EvoprintHD of Kr

BIOLOGICAL OVERVIEW

Krüppel is a gap gene. The term gap comes from the effect of mutation: gap genes cause the loss of central segments from the embryo, thus creating gaps in the developing structure.

The domain of early Krüppel expression is in the center of the embryo. Gap genes like knirps, giant and Krüppel are among the earliest genes expressed during development. They subdivide the embryo along the anterior/posterior axis, creating a framework for the subsequent expression of pair-rule genes. In turn, pair-rule genes are expressed in seven symmetrical stripes under the control of gap genes and a number of genes expressed in the egg before fertilization, such as bicoid, hunchback and nanos.

Krüppel represses transcription of other genes, not unlike the subduing effects adults might have on an otherwise noisy adolescent party. It represents one of the earliest calls to reason, assuring that development remains an orderly process.

After gastrulation the party is only just beginning. One example of the part Kr plays in tissue differentiation will be given. In each Malpighian tubule of Drosophila, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells, each expressing Krüppel in each tubule primordium. Each cluster is marked out by the expression of proneural genes, and the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. Here achaete is responsible for tip cell allocation, but Kr acts as the selector gene, responsible for tip cell fate. The tip cell directs the growth of the Malpighian tubules and organizes the mitotic response and migration of the other cells forming each tubule (Hoch, 1994). Therefore Krüppel is responsible for cell fate in the Malpighian tubules, a role quite distinct from Krüppel's role as a gap gene.

The available in vivo evidence suggests that Kruppel acts as a transcriptional repressor; however, conclusive in vivo evidence demonstrating that Krüppel can additionally function as an activator of gene expression has only recently been found. Krüppel binds to the consensus sequence AAAAC/GGGGTTAA (Rosenberg, 1986 and Pankratz, 1989). The zinc finger domain of the Kr protein is framed by two evolutionarily conserved transrepressor domains [an N-terminal TR1 (transrepressor domain 1) and C64 (C-terminal repressor domain)] and a single, weak transactivator domain (TA1). C64 was initially identified when transferred to the DNA-binding domain of the yeast transcriptional activator GAL4: all three transacting domains, TR1, TA1 and C64, have been shown to confer their activities to the bacterial LacI protein. The two independent and transferable repressor domains of Kruppel have been shown to control expression of the pair-rule gene hairy; the minimal cis-acting element of hairy stripe7 mediates either Kruppel-dependent activation or repression in different regions of the blastoderm embryo (La Rosee-Borggreve, 1999).

In Drosophila cultured cells, TA1 alone is incapable of acting as a weak transactivator domain. TA1 is however, activation-competent in the presence of the adjacent stretch of 51 amino acid residues of the Kr protein. This 51 amino acid region contains sequence motifs similar to those observed in the transactivation domains of CTF/NF1, Sp1 and Pit1, but since this sequence alone fails to mediate gene activation, it is referred to as the co-activating domain (CAD). Combined TA1 and CAD causes reporter gene activation even in the presence of TR1. Together, these two domains override the TR1-dependent transrepression activity. In contrast, when TA1 and CAD are directly fused with the C64 repressor domain of Kr, reporter expression is nullified. Therefore, the opposite regulatory activities of the TA1/CAD and C64 domains are extinguished when fused. Thus, it appears necessary that, as in the full-size Krüppel protein, these domains are separated in order to exert opposite regulatory functions on transcription (La Rosee-Borggreve, 1999).

The hairy stripe7 enhancer element, decodes the activity of three activators: the maternal homeodomain proteins Caudal and Bicoid, and the zinc finger protein Kruppel. Caudal and Krüppel activities are necessary, and sufficient, to activate h7-mediated lacZ reporter gene (h7-lacZ) expression but Bicoid activity is additionally required to achieve wildtype expression levels. Absence of Kr activity not only significantly reduces the level of h7-dependent reporter gene activation in the posterior region of the embryo, but also results in the appearance of a second and novel expression domain in a position corresponding to the highest levels of Krüppel in wildtype blastoderm embryos. Thus, h7 not only mediates gene expression in response to low levels of Krüppel in the posterior region of the blastoderm embryo, but it simultaneously prevents reporter gene expression at high concentrations in the central region of the embryo. To determine the ability of Kr to directly interact with the h7 element, DNaseI footprinting experiments were performed using bacterially produced Kr and subfragments of the h7 element. The h7 element has been shown to contain five in vitro Krüppel binding sites; this opens the possibility that Krüppel may act through multiple binding sites within the h7 element (La Rosee-Borggreve, 1999).

The C-terminal region of Krüppel that encompasses the predominant repressor domain is not essential for activation, but is required to fully suppress h7-mediated transcription in response to high levels of Krüppel activity. This domain contains an interaction motif for dCtBP, a homologue of the human co-repressor CtBP. dCtBP activity is, however, dispensable for Krüppel-mediated repression in the embryo since Krüppel-mediated repression functions in the absence of dCtBP (La Rosee-Borggreve, 1999).

In vitro experiments have shown that C64 provides a homodimerization surface that permits Krüppel homodimer formation at high protein concentrations. The homodimer acts exclusively as a transcriptional repressor, whereas the Krüppel monomer has been shown to function as a transcriptional activator both in vitro and in Drosophila tissue culture assays (Sauer, 1993). Based on the in vitro results, it has been proposed that Krüppel acts as a transcriptional repressor in the central region of the blastoderm embryo and may function as an activator of target genes outside the central region where the concentration of Krüppel gradually decreases (Sauer, 1993). The h7-mediated expression pattern in Kr^V mutant embryos is consistent with this proposal: the lack of the C-terminus, and hence the dimerization domain, does not affect Krüppel's ability to co-activate h7-mediated gene expression in a position of low Krüppel concentration in the embryo, but rather, strongly reduces its repressor function at high concentrations (La Rosee-Borggreve, 1999).

Gray and Levine (1996) proposed two models to explain Krüppel-mediated repression. One model suggests that Krüppel possesses two separate activities, one interfering with enhancer-bound activators by quenching, and the other directly inhibiting transcription by interacting with components of the basal transcription machinery. The second model proposes that Krüppel recruits a repressor complex that only functions locally. Some aspects of the results presented here fit with the first model, others with the second. For example, TR1 could be the repressor domain that acts through quenching. In this case, TR1 would interfere with Bicoid-dependent activation mediated by h7 in the central region of the embryo, but not with Caudal-dependent activation, which is predominant in the posterior region of the embryo. This assignment is consistent with the finding that repression of h7-mediated gene expression is strongly reduced in the central region of the Kr^V mutant embryo but no effect is observed in the posterior region of the embryo when Krüppel lacking the C-terminal region is expressed throughout the embryo (La Rosee-Borggreve, 1999).

Alternatively or additionally, C64 could act either by blocking activation via inhibiting basal transcription, or it may interfere with, and thereby extinguish, both Caudal and Bicoid activities directly. Direct inhibition of the basal transcription machinery would be consistent with in vitro data showing that C64 prevents transcription by interacting with the general transcription factor TFIIEbeta (Sauer, 1995a). This proposal would, however, be consistent with the recent finding that the C-terminal repression region of Krüppel inhibits certain activators (Hanna-Rose, 1997) only if the subset of affected activators would target TFIIEbeta to exert their function. The second model which explains transcriptional repression via a repressive complex formation is consistent with the observation that the C-terminal domain enables Kr to form heterodimer complexes with other transcription factors such as Knirps (Sauer, 1995a). A further possibility is that the C-terminal domain could serve to recruit more general co-repressors such as Groucho or CtBP to template DNA. A CtBP-binding motif has indeed been noted in the C- terminal repressor region of Krüppel (Nibu, 1998). The Drosophila homolog, dCtBP, has been shown to interact in vitro with the gap gene product Knirps and gene-dosage interaction studies with dCtBP and knirps mutants have suggested that Knirps-dCtBP interactions are also able to occur in vivo (Nibu, 1998). The recruitment of dCtBP by short-range repressors, such as Knirps and Krüppel, may theoretically be able to alter the chromatin structure, its status of acetylation or the presence of transcriptional activators bound to a nearby site within the enhancer. Nevertheless, the weakest known knirps mutant, knirps^14F, which lacks the dCtBP-interaction motif, develops an almost normal abdominal segment pattern with the exception that the abdominal segment 4 is consistently missing. This suggests that dCtBP may possibly be important for some specific but not all aspects of Knirps-dependent repressor function. The results shown here indicate that dCtBP is neither required for Krüppel-dependent repression of h7-mediated activation in the central region of the embryo, nor for Knirps-dependent repression of the expression domain in the posterior region of the embryo. Furthermore, dCtBP is also not required for repression of this expression domain in response to ubiquitously expressed Krüppel (La Rosee-Borggreve, 1999 and references therein).

The results shown here describe a previously missing piece of information surrounding Krüppel function; namely, that Krüppel possesses both activator and repressor function in vivo. The switch between activator and repressor functions is dependent on the concentration of Krüppel protein and is mediated by the C-terminus. The precise mechanism by which this mode of switching is regulated and potential cofactors of Krüppel are still unknown and need to be addressed by future studies (La Rosee-Borggreve, 1999).

GENE STRUCTURE

Exons - two

Bases in 3' UTR - 264

PROTEIN STRUCTURE

Amino Acids - 466

Structural Domains

Krüppel is a zinc finger protein with four tandomly repeated zinc finger domains (Rosenberg, 1986). A subset of zinc finger transcription factors contain amino acid sequences that resemble those of Krüppel. They are characterized by multiple zinc fingers containing the conserved sequence CX2CX3FX5LX2HX3H (X is any amino acid, and the cysteine and histidine residues are involved in the coordination of zinc) that are separated from each other by a highly conserved 7-amino acid inter-finger spacer, TGEKP(Y/F)X, often referred to as the H/C link.

Each 30-residue zinc finger motif folds to form an independent domain with a single zinc ion tetrahedrally coordinated beween an irregular, antiparallel, two stranded ß-sheet and a short alpha-helix. Each zinc finger of mouse Zif268 (which has three fingers) binds to DNA with the amino terminus of its helix angled down into the major groove. An important contact between the first of the two histidine zinc ligands and the phosphate backbone of the DNA contributes to fixing the orientation of the recognition helix. Although the two fingers of Drosophila Tramtrack interact with DNA in a way very similar to those of Zif268, there are important differences. Tramtrack has an additional amino-terminal ß-strand in the first of the three zinc fingers. The charge-relay zinc-histidine-phosphate contact of Zif268 is substituted by a tyrosine-phosphate contact. In addition, for TTK, the DNA is somewhat distorted with two 20 degree bends. This distortion is correlated with changes from the rather simple periodic pattern of amino base contacts seen in Zif268 and finger 2 of TTK (Klug, 1995 and references).

To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, the Kruppel homolog was cloned from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that is most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encodes a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments have demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region is able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibits transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development (Hanna-Rose, 1997).

date revised: 5 January 99

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