BIOLOGICAL OVERVIEW

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, a yeast interaction screen was carried out and a Hairy-interacting protein was identified, the Drosophila homolog of the human C-terminal-binding protein (CtBP) (Poortinga, 1998). CtBP is the subject of this overview, but to put its function in perspective, it will be necessary to first discuss Hairy.

The Hairy and Enhancer of split proteins are characterized by two conserved domains, the Orange domain and the C-terminal conserved tetrapeptide WRPW. Functional studies have shown that both of these domains, as well as the bHLH domain, are needed for the proper function of these proteins. The Orange domain contributes to the functional specificity of Hairy/E(spl) proteins (Dawson, 1995 and Giebel, 1997). The WRPW motif has been shown to be necessary and sufficient for the recruitment of Groucho, a WD repeat-containing protein that is not able to bind DNA on its own but, when brought to an endogenous or heterologous promoter, serves as a strong repressor of transcription. Together, these results have led to the prevailing view that Hairy functions as a promoter-bound repressor. An intact bHLH region is required for Hairy to bind to specific DNA sites; once bound, Hairy then recruits the Groucho co-repressor protein (Poortinga, 1998).

Recruitment of Groucho does not account for all of Hairy's repressor properties. Hairy can function genetically as a repressor in the absence of the WRPW motif, and presumably in the absence of the Groucho co-repressor. The Orange domain has been shown to be required for proper function of Hairy and E(spl)m8. This suggests that Hairy is involved in separable repression mechanisms: repression in some cases requiring the bHLH and Orange domains, and in other cases, requiring the bHLH and C-terminal WRPW motif (Dawson et al., 1995). Thus, Hairy may function modularly, with the scope and specificity of its interactions dependent on the proteins recruited to its various conserved domains. Based on the expectation that Hairy works as part of a multiprotein complex, the yeast two-hybrid protein interaction system was employed to identify a Hairy-interacting protein, the Drosophila homolog of human C-terminal binding protein (CtBP), that interacts with a small, previously uncharacterized C-terminal region of Hairy (Poortinga, 1998).

Drosophila CtBP plays a role in embryonic segmentation. Reduction of maternal dCtBP results in severe segmentation defects visualized by cuticle preparations or engrailed staining. Expression of the primary pair-rule genes, eve and runt, as well as expression of hairy itself, is disrupted in embryos lacking maternal dCtBP, whereas the level and spatial positioning of gap gene expression appears normal. The primary pair-rule genes are required to establish each other's expression as well as to direct the striped expression of downstream secondary pair-rule genes, such as fushi tarazu. Similar to what is seen in hairy mutant embryos, Ftz stripes are expanded throughout the trunk region in embryos lacking maternal dCtBP. The physical interaction of CtBP with Hairy and CtBP's genetic interaction with hairy indicate a role for dCtBP in Hairy-mediated repression. The domain of Hairy that interacts with CtBP contains a five amino acid repeat (PLSLV) that bears sequence similarity to a six amino acid motif in the E1a C-terminus (PXDLSX) which interacts with mammalian CtBP (Poortinga, 1998).

CtBP is involved in transcriptional repression mediated by Knirps and Snail. Knirps and Snail are thought of as short-range repressors, acting over distances of less than 100 base pairs to quench upstream activators or the core transcription complex. This form of repression allows enhancers to work independently of one another to direct complex, additive patterns of gene expression, including the seven-stripe patterns of even-skipped and hairy expression. A P-DLS-K sequence is present in the repression domains of Knirps and Snail, and the latter protein also contains the related sequence P-DLS-R. Gene dosage assays suggest that Knirps and Drosophila CtBP interact in vivo. Embryos that are heterozygous for the knirps⁹ null mutation exhibit occasional defects in the even-skipped expression pattern, including reduced staining of stripe 5. Combining the Drosophila CtBP and knirps⁹ mutation results in more severe disruptions in the eve pattern, including the fusion or loss of stripes 4 and 6. The latter knirps⁹/CtBP transheterozygous phenotype is virtually indistinguishable from that observed for knirps mutant embryos. These results raise the possiblility that Drosophila CtBP is part of a larger co-repressor complex that assembles on the Knirps/DNA template. Snail might also require CtBP, because embryos derived from dCrBP mutant oocytes exhibit dorsoventral patterning defects. The C-terminal repression domain of a third short-range repressor, Kruppel, contains a sequence that is related to the mammalian and Drosophila CtBP interaction sequence -- P-DLS-H. Mutations in this sequence nearly abolish Kruppel-mediated repression in human osteocarcinoma cells (Nibu, 1998a).

CtBP may mediate repression throught the enzymatic modification of chromatin because it sequence is related to D-isomer 2-hydroxy acid dehydrogenases. Despite this rather unexpected homology, immunolocalization assays indicate that the Drosophila CtBP protein accumulates in nuclei. Perhaps CtBP cause local changes in chromatin structure by introducing subtle changes in core histones. Alternatively, it is possible that CrBP is a component of an enzymatic cascade that modulates the activities of histone deacetylases or other co-repressor proteins (Nibu, 1998a).

GENE STRUCTURE

By screening early embryonic cDNA libraries with the two-hybrid cDNA insert as a probe, four different cDNA classes have been identified. Each of these four classes encodes the first 376 amino acids of dCtBP then undergoes alternative splicing so that the most C-terminal amino acids (5-83 amino acids), as well as the 3' non-coding regions, are different (with the two-hybrid cDNA having an additional 10 amino acids). cDNAs corresponding to all transcripts identified by Northern analysis have not yet been identified. However, a dCtBP subclone containing only the ATG to the junction where the sequence divergence occurs (amino acids 1-376) retains full interaction with Hairy, indicating that all protein isoforms should interact with Hairy (Poortinga, 1998).

PROTEIN STRUCTURE

Amino Acids - 383 (Nibu, 1998a) 381 and 459 (Poortinga, 1998)

Structural Domains

A sequence database search reveals 63% sequence identity over the entire h-C28 clone (amino acids 93-343), as compared with the human CtBP, a 48 kDa phosphoprotein. Based on the high degree and extent of this homology, it is concluded that h-C28 encodes the Drosophila homolog of the human CtBP protein. Human CtBP was identified as a protein that binds the C-terminus of the adenovirus E1a oncoprotein (Boyd, 1993, Schaeper, 1995 and Poortinga, 1998).

date revised: 5 June 98

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