BIOLOGICAL OVERVIEW

Bearded null mutants are viable and exhibit no mutant phenotypes, but dominant mutant alleles cause phenotypic effects on adult sensory organ development that closely mimic those resulting from loss-of-function mutations in Notch (N) pathway genes (Leviten, 1996). These include the commitment of extra proneural cluster cells to the sensory organ precursor (SOP) fate, and the appearance of additional sheath cell/neuron pairs at the expense of the socket and shaft cells in the sensory organ lineage. These dominant phenotypic effects of Brd gain-of-function alleles are sensitive to the dosage of two neurogenic genes (Notch and neuralized), as well as Hairless (H), a negative regulator of N pathway activity. Both Brd and the genes of the E(spl)-C are directly activated in the proneural clusters of the wing imaginal disc by Achaete and Scute, and the same genes are expressed in the vicinity of the morphogenetic furrow of the eye imaginal disc under ac/sc-independent control (Leviten, 1997 and references).

The Bearded gene encodes a novel small protein of only 81 aa that is predicted to include a highly basic amphipathic a-helix. The Brd and E(spl) region transcript m4 (E(spl)m4) proteins are somewhat related. Structurally, both E(spl)m4 protein and Bearded appear to have similar protein structures; each sequence suggests the ability to form a basic amphipathic alpha-helix. Loss of function mutations in Enhancer of split complex genes cause a neurogenic phenotype. E(spl)-C genes function to antagonize the proneural activity of achaete and scute. E(spl)m4 might be expected to act similarly. Paradoxically, the simplest deduction from the Bearded gain-of-function phenotype (Leviten, 1996), is that Bearded normally acts to promote commitment to neural cell fates, exactly the opposite function attributed to E(spl)m4. Thus, even as the Brd and E(spl)m-4 proteins rely on similar biochemical mechanisms of action (as suggested by their structural similarities), it is a distinct possibility that they have antagonistic, rather than cognate, regulatory effects. For example, both proteins might interact with a common protein(s), but have opposite effects on the activity of these factors (Leviten, 1997).

In common with several transcription units of the E(spl)-C, including E(spl)m4, Bearded contains two novel heptanucleotide sequence motifs in its 3' untranslated region (UTR), suggesting that all these genes are subject to a previously un-recognized mode of post-transcriptional regulation. These sequence motifs are called the Brd box (AGCTTTA) and the GY box (GTCTTCC). Like known sequence elements that function in post-transcriptional regulation, both of these motifs are found in a single orientation and specifically in the UTRs of the genes that include them. Genetic evidence from Brd gain-of-function alleles implicates its 3' UTR in post-transcriptional negative regulation. Specifically, mutant Brd transcripts that are truncated in the 3' UTR due to a transposon insertion are present at elevated steady-state levels. The region deleted in the mutant transcripts includes two of the three Brd boxes and the GY box normally found in BRD mRNA, suggesting that these motifs may function to destabilize the wild-type transcript (Leviten, 1997).

UTRs can also control the polyadenylation state of the transcript. Many mRNAs are translationally inactive until they undergo additional cytoplasmic polyadenylation, a process controlled by cytoplasmic polyadenylation elements (CPEs). Polyadenylation is implicated in Brd box function. Negative regulation by the Brd box motif affects steady-state levels of both RNA and protein. It is noteworthy that relative transcript levels differ between the total and poly(A) + RNA populations. Mutation of the Brd boxes causes a modest increase in the amount of total gene transcript (~1.5-fold), but a greater increase (~2.3-fold) in the relative amount of polyadenylated transcript. These results demonstrate that Brd box-mediated regulation affects steady-state transcript levels and suggest that deadenylation or inhibition of polyadenylation is one component of this regulation. These results are consistent with a post-transcriptional function in promoting RNA turnover. Brd box activity has an even greater effect on reporter protein accumulation. Specific mutation of these elements causes a 3- to 5-fold increase in steady-state protein levels. This result indicates that Brd boxes have an additional role in regulating translation, beyond the effect attributable to transcript level differences (Lai, 1997)

Thus, the Brd 3' UTR confers negative regulatory activity in vivo. This activity is spatially and temporally general, in that most or all cells are able to respond to Brd boxes. This suggests that some genes expressed outside of proneural clusters may be regulated by these motifs as well. Three other genes that encode negative regulators of PNS development also contain these sequences in their 3' UTRs. In particular, kuzbanian (kuz) and extramacrochaetae (emc) each include single Brd boxes, while hairy (h) contains a GY box. emc also includes four copies of a GY box-related sequence (GTTTTCC) in its 3' UTR, which may be relevant for its regulation. kuz has functions in SOP selection and lateral inhibition, so its expression certainly includes proneural clusters. However, emc and h are expressed in spatial patterns that are largely complementary to proneural clusters in the leg and wing imaginal discs, and are thus possible examples of genes regulated by the Brd box (and possibly the GY box) in territories outside the clusters. Interestingly, the Emc and H proteins, as members of the HLH family, are structurally related to the E(spl)-C bHLH proteins. In contrast, kuz encodes a metalloprotease/disintegrin protein of the ADAM family (Lai, 1997 and references).

It is not yet known how these RNA sequences function to regulate translation and mRNA stability. Two possible models are being considered, one in which regulatory proteins bind to these sequences and regulate messenger activity or stability, and another in which RNA:RNA regulates the mRNA function. What is clear, however, is that cell fate is highly regulated by systems of opposing activity, and that much has yet to be discovered about how these systems integrate at both a cellular and molecular level.

Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. An investigation is described of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic alpha-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, they are referred to as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, it has been shown that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. Overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling (Lai, 2000a).

The sequences of Drosophila ESTs encoding apparent paralogs of both Brd and E(spl)m4 have been deposited in the GenBank database. PCR products containing these EST sequences were used as probes to isolate full-length cDNA and genomic DNA clones for both genes, which have been named Bob (Brother of Brd) and Tom (Twin of m4), respectively. In addition, genomic DNA has been cloned and sequenced that includes the previously identified E(spl)malpha locus: its predicted protein product is also strongly related to that of E(spl)m4. The predicted amino acid sequences of what is referred to as Brd family proteins can be classified as Brd-like (Brd and Bob) or m4-like (m4, malpha, and Tom), based on their relative sizes and degree of amino acid similarity. Although there are a few well-conserved regions in these proteins, particularly within the C-terminal half of the longer m4-like proteins, it is obvious that Brd family members are not in general highly related at the primary structure level. Brd and m4 are related by secondary structure, since a domain located near the N-terminus in both proteins is predicted to form a basic amphipathic helix. Similar N-terminal domains found in Bob and E(spl)malpha are likewise strongly predicted to form basic amphipathic helices, while a proline residue in the center of the corresponding region of Tom suggests that its 'helix' may be kinked or separated into two helices. The strong basic amphipathic character of these N-terminal domains of Brd family proteins may be considered a defining structural feature. Brd family proteins also share certain classes of consensus phosphorylation sites, namely protein kinase C (PKC) sites in their N-terminal regions and casein kinase II (CK II) sites in their C-terminal portions. The similar placement of these consensus sites in the context of otherwise weakly related amino acid sequences suggests that they may be relevant for the regulation of Brd family protein function (Lai, 2000a).

Bob and Tom genes were localized to the Drosophila genome physical map using a filter grid library of P1 clones. Interestingly, Bob and Tom map to a set of P1 clones covering cytological region 71A1-2, the known chromosomal location of Brd. Characterization of this genomic region shows that Tom gene is found to lie only about 2 kb upstream of the previously described Brd transcription unit, with Bob about 20 kb upstream of Tom. Surprisingly, the genomic DNA corresponding to the Bob EST and cDNA clones is triplicated, such that three distinct, tandemly arranged genomic loci have the capacity to encode nearly identical Bob transcripts. This triplicated structure has been observed in two independent, overlapping lambda phage genomic DNA clones. Significantly, small sequence differences in the transcribed portions of the different Bob genomic loci are also represented in the cDNA clones, allowing for the conclusion that at least two copies are transcriptionally active in wild-type flies. The three gene copies have been designated Bob A, B, and C, the encoded transcripts and proteins are referred collectively as 'Bob'. Thus, a minimum of five Brd family genes are contained within an approximately 30-kb interval that is referred to as the Brd Complex (Brd-C), and this complex contains both Brd-like (Brd and Bob) and m4-like (Tom) genes. The E(spl)-C contains at least two m4-like genes, m4 and malpha. Promoter regions of Brd family genes within the Brd-C and E(spl)-C contain consensus binding sites for proneural proteins and for Suppressor of Hairless (Lai, 2000a).

Postembryonic expression patterns of Brd family genes were examined by in situ hybridization. In wing imaginal discs of third-instar larvae, Brd and E(spl)m4 transcripts accumulate specifically in the full complement of sensory organ proneural clusters. Similarly, the complex pattern of E(spl)malpha expression in the wing disc includes proneural clusters, although malpha transcript accumulation in the clusters consistently appears broader and more diffuse than that of Brd or m4. In addition, malpha transcripts appear in a narrow stripe along the dorsoventral boundary of the wing pouch, as well as along wing vein borders. In contrast, neither Bob nor Tom exhibit any patterned expression in the wing disc, although Tom may be generally expressed at a very low level in this tissue. In the eye imaginal disc, four of the five Brd family genes in this study are expressed in the vicinity of the morphogenetic furrow, the exception being Bob, which is not detectably expressed in either the eye or antenna discs. Transcripts from the different Brd family genes accumulate with distinct spatial profiles relative to the morphogenetic furrow. Brd is expressed in two closely spaced stripes, one just anterior to, and one within and posterior to, the dpp furrow stripe. Transcripts from m4, by contrast, appear in a strong band that is largely just anterior to the zone of dpp-lacZ expression. malpha shows expression in a pattern that overlaps, and extends posterior to, the marker stripe. Finally, Tom expression somewhat resembles that of Brd, in that its transcripts accumulate in two stripes lying anterior and posterior to the dpp-lacZ stripe (Lai, 2000a).

The patterns of transcript accumulation from these genes were examined during pupal wing development, to assess the possible expression of these transcripts in sensory organ lineages and in the vicinity of the developing wing veins. The members of the Brd-C are not expressed at detectable levels in the pupal wing at 8 hours after puparium formation (APF), although Brd and Tom transcripts are present in the large clusters of proximal campaniform sensilla at this time. By contrast, m4 and malpha in the E(spl)-C display both proximal campaniform expression and specific wing margin expression at 8 hours APF. m4 transcripts accumulate in a set of anterior wing margin cells at this stage. Based on their spacing, they are likely to represent cells in the lineage of the chemosensory organs that appear in dorsal and ventral rows on the margin. Since transcripts from E(spl)mgamma accumulate in these organs, it appears that at least one Brd family member and at least one bHLH gene in the E(spl)-C share this aspect of their expression. It has been found that malpha is expressed at this time (8 hours APF) in a broad domain of wing margin cells that includes cells of the posterior as well as the anterior margin, and also in an incomplete wing vein boundary pattern. This latter observation prompted an examination of the accumulation of malpha transcripts in later pupal wing discs. At 24 hours APF, malpha is indeed expressed in a largely complete pattern consisting of thin rows of cells at all vein/intervein boundaries. This is highly reminiscent of the pattern of transcript accumulation from both Notch and the bHLH gene E(spl)mbeta at approximately the same time. In addition, malpha transcripts remain present throughout the wing margin (both posterior and anterior) at this stage. malpha expression in the pupal wing is highly dynamic, however: by 30 hours APF, its transcripts have nearly gone from the margin, are excluded from vein/intervein borders, and appear instead in the veins themselves and in non-vein wing blade tissue. Taken together, these observations strongly suggest that at least one Brd family member may have a role in wing vein development. In summary, in developing imaginal tissue, Brd family members are expressed specifically in multiple territories in which Notch signaling-dependent cell fate decisions take place (Lai, 2000a).

Overexpression of Brd causes adult phenotypes closely resembling those conferred by loss-of-function mutations in N pathway genes. These phenotypes include both bristle multiplication and bristle loss; the former is due to the specification of supernumerary SOPs, while the latter is caused by inappropriate allocation of cell fates within the bristle lineage. Likewise, overexpression of other Brd family genes similarly interferes with cell fate specification events controlled by N pathway activity. By comparison with the results with Brd, m4, and malpha, overexpression of Bob and Tom each cause much stronger mutant phenotypes. With one copy of UAS-Bob, a strong tufting or lethal phenotype is observed, with bristle tufting extending to most macrochaetes and microchaetes, as well as occasional bristle loss. UAS-Tom causes the most severe effects of all Brd family members when expressed under the control of sca-GAL4, with most lines giving high percentages of lethality at late pupal/pharate adult stages. The relatively infrequent escapers typically exhibit strong tufting of nearly all macrochaetes and microchaetes and frequently display some degree of bristle loss, especially on the legs (Lai, 2000a).

The commonality of an N-terminal basic domain in all Brd family proteins, along with their similar phenotypic effects when over- or mis-expressed, suggests that they may have a common biochemical mechanism of action and may interact with a common target or targets. The conserved C-terminal extension found in the m4-related proteins (m4, ma, and Tom) further suggests that this subfamily may have additional functions that are not shared by the shorter Brd-related proteins (Brd and Bob). In particular, it is possible that the terminal DRWV/AQA motif in these proteins, by analogy with the conserved C-terminal domain of the E(spl)-C bHLH proteins (which recruits the co-repressor Groucho), may also mediate protein-protein interactions. A similar possibility exists for the shared PVXFXRTXXGTFFWT motif (Lai, 2000a).

If the gain-of-function effects reported here are indicative of the normal direction of Brd family protein function, and if all members of the Brd gene family are indeed targets of transcriptional activation by this pathway, as has been hypothesized, then Brd family proteins are excellent candidates to mediate a negative feedback mechanism in N signaling. However, a full understanding of Brd family protein function must ultimately incorporate loss-of-function genetic data, which, owing to apparent functional overlap among these genes, is not available. Thus, it is entirely possible that overexpression of Brd family proteins, rather than reinforcing or exaggerating their wild-type activity, instead causes a 'dominant negative' effect; in this case, these proteins may normally function as positive effectors of N signaling (Lai, 2000a).

An important issue concerning the function of Brd family proteins is whether they exert their effects on N signaling on the sending or receiving side of the process, or both. Preliminary evidence suggests that overexpression of Brd family genes is able to exert a cell non-autonomous effect on lateral inhibition in proneural clusters, consistent with the possibility that these proteins can antagonize the ability of a cell to send an inhibitory signal. This is of considerable interest, since relatively little is known about the detailed structure and function of the N pathway upstream of the N receptor (Lai, 2000a).

GENE STRUCTURE

Brd and E(spl)m4 genes exhibit an unusual degree of nucleotide sequence identity in their 3' UTRs. An alignment of a continuous 40- nt region of the 3' UTRs for each gene shows that 31 nt are shared between Brd and m4. This region includes a 14/14 identity containing the third (most 3') Brd box in Brd, as well as an adjacent 7/7 identity referred to as the "GY box." Both sequence elements are present in the 3' UTRs of most genes of the Complex, sometimes in multiple copies. The Brd box is most often represented by the 7-nt core sequence AGCTTTA, although in two cases [E(spl)m4-B1 and E(spl)m5-B3], the full 9-nt element CAGCTTTAA (repeated three times in Brd) is found. In both of these cases, the Brd box motif is part of a longer sequence identity with Brd, that is, the 14/14 identity noted above that includes Brd-B3 and E(spl)m4-B1, and a 12/12 identity that includes Brd-B2 and E(spl)m5-B3. Overall, of the nine transcription units in the E(spl)-C for which appropriate sequence data are available [seven bHLH repressor-encoding genes plus E(spl)m4 and groucho (gro)], the Brd box occurs in the 3' UTRs of six of them. The exceptions are E(spl)m beta, E(spl)m8 and gro. The 7-nt GY box (GTCTTCC) is also present in the 3' UTRs of multiple E(spl)-C genes. As with the Brd box, GY boxes are also frequently part of longer sequence identities between genes, such as the 11/11 block that includes E(spl)m3-G1 and E(spl)m5-G3, or the 10/10 identity that contains Brd-G1 and E(spl)m3-G1. No gene was identified that contains a GY box without an accompanying Brd box. No consistent positional relationship can be discerned between the Brd and GY boxes in the different genes, or between these elements and the stop codon, polyadenylation signal or polyadenylation site (Leviten, 1997).

The exact structure of the Brd mutant transcription unit was determined by DNA sequence analysis. Brd gain-of-function alleles are associated with an insertion of a non-P repetitive element retrotransposon of the blood family into position +399 in the 3' untranslated region (UTR) of the gene. Upstream of the point of insertion of this element, the mutant genomic DNA is identical to the wild-type cDNA sequence. The blood family of retrotransposons was first recognized in association with the blood allele of the white locus. The blood element in Brd mutants is oriented such that a polyadenylation signal is located just 3 bp inside the LTR proximal to the Brd transcription start. Efficient use of this blood polyadenylation signal could account for the truncation of the transcript in the Brd mutants. To confirm this interpretation, the 3' ends of the Brd transcript were cloned from both wild-type and Brd 1 mutant larvae. The Brd 1 transcript does include blood element LTR sequences at its 3' terminus, and is polyadenylated 15 nt downstream of the blood polyadenylation signal. This generates a mutant transcript that is approximately 100 nt shorter than the wild-type larval transcript, which itself terminates at exactly the same position as indicated by the embryonic cDNA clones. The blood transposon present in the gain-of-function alleles is inserted between the first and second of the Brd box elements, and causes the two most 3' Brd boxes to be excluded from the mutant transcript (Leviten, 1997).

Bases in 5' UTR - 70

Exons - 1

Bases in 3' UTR - 233

PROTEIN STRUCTURE

Amino Acids - 81

Structural Domains

Brd encodes a novel small protein that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic a-helix, suggesting that the two proteins have related biochemical functions. While the protein overall is slightly basic (predicted isoelectric point 8.65), it has a distinctly basic N-terminal half and a relatively acidic C-terminal half. The N-terminal region contains a high proportion of residues potentially subject to phosphorylation, including eight threonine and three tyrosine residues in the first 24 aa. In addition, there is a clear casein kinase II consensus phosphorylation site near the Brd C terminus (Leviten, 1997).

Although Brd is not strongly related to any known protein in the GenBank database, Brd shares weak sequence similarity with the product of the m4 transcription unit of the E(spl)-C. Alignment of the Brd and E(spl)m4 sequences reveals two regions of limited similarity, one in the N-terminal portion of both proteins, and a second within acidic portions of both proteins. The region of highest identity overlaps the Brd amphipathic helix. Residues 24-38 of E(spl)m4 can likewise be expected to form such a structure (Leviten, 1997).

date revised: 5 February 2000 

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