodomain-interacting protein kinase. This downregulation of Gro activity has been explained in terms of reducing the activity of specific repressors in specific tissues, such as E(Spl) factors during wing vein formation. This appears to be a somewhat illogical way to downregulate the activity of specific repressors, as there are almost certainly many other TFs utilizing Gro in the same cells and in other tissues exposed to RTK signaling and their activity might be compromised. There are no data indicating whether the downregulation of Gro activity in follicle cells serves any purpose and could simply be a consequence of the decision to downregulate Gro activity by this means in other tissues. However, this has serious implications for Brk and has required Brk to be versatile in its mechanisms of repression. Of course, the possibility has not been ruled out that downregulation of Gro activity does serve a purpose for Brk in follicle cells; for example, if Gro were available here it might provide Brk with too much activity or allow it to inappropriately repress a target that CtBP or 3R cannot. This might be tested by assessing egg shell phenotypes after driving unphosphorylatable Gro at physiological levels in a brkΔ3RCM mutant, but currently this is technically challenging (Upadhyai, 2013).

The idea that repressors need to be versatile in their repressive mechanisms because of variable CoR availability presumably extends beyond Brk and Hairless, Hairy and Knirps. In fact, other repressors in Drosophila possess both CtBP- and Gro-interaction motifs, including Snail. This might not be simply related to downregulation of Gro activity, as CtBP activity can also be modulated; for example, SUMOylation and acetylation of mammalian CtBPs is implicated in regulating their nuclear localization. In addition, other CoRs might similarly be available only in some cells; MAPK activity has been shown to phosphorylate and lead to the nuclear export and inactivation of the SMRT CoR complex. Finally, a further consideration raised by the present study is that care should be taken in assuming that a TF requires and can use a specific CoR to repress its targets in a particular tissue simply because it possesses an interaction motif for that CoR (Upadhyai, 2013).


PROTEIN STRUCTURE

Amino Acids - 704

Structural Domains

The protein sequence of Brk contains several regions of repeated amino acids. There are three glutamine-rich regions, an alanine- and histidine-rich region, a serine-rich region, and one run of eight histidines. Blast database searches that filter out these repeats fail to identify significant homology to any protein in the data banks. However, searching specifically with the N-terminal 100 amino acids of Brk reveals a weak homology between amino acids 44-99 and the homeobox domains from several proteins (21%-23% identities). The predicted secondary structure of this region of Brk contains two alpha helices, one from residues 10-16 and another from residues 31-56, and a less structured region in between them. Noticeably, the conserved amino acids (R-43, Q-44, W-48, Q-50), which are part of the DNA recognition helix in the homeodomain, are also present in the C-terminal alpha helix in this region of Brk. Although speculative, this suggests that Brk may bind to DNA. The sequence PMDLSLG at 377-383 is similar to a motif (P-DLS-K) present in several proteins known to act as transcriptional repressors that have been shown to interact with the corepressor CtBP (Nibu, 1998). In addition, there is a short stretch of basic amino acids (KKQRRLKKK) at position 517-525 that is reminiscent of the nuclear localization signal of SV40 large T antigen. The presence of these basic residues and the putative DNA-binding domain and transcriptional repression domain, together with the genetic data that Brk inhibits the pathway downstream of the SMADs, suggests that Brk functions as a repressor of Dpp target genes (Jazwinska, 1999a).

The most striking features of the amino acid sequence are homopolymer stretches encoded by opa (CAX) repeats; such repeats have no clear function. A search of databases for similarity to the nonrepetitive regions of Brk failed to identify any potential homologs or any regions of strong similarity to any regions of other proteins. As a first attempt to determine the possible function of this protein, its cellular localization was determined by immunofluorescence using a specific antibody to the protein. This shows that Brk protein expression in the wing disc is indistinguishable from that of the RNA and that the protein is localized to the nucleus. The sequence contains three potential nuclear localization signals. Although no obvious homologs have been identified by standard similarity searches, potential functional domains can be predicted by other means, including a method to identify potential helix-turn-helix (HTH) DNA-binding motifs. This procedure predicts Brk protein to have an HTH motif in the N-terminal region at positions 68-89. Another feature of the primary sequence is a central domain of about 150 amino acids, almost half of which are charged. Several repressor domains in transcription factors are characterized by having a similar high percentage of charged residues, suggesting this domain in Brk may play a similar role (Campbell, 1999).


brinker: | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 December 2013

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