broad: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

Gene name - broad

Synonyms - Broad Complex

Cytological map position - 2B5

Function - transcription factor

Keywords - molting

Symbol - br

FlyBase ID:FBgn0283451

Genetic map position - 1-0.28

Classification - C2H2 zinc finger, BTB motif

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |

Recent literature
Jia, D., Bryant, J., Jevitt, A., Calvin, G. and Deng, W. M. (2016). The ecdysone and Notch pathways synergistically regulate Cut at the dorsal-ventral boundary in Drosophila wing discs. J Genet Genomics [Epub ahead of print]. PubMed ID: 27117286
Summary:
Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal-ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. This study shows that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, it was found that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, these findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, br was shown to promote Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc.
Guo, Y., Flegel, K., Kumar, J., McKay, D.J. and Buttitta, L.A. (2016). Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells. Biol Open [Epub ahead of print]. PubMed ID: 27737823
Summary:
During development cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. This study examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa. In the wing, ecdysone signaling at the larva to puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva to puparium pulse during early metamorphosis, Broad expression plummets allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing.

Hitrik, A., Popliker, M., Gancz, D., Mukamel, Z., Lifshitz, A., Schwartzman, O., Tanay, A. and Gilboa, L. (2016). Combgap promotes ovarian niche development and chromatin association of EcR-binding regions in BR-C. PLoS Genet 12: e1006330. PubMed ID: 27846223
Summary:
The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. This study shows that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling Ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.
Syed, M. H., Mark, B. and Doe, C. Q. (2017). Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity. Elife 6 [Epub ahead of print]. PubMed ID: 28394252
Summary:
An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.
Kaieda, Y., Masuda, R., Nishida, R., Shimell, M., O'Connor, M. B. and Ono, H. (2017). Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster. Dev Biol 430(1): 166-176. PubMed ID: 28782527
Summary:
Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. This study manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. Expression was monitored of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, it was found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. It is suggested that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression.
BIOLOGICAL OVERVIEW

The temporally ordered expression of transcription factors is one process that can determine the course of development. In this model, genes are activated or repressed according to a genetically based, hierarchical time table. Alternative splicing, regulated both temporally and spatially, can likewise be a determining factor in developmental fate. Through alternative splicing, the RNA maturation machinery of the cell can generate different messenger RNAs from identical pre-messenger RNA transcripts. Alternative splicing is the primary mechanism used by the cell to regulate the function of Broad.

Before describing BRC, and the role of alternative splicing in BRC function, a word about genetic complementation is in order. Crossing two different mutant alleles of a gene in the same organism often gives rise to one of two alternative results. If the two alleles are part of the same complementation group, then the alleles do not complement, and a mutant phenotype results. If the two alleles are part of different complementation groups, then a wild type phenotype results. A test for complementation is the classic means that geneticist use to define the concept of a gene. When two alleles do not complement then they are part of the same functional genetic unit, or gene.

Broad Complex (BR-C), now know as broad is the apt term for a very complex gene that contains up to four classical complementation groups (broad, reduced bristles on palpus (rbp), l(1)2Bc and l(1)2Bd), as well as a group of non-pupariating (npr1) alleles that do not complement these functions. The original BR-C allele, br1, is viable when homozygous in females or hemizygous in males; here, the phenotypical wings are slightly shorter and wider than in wild type. Mutations of the l(1)2Bc complementation group causes late prepupal or early pupal developmental arrest; no head or dorsal thorax form because fusion of the epidermis in the thorax and head regions is defective. npr1 alleles produce the most severe phenotypes, resulting in a failure to pupariate and imaginal discs that form swollen vesicles (Kiss, 1988).

Molecular genetics has clarified the complex nature of Broad Complex. This single gene codes for multiple protein isoforms; they share a common (core) amino terminus fused to any one of four pairs of C2H2-type zinc-finger domains (Z1, Z2, Z3 and Z4). BR-C protein products are widely distributed among all tissues examined in the late larval to prepupal stages of development. All tissues studied to date contain all BR-C isoforms. However, their relative abundance differs greatly among tissue types, suggesting that the various members (or combinations) of the BR-C family of proteins function in different developmental pathways. The messenger RNA species coding for alternative isoforms are generated by alternative splicing of a single pre-messenger RNA transcript (Bayer, 1996).

Although the BR-C transcript and protein isoforms have been detected in every tissue examined during metamorphosis (Emery, 1994 and Huet, 1993), the relative ratio of zinc-finger isoforms differs among tissues, implying that differential expression of isoforms may contribute to the diversity of ecdysone responses among tissues. For example, Z1 and Z3 are the predominant isoforms expressed in late third instar larval salivary glands, while at the same stage Z2 predominates in instar discs. The relative ratios of zinc-finger mRNA isoforms also change over time. Tested for induction in tissue culture, the Z2, Z3, and Z4 mRNA isoforms accumulate rapidly in late third instar larval discs in response to ecdysone but abruptly disappear after 6 hours of culture. In contrast, the Z1 mRNA accumulates more slowly, to become the predominant isoform expressed after 6 hours. Thus, the expression of the four zinc-finger mRNA isoforms is dynamic, changing with time in a hormonally regulated sequence through the course of metamorphosis. (Bayer, 1996).

At least some of the changes in BR-C isoform expression appear to be posttranscriptional. For example, the switch in zinc-finger isoform expression does not result from differential use of the two BR-C promoters. Each can drive synthesis of the full repertoire of zinc-finger isoforms. In addition, sequences from the intronic regions upstream of the Z1 exon and Z4 exon are present in BR-C mRNAs of the largest size class. This suggests that splicing of some mRNA forms is regulated. It is thought that transcription of the early isoforms must involve transcription through the Z1 exon, located closest to the core DNA sequence. The Z1 exon could initially be eliminated from the pool of early BR-C mRNAs by splicing (Bayer, 1996).

The switch in BR-C mRNA isoform expression seen in cultured imaginal discs is also manifest at puparium formation, as a dramatic shift occurs in isoform expression, from Z2 to Z1 (Emery, 1994). The precipitous nature of the switch in zinc-finger isoform expression at a key stage in metamorphosis suggests that alterations in the function of BR-C are critical for the temporal regulation of downstream target genes during metamorphosis (Bayer, 1996).

It was originally proposed (DiBello, 1991) that the complementation groups represent subfunctions corresponding to specific zinc-finger proteins; that Z1 mediates rbp+ functions, Z2 mediates br+ functions and Z3 mediates 2Bc+ functions. This view is now amply supported by current studies. No genetic correlation has yet been made for the newly described Z4 isoform (Bayer, 1996).

Genetic and transgenic analysis of BR-C regulation of downstream genes shows that, in at least three cases the one isoform provides a regulatory function opposite that of the other isoforms. In one case the Z1 isoform activates a gene and the Z2 protein represses it. Thus a switch from predominant Z2 to Z1 isoform expression may mediate the precise temporal control of gene expression. In another case, expression of a salivary gland specific gene Sgs4 Z3 acts as a repressive isoform, while Z1 activates. However, since all isoforms can bind Sgs4 regulatory regions (von Kalm, 1994) it is suggested that Z1 and Z3 may compete for control of the Sgs4 regulatory element. For Dopa decarboxylase (Andres, 1993 and Hodgetts, 1995), Z2 transgene activates transcription, while either Z1 or Z4 represses this gene. Thus the primary role of BR-C appears to be the restriction of temporal expression of downstream genes to discrete developmental periods (Bayer, 1996).

Another example of the importance of alternative splicing in regulation of cell fate is the spatial variation in splicing of Ultrabithorax mRNA. Splicing factors are pivotal to the success of this process. These factors can recognize specific sequences in RNA, and based on sequence recognition, specifically remove unwanted introns and splice together the desired exons. In RNAs subjected to alternative splicing (both BR-C and UBX provide good examples), different introns and exons are removed depending on particular tissue types. The result is different tissue specific splice variants, a total of six different protein isozymes coded for by the different splice variants of UBX. The UBX splice variants differ in the distance between the homeodomain and a domain responsible for interaction with Extradenticle, required as a coactivator on UBX target genes. Different UBX isozymes function effectively with EXD on different target genes depending on the distance between the homeodomain and the interactive domain (Johnson, 1995).

In the case of UBX and BR-C it seems that cell fate depends primarily on the nucleoplasmic environment provided by splicing proteins, and not on the ordered expression of transcription factors. In the case of UBX, the pivotal role played by splicing is spatial, while in the case of BR-C, the pivotal role of splicing on cell fate determination is both spatial and temporal. The importance of alternative splicing in cell fate determination is a radical shift in paradigm for those that think that temporally ordered expression of transcription factors is the key determiner of cell fate.

Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. This study shows that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). Ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, this study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation (Moeller, 2013).

Although extensive studies have made it clear that transition to the adult stage in insects requires a high-level pulse of ecdysone, the mechanism that shapes the pulse, by determining its duration and amplitude, has remained unclear. These experiments show that the maturation-inducing pulse that coordinates the juvenile-adult transition in Drosophila is generated by ecdysone feedback control of PG steroidogenic activity. At the end of the third larval instar, ecdysone acts through EcR in a feed-forward circuit to produce the high-level pulse that triggers pupariation in response to PTTH. This illustrates an EcR-dependent positive feedback operating downstream of PTTH to generate a sustained output in terms of biosynthesis in response to neuropeptide signaling (Moeller, 2013).

The feed-forward loop described in this study provides an explanation for a number of previous observations. These studies have indicated that ecdysone can modulate PG steroidogenic activity and that PG cells undergo autonomous activation under long-term culture conditions. Interestingly, autonomous activation is prevented by juvenile hormone (JH), which inhibits br expression. During the last larval instar of holometabolous insects, a drop in JH levels eventually leads to the production of a high-level ecdysone pulse that triggers metamorphosis, although the mechanism underlying this is poorly understood. Since the decline of JH is permissive for br expression, the fact that Br promotes PG steroidogenic activity is likely to explain how the drop in JH results in the production of a high-level ecdysone pulse initiating metamorphosis. Thus, the data provide a link between JH and ecdysone that might explain how the presence of JH prevents metamorphosis (Moeller, 2013).

Observations clearly show that positive feedback is required for the transcriptional upregulation of phantom (phm), disembodied (dib) and shadow (sad), all of which encode enzymes that act at late steps in the ecdysone biosynthetic pathway. By contrast, EcR and Br activity are not necessary for the normal activity of spookier (spok), which is involved in an earlier step in the pathway and whose transcription is regulated by Molting defective, a factor that is not involved in the regulation of the other identified biosynthetic enzymes. In addition, in contrast to the other ecdysone biosynthetic enzymes, Spok is also likely to be regulated at the level of translation and phosphorylation in response to PTTH signaling. Furthermore, expression of PTTH receptor-encoding torso is not EcR and Br dependent, consistent with levels of torso not being synchronized with the ecdysone peaks. Together with the results demonstrating that the feedback is required downstream of Ras in the PG, this shows that the feed-forward loop functions downstream of PTTH to amplify the signal and not for endowing the PG with competence to respond to PTTH (Moeller, 2013).

The findings raise an important issue that challenges the classical view that ecdysone released from the PG is converted to its more active metabolite 20-hydroxyecdysone (20E) in peripheral target tissues, where it interacts with EcR. Although 20E may travel back and inform the PG, a more direct route would be that ecdysone produced by the PG acts on the gland itself or that the PG produces small amounts of 20E that control the activity of the gland. Consistent with these possibilities, reduced expression of shade, which encodes the enzyme that converts ecdysone to 20E, in the PG leads to a developmental arrest in the larval stages and all three Drosophila EcR isoforms can induce transcription in response to ecdysone. Interestingly, recent reports have demonstrated the essential function of E75, DHR3 (Hr46 – FlyBase), βFTZ-F1 and DHR4 in regulating the production of ecdysone in the PG. Although nitric oxide and PTTH regulate the activity of some of these factors, these signals alone are unlikely to explain the regulation of their function in the PG. Based on the results, an obvious possibility is that EcR controls the expression of these classical ecdysone-inducible genes in the PG. Extensive studies on these ecdysone target genes have led to the elucidation of an early response network for steroid hormone action and the molecular characterization of the genetic architecture underlying the cellular responses to steroids. Surprisingly, this study shows that this genetic program that guides the downstream cellular decisions in response to regulatory ecdysone pulses is utilized upstream to shape the pulse by setting its duration and amplitude. Thus, the same genetic components are used for coordinating the production and reception of the steroid signals that drive directional developmental progression (Moeller, 2013).

Previous experiments demonstrated that ecdysone, produced by the PG, induces an inactivation enzyme responsible for clearance of circulating ecdysone (Rewitz, 2010). This study shows that termination of the pulse requires negative feedback that represses PG steroid production activity in coordination with peripheral clearance. How does ecdysone stimulate and repress biosynthesis in the PG through EcR? The results show that EcR induces different Br isoforms, forming circuits that either increase or inhibit the activity of the biosynthetic pathway by regulating the levels of the enzymatic components. Br is required specifically for the juvenile-adulti transition and is expressed during the last instar. This study shows that the appearance of Br in the PG requires EcR and correlates with the ecdysone peak. The positive effect of EcR on ecdysone biosynthesis is mediated largely through Br-Z4, which has previously been shown to induce transcription of Niemann-Pick type C-1a (Npc1a), which encodes a key cellular component required in the PG for the delivery of cholesterol as a substrate for steroid synthesis. Together, this suggests that ecdysone-mediated positive feedback coordinates increased substrate delivery with upregulation of the biosynthetic machinery in order to produce the maturation-inducing ecdysone pulse. Conversely, the Br-Z1 isoform inhibits ecdysone synthesis, forming a negative feedback that is important for the decline of the ecdysone titer during the prepupal stage. Thus, the temporal control of these circuits relies on a dynamic switch in the PG from Br-Z4 to Br-Z1. A similar switch has been found in the imaginal discs, where Br-Z4 rapidly accumulates in response to ecdysone and then disappears several hours later when Br-Z1 is upregulated. It has been suggested that the switch from Br-Z4 to Br-Z1 is regulated at the level of alternative splicing of br transcripts. The data suggest that the switch is a hard-wired genetic timing mechanism rather than being dependent on ecdysone concentrations. This switching might also occur at the enhancer level through competition of binding to overlapping Br-Z1/Z4 regulatory sites, as was found in the phm promoter. Importantly, coupling a negative with a positive feedback through a common regulatory site ensures a self-limiting response by preventing 'run away' synthesis that would otherwise result from positive-feedback amplification alone (Moeller, 2013).

In conclusion, this study shows that the maturation-inducing ecdysone pulse is shaped by an autonomous feed-forward and feedback circuitry within the endocrine tissue that modulates the rate of hormone synthesis. The coupling of these feedback circuits ensures rapid, self-limiting hormone production that translates neuropeptide signaling into a regulatory steroid pulse which functions as a switch to drive developmental progression (Moeller, 2013).


GENE STRUCTURE

Mapping of 31 cDNA clones indicates that approximately 100 kb of the genome is devoted to the synthesis of many BR-C RNAs. The initial molecular analysis by DiBello (1991) revealed that BR-C encodes a family of DNA-binding proteins with an amino-terminal core region linked by alternative splicing to one of three pairs (Z1, Z2 and Z3) of C2H2 zinc-finger domains. Since that time, an additional zinc-finger sequence has been found (Z4), which encodes a pair of zinc-finger motifs similar to the other three. Z1 shares a start site with Z2, located 55 kb upstream of the DNA coding for the core sequence. For Z2, this start site is used alternatively with another start site shared with Z3 and Z4 as well as Z1, located only 10 kb from the shared core sequence. The four different zinc-finger sequences characterizing Z1, Z4, Z2 and Z3 are located on the other side of the core sequence in the order given. Each of the zinc-finger pairs shows a high degree of similarity among the four BR-C protein isoforms. The 68% similarity between Z1 and Z4 domains is unusually high and suggests a similar specificity in DNA binding (Bayer, 1996).

Using a Z1 specific probe, 4.4-kb mRNA species are detected plus a faint, diffuse band in the 10-kb size range. In contrast, the Z2-Z3 and Z4 specific probes do not detect the 4.4 kb transcript. Each hybridizes instead to the three large RNA size classes of 10, 9 and 7 kb forms. The predominant Z2 and Z3 transcripts are the 9 and 7 kb forms, while the predominant z4 transcript is the 7 kb form (Bayer, 1996).

Exons - 11 shared in different combinations in different proteins


PROTEIN STRUCTURE

Amino Acids - 877 (Z4 transcript)

Structural Domains

Sequence analyses of cDNA clones show that the BR-C encodes a family of related proteins characterized by a common core amino-terminal domain fused to alternate carboxy domains each containing a pair of zinc fingers. Most proteins also contain domains rich in distinctive amino acids located between the common core and C-terminal zinc finger regions (DiBello, 1991).

The J element is a novel DNA sequence involved in the regulated expression of class II major histocompatibility complex genes. DPA, a J element binding protein, contains 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The H/C motif is a stretch of seven amino acids connecting the final histindine of one finger to the first cysteine of the next finger. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box). This N-terminal region shares sequence homology with Drosophila proteins Tramtrack, Broad Complex and Kelch, a structural component of ring canals. It has been suggested that the TAB is a protein-protein interaction domain (Sugarawa, 1994).


broad: Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

date revised: 3 Jan 97 

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