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.

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).

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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