Krüppel: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - Krüppel

Synonyms -

Cytological map position - 60F3

Function - transcription factor

Keywords - gap gene

Symbol - Kr

FlyBase ID:FBgn0001325

Genetic map position - 2-107.6

Classification - zinc finger

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene
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 KrV 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 KrV 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, knirps14F, 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).

Drosophila Polycomb complexes restrict neuroblast competence to generate motoneurons

Similar to mammalian neural progenitors, Drosophila neuroblasts progressively lose competence to make early-born neurons. In neuroblast 7-1 (NB7-1), Kruppel (Kr) specifies the third-born U3 motoneuron and Kr misexpression induces ectopic U3 cells. However, competence to generate U3 cells is limited to early divisions, when the Eve+ U motoneurons are produced, and competence is lost when NB7-1 transitions to making interneurons. This study found that Polycomb repressor complexes (PRCs) are necessary and sufficient to restrict competence in NB7-1. PRC loss of function extends the ability of Kr to induce U3 fates and PRC gain of function causes precocious loss of competence to make motoneurons. PRCs also restrict competence to make HB9+ Islet+ motoneurons in another neuroblast that undergoes a motoneuron-to-interneuron transition, NB3-1. In contrast to the regulation of motoneuron competence, PRC activity does not affect the production of Eve+ interneurons by NB3-3, HB9+ Islet+ interneurons by NB7-3, or Dbx+ interneurons by multiple neuroblasts. These findings support a model in which PRCs establish motoneuron-specific competence windows in neuroblasts that transition from motoneuron to interneuron production (Touma, 2012).

This study used multiple genetic approaches to investigate the timing and specificity of competence restriction by PRCs in Drosophila neuroblasts. The data show that PRCs establish motoneuron competence windows in two distinct neuroblast lineages, regulating the production of both Eve+ and HB9+ Islet+ motoneurons. This provides a mechanistic explanation for the loss of competence that has been previously described in NB7-1 and NB3-1. The experiments manipulating the timing of Pdm and Cas expression show that this mechanism is not limited to fate specification by Kr but is involved in establishing a broad motoneuron competence window. Consistent with this model, there appears to be little restriction of competence in a lineage that produces exclusively interneurons (NB3-3) and, correspondingly, PRC activity does not affect the ability of Kr to alter interneuron fates in this lineage. In addition, whereas Ph gain-of-function is sufficient to inhibit production of HB9+ Islet+ motoneurons by NB3-1, the production of HB9+ Islet+ interneurons by NB7-3 and of Dbx+ interneurons by multiple neuroblasts are unaffected (Touma, 2012).

The initial screen revealed a requirement for a subset of PRC1 and PRC2 genes in the regulation of competence. Lack of a statistically significant phenotype for other genes might be due to dosage: all embryos are heterozygous for the mutant allele and there is maternal contribution of Polycomb group and Trithorax group transcripts. Subsequent studies primarily used the Su(z)123 (null allele) and phd401, ph-p602 (ph-d401 is hypomorphic, ph-p602 is null) mutants. Su(z)12 is a component of PRC2 and Ph is a component of PRC1, allowing assessment the roles of each PRC complex. Su(z)12 loss-of- function extended competence to the end of the NB7-1 lineage. Su(z)12 is an essential co-factor of the E(z) H3K27 methyltransferase and levels of Su(z)12 activity correlate with the extent of H3K27 methylation at target genes. This suggests that the degree of competence restriction is determined by the levels of H3K27 methylation at genes required for motoneuron production. Progressive restriction of competence was still observed in the ph-d401, ph-d602 mutants, which was likely to be due to residual Ph activity. However, competence in these mutants is not completely lost until nearly twice the number of neuroblast divisions have occurred than are normally associated with loss of competence (nine divisions in ph mutants versus five in wild type). It is hypothesized that PRC-induced chromatin modifications accumulate over multiple neuroblast divisions and must reach some threshold for inhibiting motoneuron fates, similar to the accumulation of H3K27 trimethylation at the Neurog1 locus during competence restriction in mammalian cortical progenitors. Without testing additional Polycomb group and Trithorax group genes as homozygous mutants and generating maternal nulls (which in some cases might not survive to the relevant stages of neurogenesis), precisely which Polycomb group proteins are necessary for the restriction of competence cannot be precisely identifed. The core components of PRC1 and PRC2 are likely to be ubiquitously and constitutively expressed throughout neurogenesis, and this has been confirmed for Pc and Ph. However, cell type-specific PRC complexes and developmentally regulated changes in PRC composition have been described previously, suggesting that PRC1 or PRC2 co-factors might regulate the timing of competence restriction. It will be interesting to test the role of co-factors that are known or predicted to recruit PRC2 to specific genes, such as the PhoRC complex, Pipsqueak and Grainy head (Touma, 2012).

The sequential generation of motoneurons followed by interneurons has been observed during nervous system development of many insects. Clonal analysis of Drosophila neuroblasts suggests that motoneurons are always produced first, as demonstrated for NB7-1 and NB3-1, although precise birth order data are lacking for most other lineages. In the mammalian spinal cord, motoneurons and interneurons are produced from spatially segregated populations of progenitors that develop along the dorsal-ventral axis of the neural tube. Drosophila lacks this spatial segregation of motoneuroncommitted or interneuron-committed progenitors. Instead, temporal changes allow single progenitors to produce mixed lineages. PRCs appear to work in parallel to the temporal identity transcription factors by establishing competence windows in which temporal identity factors can specify motoneuron fates. Competence windows might represent a 'quality control' mechanism in which PRCs reinforce the timing of fate specification, similar to the role proposed for miRNAs during Drosophila development. Competence windows might also allow temporal identity factors to be 'redeployed' at later divisions. The majority of neuroblasts express Kr and Cas a second time and this study has confirmed that NB7-1 re-expresses Kr when interneurons are being produced. The function of Kr during later neuroblast divisions remains to be determined. If PRC activity alone were responsible for blocking a Kr-specified motoneuron late in the NB7-1 lineage, at least one ectopic U3 might be expected in ph-d, ph-p hemizygous or Su(z)12 homozygous mutant embryos. However, no altered U motoneuron fates were observed in such mutants. There are at least two potential explanations for this result. First, residual PRC activity in these mutants might allow sufficient changes in chromatin states to block endogenous Kr from specifying a motoneuron. This possibility is supported by data showing a dosage-sensitive relationship between Kr and PRC levels in specifying U3 fates, and the eventual loss of competence in ph mutants subjected to heat shock-induced pulses of Kr. Alternatively, there might be an additional transcription factor (or factors) that specifies interneuron fates in the NB7-1 lineage. This interneuron fate determinant could have a dominant effect, such that even when PRC activity is reduced, interneuron fates (or an Eve- 'hybrid' fate) prevail. Conversion to an Eve+ motoneuron might therefore only occur in a combined PRC loss-of-function and Kr gain-of-function background (Touma, 2012).

In both NB7-1 and NB3-1, later-born motoneuron fates are preferentially inhibited in Ph gain-of-function experiments, supporting a link between the number of neuroblast divisions and the restriction of motoneuron competence. The timing of competence restriction might also be regulated by the temporal identity factors themselves. Previous studies of competence in NB7-1 and NB3-1 have shown that constitutive expression of Hb can maintain neuroblasts in a fully competent state. In addition, precocious Pdm expression can inhibit Kr expression and block U3 fates in NB7-1 and RP3 fates in NB3-1. How Hb or Pdm might interact with Polycomb or Trithorax complexes during the regulation of competence remains to be determined (Touma, 2012).

In an attempt to identify PRC target genes that affect competence, NB7-1 fates were analyzed in embryos with wor-GAL4 driving expression of Kr in combination with the following candidates: the anterior-posterior patterning Hox genes Ultrabithorax, abdominal A, Antennapedia and Abdominal B, the nervous system-expressed Hox gene BarH1, the neuroblast fate determinant gooseberry, and the cell cycle regulator Cyclin A. No extension of competence was detected when these PRC targets are coordinately overexpressed with Kr. It would be technically very challenging and beyond the scope of this work to identify direct PRC targets in NB7-1 or NB3-1. However, clues are provided by previous studies that identified PRC targets in Drosophila embryos). One interesting set of PRC targets is a group of genes involved in motoneuron formation or function: eve, islet, HB9, Nkx6 (HGTX - FlyBase), zfh1 and Lim3. All motoneurons that innervate dorsal muscles express Eve, most motoneurons that innervate ventral muscles express some combination of Lim3, Islet, HB9 and Nkx6, and all somatic motoneurons express Zfh1. None of these genes is sufficient to confer motoneuron fates on their own, and some (eve, HB9, islet) are also expressed in subsets of interneurons. It is possible that PRCs silence the transcription of multiple genes that establish motoneuron fate 'combinatorial codes.' Relevant PRC target genes might be coordinately regulated by the temporal identity transcription factors (as suggested by the ability of high levels of Kr to partially overcome competence restriction) or transcription of these targets might depend on indirect interactions (Touma, 2012).

In mammalian embryonic stem cells, PRCs maintain pluripotency by inhibiting transcription of developmental pathway genes. These genes contain 'bivalent' histone modifications, with PRC-associated H3K27 methylation and Trithorax-associated H3K4 methylation keeping developmental regulators silenced but poised for activation. During differentiation of embryonic stem cells into neural progenitors, neural development genes lose PRC-associated modifications but retain H3K4 methylation, resulting in increased transcription. Although PRC silencing maintains pluripotency in embryonic stem cells, PRCs are likely to have an additional role in restricting fate potential once a progenitor becomes lineage committed. This was recently demonstrated for mouse embryonic endoderm progenitors, which undergo a fate choice for liver or pancreas development. The regulatory elements of liver and pancreas genes have distinct chromatin patterns prior to commitment to either lineage, and EZH2 [an ortholog of Drosophila E(z)] promotes liver development by restricting the expression of pancreatic genes. Similar chromatin 'prepatterns' might exist for motoneuron and interneuron genes in newly formed Drosophila neuroblasts, with subsequent PRC activity selectively silencing motoneuron genes in NB7-1 and NB3-1. PRC activity has also been shown to regulate the timing of terminal differentiation in mouse epidermal progenitors and the transition from neurogenesis to astrogenesis in mouse cortical progenitors. The identification of a related mechanism in Drosophila neuroblasts suggests that temporal restriction of fate potential is a common function of PRCs. Drosophila embryonic neuroblasts will provide a useful system for addressing several outstanding questions regarding PRC regulation of fate potential, including how PRCs are recruited to target genes, the composition of the relevant silencing complexes, and how PRC activity is temporally regulated (Touma, 2012).


GENE STRUCTURE

Bases in 5' UTR - 186

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).


Krüppel: Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised:15 November 2012

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