Drosophila lola encodes a family of BTB-transcription regulators with highly variable C-terminal domains containing zinc finger motifs
Introduction
The human genome sequence contains approximately 32,000 genes, only twice as many as that of Caenorhabditis elegans or Drosophila melanogaster (International Human Genome Sequencing Consortium, 2001). Therefore, the complexity of organisms is not simply achieved by an increase in the number of genes. They seem to utilize a single gene in a variety of ways, through co- or post-transcriptional mechanisms, which include multiple transcription start or termination sites, alternative splicing and RNA editing. Among them, alternative splicing is rather common in higher eukaryotes (Graveley, 2001). It has been estimated that 40–60% of human genes have at least one alternatively spliced form (Modrek and Lee, 2002). However, the mechanisms of alternative splicing have only been elucidated for a limited number of genes (López, 1998; Smith and Valcárcel, 2000).
A family of transcription factors containing the BTB domain and zinc finger motif are involved in a wide variety of developmental pathways and often produce multiple isoforms via alternative splicing. In Drosophila, the tramtrack (ttk) gene is required for development of the nervous system (Guo et al., 1995). Two alternatively spliced isoforms that share a common N-terminal region, including the BTB domain, have distinct C-terminal regions, each with a unique pair of ‘classical’ TFIIA-type C2H2 zinc fingers that recognize different DNA sequences (Read and Manley, 1992). The Broad Complex (BR-C) gene belonging to the same family is one of the few genes directly induced by the ecdysteroid hormone, and regulates the expression of subordinate genes involved in the metamorphosis. There are four isoform-specific exons encoding pairs of classical C2H2 zinc finger motifs (DiBello et al., 1991). The modifier of mdg4 (mod(mdg4)) gene, also known as E(var)3-93D, is involved in a variety of processes, such as silencing in position effect variegation, control of the gypsy insulator sequence, regulation of homeotic gene expression, and programmed cell death (Dorn et al., 1993; Gerasimova and Corces, 1998; Harvey et al., 1997). The mod(mdg4) gene produces 26 different isoforms generated by alternative splicing (Büchner et al., 2000; Dorn et al., 2001). The common exons encoding the BTB domain are alternatively spliced to one of the 3′-variable exons encoding a modified version of C2H2 that potentially forms a finger structure (Büchner et al., 2000; Dorn et al., 2001).
The longitudinals lacking (lola) gene is another member of the BTB-zinc finger protein genes and has been shown to be required for proper pathfinding and target recognition of the SNb motor axons in the Drosophila embryo (Crowner et al., 2002; Giniger et al., 1994; Madden et al., 1999). Giniger et al. (1994) described two transcripts, lola 3.8 and 4.7, which were generated from the lola locus by the use of two promoters and alternative splicing. Two transcripts encode the isoforms sharing a large common N-terminal region, including the BTB domain, and a C-terminal region unique to each isoform. The C-terminal region of the long isoform encoded by lola 4.7 contains a pair of classical C2H2 zinc finger motifs, with a minor modification: the second histidine in the first finger is replaced by cysteine (hereafter, referred as C2HC–C2H2 motif). The shorter isoform lola 3.8 does not encode zinc finger motifs or any other known motifs.
Here, we show that lola is a large and complex locus, extending over 60 kb, and encodes a total of 80 variants through multiple promoter activity and alternative splicing. Lola isoforms consist of a BTB-containing N-terminal region and a C-terminal variable region encoded by alternatively spliced exons among 20 groups, 17 of which have a unique zinc finger motif. Since the BTB domain mediates dimerization, lola could theoretically encode a family of transcription regulators with a large variety of DNA- or protein-binding specificities.
Section snippets
EST clones
The BDGP EST database (http://www.fruitfly.org/) was searched for cDNA clones that matched lola cDNA (Giniger et al., 1994; Toba et al., 1999) as queries. Five clones, LD03274, LD11722, LD13249, LD15869 and GM01463 were obtained from the BDGP. The cDNA inserts were sequenced using a BigDye terminator cycle sequencing kit (Perkin–Elmer) and a Perkin–Elmer ABI PRISM genetic analyzer 310.
RACE
The 5′ ends of the lola transcripts were determined using the 5′ rapid amplification of cDNA ends (RACE)
cDNAs of lola
The gene misexpression system involving modified P-elements is widely used in Drosophila as a tool for identifying new genes. We identified lola as a gene whose misexpression could induce homeotic transformation of aristae to tarsi (Toba et al., 1999). Molecular analysis of forcibly expressed transcripts suggested that lola produced multiple forms of transcripts that appeared to be generated through alternative splicing. To reveal the variability of the lola transcripts, we sequenced five EST
lola encodes a large family of alternatively spliced transcripts
We have explored the variability of the Drosophila lola cDNA structure by means of 5′ RACE, 3′ RACE, genome sequence searches, and EST sequencing, and demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5′ and 3′ variable sequences and constitutive common exons. The common exons encode the N-terminal region containing the BTB domain, and the variable exons encode the C-terminal variable domain,
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) ‘Genome Science’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#12202002).
References (33)
- et al.
Key residues characteristic of GATA N-fingers are recognized by FOG
J. Biol. Chem.
(1998) - et al.
Polycomb and trithorax group proteins mediate the function of a chromatin insulator
Cell
(1998) Alternative splicing: increasing diversity in the proteomic world
Trends Genet.
(2001)- et al.
tramtrack acts down stream of numb to specify distinct daughter cell fates during asymmetric cell division in the Drosophila PNS
Neuron
(1995) - et al.
Zinc finger proteins: new insights into structural and functional diversity
Curr. Opin. Struct. Biol.
(2001) - et al.
lola has the properties of a master regulator of axon-target interaction for SNb motor axons of Drosophila
Dev. Biol.
(1999) - et al.
Alternative pre-mRNA splicing: the logic of combinatorial control
Trends Biochem. Sci.
(2000) - et al.
The genome sequence of Drosophila melanogaster
Science
(2000) - et al.
Crystal structure of BTB domain from PLZF
Proc. Natl. Acad. Sci. USA
(1998) - et al.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucleic Acids Res.
(1997)
Promoter elements in Drosophila melanogaster revealed by sequence analysis
Genetics
The POZ domain: a conserved protein-protein interaction motif
Genes Dev.
Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila
Genetics
Lola regulates midline crossing of CNS axons in Drosophila
Development
The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers
Genetics
The enhancer of position-effect variegation of Drosophila
E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators. Proc. Natl. Acad. Sci. USA
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Present address: Takashi Ohsako, Drosophila Genetic Resource Center, Kyoto Institute of Technology, Kyoto 616-8354, Japan.