Tup1, a yeast WD repeat protein involved in transcriptional silencing

Repression of yeast mating-type regulated genes by the global repressor Ssn6/Tup1 has been linked to a specific organization of chromatin. Tup1 directly interacts with the amino-terminal tails of histones H3 and H4, providing a molecular basis for this connection. This interaction appears to be required for Tup1 function because amino-terminal mutations in H3 and H4 that weaken interactions with Tup1 cause derepression of both cell-specific genes and DNA damage-inducible genes. Moreover, the Tup1 histone-binding domain coincides with the previously defined Tup1 repression domain. Tup1/histone interactions are negatively influenced by high levels of histone acetylation, suggesting a mechanism whereby the organization of chromatin may be modulated in response to changing environmental signals (Edmondson, 1996).

The Saccharomyces cerevisiae alpha2 repressor controls two classes of cell-type-specific genes in yeast through association with different partners. alpha2-Mcm1 complexes repress a cell-specific gene expression in haploid alpha cells and diploid a/alpha cells, while a1-alpha2 complexes repress haploid-specific genes in diploid cells. In both cases, repression is mediated through Ssn6-Tup1 corepressor complexes that are recruited via direct interactions with alpha2 (Tup1 is a yeast homolog of Groucho). Nucleosomes are positioned adjacent to the alpha2-Mcm1 operator under conditions of repression and Tup1 interacts directly with histones H3 and H4 (see Drosophila Histone H4). An examination was carried out of the role of chromatin in a1-alpha2 repression to determine if chromatin is a general feature of repression by Ssn6-Tup1. Mutations in the amino terminus of histone H4 cause a 4- to 11-fold derepression of a reporter gene under a1-alpha2 control, while truncation of the H3 amino terminus has a more modest (3-fold or less) effect. Strikingly, combination of the H3 truncation with an H4 mutation causes a 40-fold decrease in repression, clearly indicating a central role for these histones in a1-alpha2-mediated repression. However, in contrast to the ordered positioning of nucleosomes adjacent to the alpha2-Mcm1 operator, nucleosomes are not positioned adjacent to the a1-alpha2 operator in diploid cells. These data indicate that chromatin is important to Ssn6-Tup1-mediated repression but that the degrees of chromatin organization directed by these proteins differ at different promoters (Huang, 1997).

Extra sexcombs is Drosophila Wd-40 repeat protein

The extra sexcombs (esc) gene is expressed maternally and its product is most abundant during the early embryonic stages. It encodes a protein of the WD-40 repeat family, which localizes predominantly to the nucleus. During germ band extension, it is expressed in a stereotypic pattern of neuroblasts. A model has been proposed in which Esc is recruited by gap proteins both to act as a corepressor that competes with the TAFII80 coactivator to block transcription and also to mediate the transition to permanent repression by Polycomb-group proteins. This model is based on a possible analogy to yeast protein Tup1, which includes tandemly repeated WD-40 domains at its C-terminal portion. Tup1 protein has been shown to act as a corepressor when recruited by DNA binding proteins. Although it is unclear how Tup1 represses transcription, it has been shown that Tup1 interferes with the general transcription machinery. The interference of Tup1 with the assembly of an active transcription complex depends on the WD-40 domains. TAFII80, one of the factors associated with the TATA binding protein in the TFIID complex includes seven C-terminal WD-40 repeats. The corepressor activity of ESC appears to be mediated through its WD-40 domains that compete with those of TAFII80 for binding to other TAFs, TBP or other parts of the basal transcription apparatus, thus displacing TAFII80 from the TFIID complex (Gutjahr, 1995).

C. elegans Groucho homologs

Groucho and Tup1 are members of a conserved family of WD repeat proteins that interact with specific transcription factors to repress target genes. Mutations in WD domains of the Groucho-like protein, UNC-37, affect a motor neuron trait that also depends on UNC-4, a paired type homeodomain protein that controls neuronal specificity in Caenorhabditis elegans. In unc-4 mutants, VA motor neurons assume the pattern of synaptic input normally reserved for their lineal sister cells, the VB motor neurons; the loss of normal input to the VAs produces a distinctive backward movement defect. Substitution of a conserved residue (H to Y) in the fifth WD repeat in unc-37(e262) phenocopies the Unc-4 movement defect. Conversely, an amino acid change (E to K) in the sixth WD repeat of UNC-37 is a strong suppressor of unc-37(e262) and of specific unc-4 missense mutations. UNC-4 expression in the VA motor neurons specifies the wild-type pattern of presynaptic input. UNC-37 is also expressed in the VAs and unc-37 activity in these neurons is sufficient to restore normal movement to unc-37(e262) animals. It has been proposed that UNC-37 and UNC-4 function together to prevent expression of genes that define the VB pattern of synaptic inputs and thereby generate connections specific to the VA motor neurons. The WD repeat domains of UNC-37 and of the human homolog, TLE1, are functionally interchangeable in VA motor neurons which suggests that this highly conserved protein domain may also specify motor neuron identity and synaptic choice in more complex nervous systems. It is unknown whether UNC-4 and UNC-37 act in parallel pathways to regulate separate sets of target genes, or whether the two regulate a common set of target genes (Pflugrad, 1997).

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). The UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific C. elegans motor neurons. C. elegans UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (putative vesicular acetylcholine transporter UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in a null unc-4 mutant confirms that SV number in the presynaptic zone is reduced (~40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, it is proposed that these effects are performed via an intermediate gene. These results are consistent with a model in which this unc-4 target gene ('gene-x') functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons (Lickteig, 2001).

unc-17 and cha-1 are arranged in an operon where they share a common promoter and first untranslated exon. A 3.2 kb promoter element from this upstream region is sufficient to drive expression of GFP in virtually all cholinergic neurons including excitatory motor neurons in the ventral cord. Expression of the unc-17-cha-1 promoter:: gfp reporter gene in unc-4 motor neurons is not altered by loss-of-function mutations in either unc-4 or unc-37. Thus, the unc-17-cha-1 promoter region does not respond to changes in unc-4 and unc-37 activity. In contrast, expression of unc-17-cha-1 promoter:: gfp in these neurons does depend on the cell autonomous activity of the UNC-3 protein that has been shown to function as a regulator of unc-17-cha-1 transcription. These findings argue against a mechanism in which unc-4 and unc-37 mutations affect unc-17 and cha-1 transcription (Lickteig, 2001).

To determine whether other vesicular proteins are also regulated at a post-transcriptional level by unc-4 and unc-37, expression of a GFP-tagged Synaptobrevin driven by the unc-4 promoter was examined. This construct (unc-4p:: SNB-1:: gfp) contains the full-length Synaptobrevin protein with a C-terminal GFP tag. Expression of this transgene produces a punctate pattern of GFP staining that is correlated with the localization of SNB-1:: GFP at the PSDs of unc-4 motor neurons. This pattern is especially well resolved in the SAB processes in the head and in VC axons that exit the nerve cord to innervate vulval muscles. To determine whether the regulation of Synaptobrevin by unc-4 and unc-37 is independent of the Synaptobrevin promoter, the unc-4p:: SNB-1:: gfp construct was placed in unc-4- and unc-37-mutant backgrounds. In these animals, expression of GFP-tagged Synaptobrevin is clearly reduced in the axonal projections of the VC and SAB motor neurons. A similar reduction is seen for UNC-17 and ChAT antibody levels in these cells in unc-4 and unc-37 mutants. Because unc-4 expression is not regulated by unc-4 or unc-37, it follows that the decreased levels of SNB-1:: GFP in these mutants is not a result of unc-4 or unc-37 regulation of the unc-4 promoter. Therefore, unc-4 and unc-37 regulation of Synaptobrevin expression does not occur via a transcriptional mechanism but must depend on some feature of the Synaptobrevin-transcribed sequence. This finding parallels the observation above that UNC-17 and ChAT are also likely to be regulated by unc-4 and unc-37 at a post-transcriptional level and therefore favors a model in which all of the affected vesicular proteins are similarly regulated (Lickteig, 2001).

C. elegans UNC-4 is the homolog of a Drosophila paired-like homeodomain protein (Unc-4). The Drosophila protein is expressed in subsets of postmitotic neurons and epidermis (Tabuchi, 1998). The C. elegans UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes; this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. It is proposed that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord (Winnier, 1999).

The observation that the eh1 domain of UNC-4 mediates interactions with UNC-37 parallels the conclusions of earlier studies showing that the Drosophila Engrailed eh1 domain is necessary for interactions with Groucho. However, the current findings also differ in one important aspect. The UNC-4 eh1 domain is not adequate for UNC-37-dependent binding or for mediating UNC-37-dependent repression. Mutant UNC-4 proteins in which the eh1 domain is intact, but are missing the flanking carboxy-terminal amino acid sequences, fail to interact with UNC-37 in the yeast two-hybrid assay and do not exhibit in vivo repressor function. In contrast, insertion of a 7 amino-acid-long eh1 core sequence within the carboxy-terminal domain of Hairy is sufficient to confer Groucho-dependent repression. At least two hypotheses may explain these differences. The UNC-4 sequence flanking the eh1 domain may have a repressor function, or alternatively, these residues may be critical for UNC-4 protein stability or conformation (Winnier, 1999).

UNC-4 repressor function is effectively eliminated by a single amino acid substitution (H539Y) of UNC-37 in the hypomorphic allele e262. This mutation, in the fifth WD repeat of UNC-37, is predicted to disrupt a hydrogen-bonding network that stabilizes the propeller-like modular structure that individual WD domains have been shown to adopt. The limited disruption of other unc-37-dependent functions by the e262 allele indicates that the principal effect of this mutation is to perturb unc-4 function. This conclusion is consistent with the observation that the strong backward movement defect that e262 mutants display can be rescued by unc-4 promoter-driven expression of UNC-37 in VA motor neurons. A simple explanation for these effects is that the e262 mutation perturbs physical interactions of UNC-37 with UNC-4 but not with other classes of transcription factors. However, this prediction has not been substantiated by yeast two-hybrid assays, which detect robust binding of UNC-4 to the UNC-37(H539Y) mutant protein. Alternatively, the e262 mutation could disrupt an UNC-37-dependent transcriptional repressor mechanism that is uniquely employed by UNC-4. Given that this critical histidine residue is conserved in all known Groucho family members, it will be important to distinguish between these possibilities (Winnier, 1999).

A different UNC-37 point mutation, E580K, restores in vivo repressor function to specific UNC-4 missense mutations, as well as physical interaction with UNC-4. This invariant Groucho residue is predicted to reside on the surface of the sixth WD propeller domain and is therefore potentially involved in protein-protein interactions. However, UNC-37 proteins bearing the E580K mutation do not display a mutant phenotype on their own; this indicates that interaction with the wild-type UNC-4 protein is not perturbed by this mutation (Winnier, 1999).

Full-length UNC-4 and UNC-37 proteins do not interact in yeast two-hybrid assays or in vitro. However, an UNC-4 carboxy-terminal fragment, lacking homeodomain and amino-terminal residues, does associate with UNC-37. These data suggest that UNC-4 has adopted an amino-terminal-dependent intramolecular mechanism to regulate its interactions with UNC-37. The Drosophila Runt DNA-binding protein appears to use a similar mechanism to regulate its associations with the Groucho corepressor. Runt exhibits both Groucho-dependent as well as Groucho-independent repressor activities. Furthermore, Runt proteins appear to function as activators in some cellular contexts, requiring strict regulation of associations with widely expressed Groucho family members. The Rel protein Dorsal, which has been shown to interact with Groucho, also exhibits activator as well as repressor functions. Thus, specific DNA-binding proteins may regulate their interactions with Groucho-like corepressor proteins to perform activator versus repressor functions. The proposed UNC-4 amino-terminal inhibition of UNC-37 interaction may indicate that UNC-4 is capable of UNC-37-independent gene regulation. Consistent with this idea, UNC-4, but not UNC-37, is required for expression of del-1::GFP in specific neurons of the retrovesicular ganglion (Winnier, 1999).

The predicted DNA-binding motif of UNC-4, the homeodomain, is 100% conserved in a nematode species (C. briggsae) that diverged from C. elegans 50 million years ago; it is 85%-90% conserved in UNC-4-related proteins from Drosophila (Tabuchi, 1998), zebrafish, mouse, and rat. In addition, a carboxy-terminal region that is similar to the eh1 repressor domain of Drosophila Engrailed is present in all of these UNC-4-like proteins. The evolutionary conservation of both of these domains within the family of UNC-4-related proteins suggests that UNC-4-dependent repressor activity may also be conserved (Winnier, 1999).

The expression patterns of Drosophila and vertebrate unc-4-related genes are consistent with these proteins having nervous system functions. The Drosophila UNC-4-like protein DPHD-1 is selectively expressed in specific postmitotic neurons (Tabuchi, 1998). Similarly, the murine unc-x-4.1 transcript is expressed along the length of the neural tube in bilaterally positioned groups of cells immediately adjacent to the floor plate. The expression domains of unc-4-related genes in Drosophila and vertebrate species are not restricted to the nervous system, however. Murine unc-4, for example, is highly expressed in the kidney. This finding indicates that UNC-4 proteins may have adopted additional functions during evolution. The Groucho-like UNC-37 corepressor has also been conserved. The WD repeat regions of a human Groucho protein and UNC-37 are functionally interchangeable in C. elegans. Thus, UNC-4/Groucho-dependent gene repression could represent a common mechanism for regulating synaptic target selection during neuronal development (Winnier, 1999).

In C. elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. CBP-1, HDA-1 and POP-1 converge on end-1, a Serpent-related GATA factor that acts as an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appears to be the activation of end-1 transcription. A molecular mechanism has been identified for the endoderm-suppressive effect of POP-1 in the MS lineage by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. Evidence is provided that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C. elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription (Calvo, 2001).

Apoptosis is essential for proper development and tissue homeostasis in metazoans. It plays a critical role in generating sexual dimorphism by eliminating structures that are not needed in a specific sex. The molecular mechanisms that regulate sexually dimorphic apoptosis are poorly understood. This study reports the identification of the ceh-30 gene as a key regulator of sex-specific apoptosis in Caenorhabditis elegans. Loss-of-function mutations in ceh-30 cause the ectopic death of male-specific CEM neurons. ceh-30 encodes a BarH homeodomain protein that acts downstream from the terminal sex determination gene tra-1, but upstream of, or in parallel to, the cell-death-initiating gene egl-1 to protect CEM neurons from undergoing apoptosis in males. The second intron of the ceh-30 gene contains two adjacent cis-elements that are binding sites for TRA-1A and a POU-type homeodomain protein UNC-86 and acts as a sensor to regulate proper specification of the CEM cell fate. Surprisingly, the N terminus of CEH-30 but not its homeodomain is critical for CEH-30's cell death inhibitory activity in CEMs and contains a conserved eh1/FIL domain that is important for the recruitment of the general transcriptional repressor UNC-37/Groucho. This study suggests that ceh-30 defines a critical checkpoint that integrates the sex determination signal TRA-1 and the cell fate determination and survival signal UNC-86 to control the sex-specific activation of the cell death program in CEMs through the general transcription repressor UNC-37 (Peden, 2007).

UNC-4 and its transcriptional corepressor UNC-37/Groucho represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans

In C. elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, a cell-specific microarray strategy was used that has identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. Expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord (Von Stetina, 2007).

Transcription factor cascades define the structure of the vertebrate motor circuit by regulating the differentiation of specific neurons that contribute to this network. A striking feature of these pathways is the frequent use of negative gene regulation to produce distinct fates between neurons generated from adjacent progenitor domains. This study shows that a similar mechanism of repression involving conserved transcriptional components distinguishes the fates of C. elegans motor neurons born as sisters from a common mother cell. These results also offer a strikingly new finding, an explicit link between this biological strategy and the choice of presynaptic partners, a developmental decision of critical importance to motor neuron function. A model is presented of transcriptional regulation of synaptic specificity in C. elegans and the possibility that related schemes may also define wiring in the vertebrate spinal cord is discussed (Von Stetina, 2007).

C. elegans mutants in the unc-4 homeodomain gene display a strong backward movement defect that results from the miswiring of VA class motor neurons with inputs normally reserved for VB motor neurons. Intriguingly, other aspects of VA cell fate (i.e., axon trajectory and process placement) are unchanged, suggesting that UNC-4 functions to control only the synaptic fate of this cell type. This study shows that this change in synaptic specificity depends in part on misexpression of the VB-specific transcription factor, CEH-12/HB9, in VA motor neurons. Normally, UNC-4 functions with UNC-37/Groucho to block ceh-12/HB9 expression in the VAs. Because HB9 is also believed to function as a transcriptional repressor in other organisms, it is proposed that ectopic CEH-12/HB9 in unc-4 and unc-37 mutants triggers miswiring by turning off genes that specify VA inputs. It is possible that ectopic CEH-12/HB9 also activates VB genes that drive the creation of VB-type inputs. These results provide strong genetic evidence for at least one additional pathway downstream from UNC-4 that functions in parallel to CEH-12/HB9. The relative contributions of these pathways to VA input specificity are biased along the anterior-posterior (A/P) axis with ectopic CEH-12 selectively driving the creation of VB inputs to posterior VA motor neurons in unc-4 mutants and the presumptive parallel pathway imposing VB inputs to anterior VAs. Finally, a third set of VB genes, glr-4, del-1, and acr-5 are negatively regulated by unc-4 but have no detectable role in the VA miswiring defect. These cell surface proteins and ion channel components could be indicative of physiologically important differences in the excitability or signaling capacity of VA versus VB motor neurons. In the future, it will be interesting to determine if ectopic ceh-12 expression contributes to the observed depletion of synaptic vesicles in unc-4 mutant neurons (Von Stetina, 2007).

Although ceh-12 is required for the imposition of VB-type inputs to posterior VA motor neurons in unc-4 mutants, inputs to most VB motor neurons apparently do not depend on ceh-12 activity. Two lines of evidence support this conclusion: (1) ceh-12 knock-out mutants do not show an obvious forward movement defect as would be expected if VB motor neurons were miswired; (2) the elimination of ceh-12 activity in these mutants does not perturb the creation of gap junctions between most VBs and AVB command interneurons. These data are consistent with the proposal that ceh-12 functions in parallel to a redundant pathway in VB motor neurons that is sufficient to retain VB-type inputs (Von Stetina, 2007).

This work describes the use of a GFP-tagged UNC-7S marker protein for visualizing gap junctions between specific neuron pairs in the C. elegans motor circuit. This assay has provided an unprecedented opportunity to score gap junction specificity in the light microscope in multiple animals and in a variety of different mutant backgrounds. These experiments indicate that the innexin, UNC-7S, is expressed in AVB command interneurons for assembly into gap junctions with B-class motor neurons. Genetic and physiological data suggest that these gap junctions are likely to be heterotypic, and also include the innexin UNC-9. The ectopic gap junctions between AVB and A-class motor neurons that appear in unc-4 mutants may have a similar subunit composition, since unc-9 is the most abundant innexin transcript expressed in A-class motor neurons. It follows that UNC-9 is also a likely candidate for assembly into gap junctions between VA and AVA command interneurons in wild-type animals. Gap junctions with AVB tend to be located on the motor neuron soma, whereas gap junctions with AVA are more often distributed along the length of the motor neuron partner. Thus, unc-4 may orchestrate the assembly of UNC-9 into gap junctions at particular locations within A-class motor neurons and with selected presynaptic partners. Although gap junctions have been previously thought to provide a largely developmental role in the generation of neural networks in higher vertebrates, recent evidence suggests that these 'electrical' synapses are also important for neural function in adult nervous systems. This view is consistent with ultrastructural and immunochemical data showing that gap junctions are widely distributed in the mature mammalian brain and spinal cord. Since the mechanisms that control the specificity of gap junction assembly in the vertebrate CNS are unknown, the discovery of downstream genes that regulate gap junction placement in C. elegans could provide targets for molecular studies in more complex nervous systems. Moreover, the joint regulation by unc-4 (or ceh-12) of the specificity of chemical and electrical synapse formation is indicative of a common nexus for pathways controlling the assembly of both types of synapses (Von Stetina, 2007).

These findings indicate that ceh-12 conspires with at least one additional pathway in VA motor neurons to control input specificity. unc-4 regulation of ceh-12 is restricted to VA motor neurons in the posterior region of the ventral nerve cord. Because anterior VA motor neurons are also miswired in unc-4 mutants, it is proposed that the presumptive downstream pathway functioning in parallel to ceh-12 may be selectively derepressed in anterior VAs. Other unc-4-regulated genes should be represented in the microarray profile of unc-37 mutant VA motor neurons. One plausible candidate in this data set that could function in parallel to ceh-12 is cog-1, the C. elegans homolog of the homeodomain transcription factor, Nkx6. In Drosophila, dHB9 and Nkx6 act together in ventrally projecting motor neurons to repress dorsal motor neuron traits. COG-1 regulates a similar decision in the C. elegans nervous system by preventing ASER sensory neurons from adopting characteristics normally reserved for ASEL. Potential COG-1 interactions with CEH-12 are suggested by the observation that cog-1::GFP is also expressed in VA and VB motor neurons. cog-1 and other candidate unc-4 target genes in the microarray data set that function in parallel to ceh-12 may be revealed by RNA interference (RNAi) tests currently underway to detect genes that enhance ceh-12-dependent suppression of the Unc-4 phenotype (i.e., improved backward locomotion). Conversely, RNAi of transcripts that are depleted in the unc-37 microarray data set and therefore potentially repressed by ectopic ceh-12 should result in an Unc-4 like movement defect if these genes are required for specifying VA-type inputs (Von Stetina, 2007).

The results showing that ceh-12 preserves VB motor neuron fate by repressing VAB-7/Eve, parallels earlier observations that HB9 regulates motor neuron differentiation in flies, birds, and mammals. In Drosophila, dHB9 is expressed in a subset of ventrally projecting motor neurons where it represses the dorsal motor neuron determinant, Eve, and blocks the adoption of a dorsal axon trajectory. Eve, in turn, opposes ventral fates in dorsal motor neurons by reciprocally repressing dHB9 in a Groucho-dependent mechanism. Interestingly, HB9 is also restricted to ventrally projecting motor neurons in the vertebrate spinal cord where it acts to prevent expression of markers for interneurons arising from the adjacent V2 progenitor domain. In this case, ectopic expression of HB9 in V2 neuroblasts is sufficient to drive expression of motor neuron markers as well as impose motor neuron-like morphological characteristics (i.e., ventral axonal projections). This dual function of HB9 to block as well as activate expression of motor neuron-specific traits is similar to the finding that CEH-12 inhibits VA motor neuron differentiation while simultaneously promoting a specific VB trait. Together, these observations suggest that the key role of HB9 function in motor neuron differentiation is evolutionarily ancient. In this regard, it is noted that the UNC-4 homolog, UNCX4.1, is strongly expressed in the V3 neural progenitor domain immediately adjacent to the MN region in which HB9 resides. It will be interesting to determine if UNCX4.1 functions in the V3 domain to block HB9 expression (Von Stetina, 2007).

The Groucho ortholog UNC-37 interacts with the short Groucho-like protein LSY-22 to control developmental decisions in C. elegans

Transcriptional co-repressors of the Groucho/TLE family are important regulators of development in many species. A subset of Groucho/TLE family members that lack the C-terminal WD40 domains have been proposed to act as dominant-negative regulators of Groucho/TLE proteins, yet such a role has not been conclusively proven. Through a mutant screen for genes controlling a left/right asymmetric cell fate decision in the nervous system of the nematode C. elegans, loss-of-function alleles were retrieved in two distinct loci that display identical phenotypes in neuronal fate specification and in other developmental contexts. Using the novel technology of whole-genome sequencing, these loci were found to encode the C. elegans ortholog of Groucho, UNC-37, and, surprisingly, a short Groucho-like protein, LSY-22, that is similar to truncated Groucho proteins in other species. Besides their phenotypic similarities, unc-37 and lsy-22 show genetic interactions and UNC-37 and LSY-22 proteins also physically bind to each other in vivo. These findings suggest that rather than acting as negative regulators of Groucho, small Groucho-like proteins may promote Groucho function. It is proposed that Groucho-mediated gene regulatory events involve heteromeric complexes of distinct Groucho-like proteins (Flowers, 2010).

Zebrafish and frog Groucho-related proteins

groucho is a Drosophila nuclear protein with structural similarity to the transcriptional repressor tup1. Drosophila Groucho forms complexes with bHLH proteins of the hairy-enhancer of split [-E(spl)] family, which then act as repressors (for example, downstream of the Notch signaling pathway). Two zebrafish groucho homologs have been cloned and the pattern of transcript distribution during embryogenesis has been examined. Both gro1 and gro2 exhibit all sequence features characteristic of the groucho family; with 79% sequence similarity at the DNA level, they can be considered orthologs of the human groucho homolog, tle3. RNA in situ hybridization shows a distinct pattern of transcript distribution for both genes during embryogenesis, suggestive of their participation in neurogenesis and somitogenesis. Maternal transcripts exist for both genes. Whereas gro2 is expressed apparently ubiquitously during gastrulation, gro1 is not expressed within a medial strip of hypoblastic cells corresponding to the axial mesoderm and connecting to the expressing cells at the epiboly. The same applies to the cells in the differentiating notochord, which contain no detectable levels of either mRNAs. Later, within the neural plate, gro1 is expressed in the prosencephalic and metencephalic regions; its pattern is strikingly similar to that of zebrafish Notch. Like zebrafish Notch, gro1 is expressed throughout somitogenesis within the posterior half of the somites (Wubeck, 1997).

During vertebrate embryonic development, the paraxial mesoderm becomes subdivided into metameric units known as somites. In the zebrafish embryo, genes encoding homologs of the proteins of the Drosophila Notch signaling pathway are expressed in the presomitic mesoderm and expression is maintained in a segmental pattern during somitogenesis. This expression pattern suggests a role for these genes during somite development. Various zebrafish genes of this group were misexpressed by injecting mRNA into early embryos. RNA encoding a constitutively active form of NOTCH1a (notch1a-intra) and a truncated variant of deltaD [deltaD(Pst)], as well as transcripts of deltaC and deltaD, the hairy-E(spl) homologs her1 and her4, and groucho2 were tested for their effects on somite formation, myogenesis and on the pattern of transcription of putative downstream genes. In embryos injected with any of these RNAs, with the exception of groucho2 RNA, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment correctly. Activation of Notch results in ectopic activation of her1 and her4. This misregulation of the expression of her genes might be causally related to the observed mesodermal defects, since her1 and her4 mRNA injections led to effects similar to those seen with notch1a-intra. deltaC and deltaD seem to function after subdivision of the presomitic mesoderm, since the her gene transcription pattern in the presomitic mesoderm remains essentially normal after misexpression of delta genes. Whereas Notch signaling alone apparently does not affect myogenesis, zebrafish groucho2 is involved in differentiation of mesodermal derivatives (Takke, 1999).

A homeobox gene, pnx, a homolog of Drosophila Slouch/S59/NK-1, is expressed in prospective posterior neurogenic regions and later in primary neurons. pnx expression is regulated by a signal from the non-axial mesendoderm and by Notch signaling. Pnx contains an Eh1 repressor domain, which interacts with Groucho and acts as a transcriptional repressor. Misexpression of pnx increases neural precursor cells and postmitotic neurons, which express neurogenin1 and elavl3/HuC, respectively. Expression of an antimorphic Pnx (VP16Pnx) or inhibition of Pnx by antisense morpholino oligonucleotide lead to the reduction in the number of a subset of primary neurons. Misexpression of pnx promotes neurogenesis independent of Notch signaling. Epistatic analyses shows that Pnx also functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that regulates posterior neurogenesis (Bae, 2003).

Pnx contains an Eh1 repressor domain and interacts with the transcriptional co-repressor Groucho2, at least in 293T cells. Reporter analysis reveals that Pnx acts as a transcriptional repressor and that the Eh1-mediated interaction with Groucho(s) is involved in this repressor activity. Furthermore, VP16-Pnx functions as an antimorphic molecule in the formation of primary neurons. These data indicate that Pnx functions as a transcriptional repressor and should repress genes that have the ability to repress the proneural genes. Candidates that are repressed by Pnx could include downstream components of the Notch signal, such as the hes/her-family genes. However, this is not the case. Misexpression of Pnx still increases the ngn1- and elavl3-expressing cells in embryos in which Notch signaling is suppressed. Furthermore, Pnx does not inhibit the expression of either her4 or her9, which are the only Hes/Her-family members reported to be expressed in the neural plate. Inhibition of the Notch signal leads to an increase in the density of neuronal cells 'within the neurogenic region', but does not lead to the expansion of neurogenic regions. By contrast, the misexpression of Pnx in either wild-type or mib mutant embryos elicits an 'expansion' of the ngn1-expressing neurogenic regions. pnx is epistatic to the Notch signaling in the formation of primary neurons, providing genetic evidence that the pnx-mediated neurogenesis does not require the Notch signal. These data indicate that Pnx can promote neurogenesis not by inhibiting the Notch signal (lateral inhibition mechanisms), but rather by expanding neurogenic regions within the neuroectoderm. To promote neurogenesis, Pnx represses the expression of certain transcriptional repressor(s), other than those downstream of the Notch signal, which inhibit the proneural gene expression and neurogenesis. The identification of targets for Pnx will shed light on the mechanisms by which the neurogenic regions are established and proneural genes are regulated (Bae, 2003).

Tcf/Lef transcription factors mediate signaling from Wingless/Wnt proteins by recruiting Armadillo/beta-catenin as a transcriptional co-activator. However, studies of Drosophila, Xenopus and Caenorhabditis elegans have indicated that Tcf factors may also be transcriptional repressors. Tcf factors are shown to physically interact with members of the Groucho family of transcriptional repressors. In transient transfection assays, the Xenopus Groucho homolog XGrg-4 inhibits activation of transcription of synthetic Tcf reporter genes. In contrast, the naturally truncated Groucho-family member XGrg-5 enhances transcriptional activation. Injection of XGrg-4 into Xenopus embryos represses transcription of Siamois and Xnr-3, endogenous targets of beta-catenin-Tcf. Dorsal injection of XGrg-4 has a ventralizing effect on Xenopus embryos. Secondary-axis formation induced by a dominant-positive Armadillo-Tcf fusion protein is inhibited by XGrg-4 and enhanced by XGrg-5. These data indicate that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho (Roose, 1998).

The zebrafish nlz gene has a rostral expression limit at the presumptive rhombomere (r) 3/r4 boundary during gastrula stages, and its expression progressively expands rostrally to encompass both r3 and r2 by segmentation stages, suggesting a role for nlz in hindbrain development. Nlz is a nuclear protein that associates with the corepressor Groucho, suggesting that Nlz acts to repress transcription. Consistent with a role as a repressor, misexpression of nlz causes a loss of gene expression in the rostral hindbrain, likely due to ectopic nlz acting prematurely in this domain, and this repression is accompanied by a partial expansion in the expression domains of r4-specific genes. To interfere with endogenous nlz function, a form of nlz was generated that lacks the Groucho binding site; this construct has a dominant negative effect. Interfering with endogenous Nlz function promotes the expansion of r5 and, to a lesser extent, r3 gene expression into r4, leading to a reduction in the size of r4. It is concluded that Nlz is a transcriptional repressor that controls segmental gene expression in the hindbrain. Lastly, additional nlz-related genes have been identified, suggesting that Nlz belongs to a family of zinc-finger proteins (Runko, 2003).

Nlz is related to Drosophila NocA as well as to an uncharacterized human protein. Nlz is also related to a second zebrafish protein (herein referred to as Nlz2), as well as to a second Drosophila protein (Elbow), two hypothetical human proteins (FLJ14299 and MGC2555), and two hypothetical mouse proteins (NM_145459 and XM_146263). Several domains are conserved within this group of proteins, including an N-terminal domain (termed the SP motif) that is also found in the Sp1 family of zinc finger transcription factors, a zinc-finger motif (Z1) that has one histidine surrounded by four cysteines and may fall into the C2HC or CHC2 class, and a second zinc-finger motif (Z2) which is of the C2H2 type. Sequence alignment and phylogenetic analysis indicate that the vertebrate proteins fall into two groups with one mouse and one human protein related to each of Nlz and Nlz2, while NocA and Elbow are more divergent. The finding that more than one Nlz-like protein exists in several organisms suggests that Nlz belongs to a family of related zinc-finger proteins. Since NocA and Elbow regulate neural and tracheal development in Drosophila, while Nlz regulates hindbrain development in zebrafish, members of this family may regulate various aspects of vertebrate development (Runko, 2003 and references therein).

Concomitant with the transition from the presomitic mesoderm (PSM) to somites, the periodical gene expression characteristic of the PSM is drastically changed and translated into the segmental structure. However, the molecular mechanism underlying this transition has remained obscure. ripply1, encoding a nuclear protein associated with the transcriptional corepressor Groucho, is required for this transition. Zebrafish ripply1 is expressed in the anterior PSM and in several newly formed somites. A structurally related gene had been identified in many other vertebrates as well as in Amphioxus, in which somites are similarly generated as they are in vertebrates; however, no related gene was found in other invertebrates, including C. elegans and Drosophila. Ripply1 represses mesp-b expression in the PSM through a Groucho-interacting motif. In ripply1-deficient embryos, somite boundaries do not form, the characteristic gene expression in the PSM is not properly terminated, and the initially established rostrocaudal polarity in the segmental unit is not maintained, whereas paraxial mesoderm cells become differentiated. Thus, ripply1 plays dual roles in the transition from the PSM to somites: termination of the segmentation program in the PSM and maintenance of the rostrocaudal polarity (Kawamura, 2005).

FoxG1 is a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon, and is expressed from the earliest stages of telencephalic development through to the adult. How the interaction with co-factors might influence the multiplicity and diversity of FoxG1 function is not known. This study shows that interaction of FoxG1 with TLE2, a Xenopus tropicalis co-repressor of the Groucho/TLE family, is crucial for regulating the early activity of FoxG1. TLE2 is co-expressed with FoxG1 in the ventral telencephalon from the early neural plate stage and functionally cooperates with FoxG1 in an ectopic neurogenesis assay. FoxG1 has two potential TLE binding sites: an N-terminal eh1 motif and a C-terminal YWPMSPF motif. Although direct binding seems to be mediated by the N-terminal motif, both motifs appear important for functional synergism. In the neurogenesis assay, mutation of either motif abolishes functional cooperation of TLE2 with FoxG1, whereas in the forebrain deletion of both motifs renders FoxG1 unable to induce the ventral telencephalic marker Nkx2.1. Knocking down either FoxG1 or TLE2 disrupts the development of the ventral telencephalon, supporting the idea that endogenous TLE2 and FoxG1 work together to specify the ventral telencephalon (Roth, 2010).

XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. This study reports an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. DIAP1 was identified as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. The data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program (Hanson, 2012).

Conversion of the Wnt transcription factor TCF/Lef from a transcriptional repressor to an activator is a critical event in Wnt signal transduction, yet understanding of how this switch occurs in cells is limited. The current model, based primarily on reconstitution studies using purified proteins, proposes direct displacement of the transcriptional corepressor Gro/TLE by the coactivator β-catenin through competition for overlapping binding sites on TCF/Lef (Hanson, 2012).

The data suggest a model in which XIAP constitutively binds and ubiquitylates non-TCF-bound Gro/TLE in the nucleus, thereby limiting the amount of Gro/TLE available to form corepressor complexes with TCF/Lef. In the presence of a Wnt signal, XIAP is recruited to TCF/Lef transcriptional complexes where it promotes dissociation of Gro/TLE. The experiments were not able to distinguish whether XIAP ubiquitylates Gro/TLE bound to TCF/Lef to promote its dissociation or ubiquitylates dissociated Gro/TLE, thereby blocking its reassociation. Regardless, ubiquitylation of Gro/TLE by TCF/Lef-bound XIAP further decreases the affinity of Gro/TLE for TCF/Lef, thereby allowing efficient recruitment and binding of the transcriptional coactivator β-catenin to TCF/Lef that is required to initiate a Wnt-specific transcriptional program. The mechanism by which XIAP is recruited to TCF/Lef transcriptional complexes is unknown, although the results demonstrating that lithium can also induce recruitment of XIAP to TCF/Lef suggest that GSK3 activity plays an important role in regulating this process (Hanson, 2012).

This proposed model for Wnt-mediated transcriptional activation parallels the findings of Sierra (2006) who proposed that inactivation of Wnt target gene transcription similarly occurs as a multistep process. That data suggest that APC and β-TRCP (an E3 ligase) mediate removal of β-catenin from Lef1 to allow for subsequent TLE1 binding. Together, these experiments and the current study have revealed that transcriptional activation and inactivation in the Wnt pathway are highly regulated processes (Hanson, 2012).

β-catenin protein levels are tightly regulated in the cell via continual synthesis and degradation by the β-catenin destruction complex. Why, then, would a cell evolve an additional layer of regulation for Wnt transcriptional activation, as is proposed in this study, as opposed to a simpler mechanism based solely on bimolecular association between β-catenin and TCF/Lef? It is proposed that this Wnt signaling circuitry provides a mechanism to dampen transcriptional noise without a corresponding loss in sensitivity. Binding of Gro/TLE to TCF/Lef allows the system to be resistant to stochastic fluxes in β-catenin levels in the absence of Wnt pathway activation. In the presence of a Wnt signal, a coincident circuit involving nuclear accumulation of β-catenin and recruitment of XIAP to TCF/Lef is established. Such circuitry ensures that transcriptional activation only occurs upon Wnt ligand binding and provides an additional mechanism for reducing spontaneous activity. Sensitivity to a Wnt signal is maintained by the facilitated removal of Gro/TLE from TCF/Lef, which ensures that even low levels of β-catenin would be sufficient to bind TCF/Lef and activate transcription (Hanson, 2012).

Support for this model comes from a study showing that β-catenin levels change only modestly (∼2- to 6-fold) upon Wnt signaling in human cells and Xenopus embryos. It is unlikely that this degree of nuclear β-catenin accumulation is sufficient to effectively displace Gro/TLE from TCF/Lef. This suggests that a facilitated mechanism for Gro/TLE removal is required prior to β-catenin-TCF/Lef complex formation (Hanson, 2012).

The data indicate that XIAP may also influence the nuclear pool of Gro/TLE that is available to form corepressor complexes with TCF/Lef. This study found that XIAP is associated with Gro/TLE in the presence and absence of Wnt signaling. Additionally, whereas ubiquitylated Gro/TLE is readily observed in total cellular lysates, only the nonubiquitylated form of Gro/TLE binds to TCF/Lef. This suggests a model in which XIAP functions to constitutively ubiquitylate free Gro/TLE to control the pool of Gro/TLE that can bind TCF/Lef. The data also suggest the presence of an as yet unidentified deubiquitylase (DUB) that facilitates removal of ubiquitin from Gro/TLE, which would allow TCF/Lef binding. Cycles of monoubiquitylation and deubiquitylation have been shown to regulate activity of the transcriptional activators Smad4, p53, and FoxO. This study provides evidence for a similar mode of regulation of a transcriptional repressor (Hanson, 2012).

Until recently, most studies have focused on transcriptional coactivator activity because it was generally believed that corepressors are abundant proteins subject to little regulation. It is becoming clear, however, that corepressor activity is highly complex and can be controlled through a variety of mechanisms. This study shows that the corepressor Gro/TLE is regulated by ubiquitylation in a manner that may be Wnt pathway specific. Gro/TLE has been shown to participate in transcriptional repression of multiple signaling pathways. The corepressor function of Gro/TLE occurs locally through its binding to DNA-bound transcription factors (primarily via its C-terminal WD40 domain) and histone deacetylase recruitment and globally via its N-terminal Q domain, which mediates oligomerization to alter chromatin structure and mediate long-range repression. The finding that XIAP ubiquitylates Gro/TLE on its N-terminal Q domain (which disrupts TCF/Lef binding) without disrupting its capacity to oligomerize suggests that XIAP modification of Gro/TLE may specifically affect its Wnt repressive function. This possibility is consistent with the observation that XIAP knockdown has no observable effect on Notch signaling. In the absence of Notch signaling, Gro/TLE normally binds to the Hairless protein to repress Notch target gene activation by the transcription factor, Suppressor of Hairless. Binding to Hairless occurs via the C-terminal WD40 domain of Gro/TLE. Thus, ubiquitin modifications of Gro/TLE on its N-terminal Q domain would not be expected to disrupt its interaction with Hairless in the Notch pathway or other pathways in which repression by Gro/TLE occurs via the WD40 domain or via Gro/TLE oligomerization (Hanson, 2012).

The identification of XIAP as a critical Wnt pathway component provides a link between apoptosis and Wnt signaling and represents a mechanism by which a cell could coordinate survival and proliferation. Wnt signaling has been shown to inhibit apoptosis and to be required for XIAP expression in cancer cells. Thus, XIAP may be part of a positive feedback loop involving Wnt pathway-induced proliferation and inhibition of apoptosis. Surprisingly, XIAP knockout mice have no obvious apoptotic or Wnt phenotypes, as would be expected given its important role in apoptotic inhibition and the findings that XIAP is required for Wnt signaling in cultured human cells and in Xenopus embryos. Only exon 1 of XIAP was deleted in the knockout mouse. Thus, it is possible that there is readthrough that permits expression of the C-terminal region of XIAP, which includes the RING domain. Alternatively, other IAP family members or E3 ligases might compensate for XIAP function when it is deleted in the mouse (Hanson, 2012).

These findings may have important clinical implications, as XIAP is upregulated in a majority of human cancers and inhibitors of XIAP are currently in clinical trials. Drug development has been largely focused on developing small molecule and peptide Smac mimetics that bind to the BIR domains of XIAP to inhibit its antiapoptotic function. This study shows that the critical role of XIAP in Wnt signaling depends on its E3 ligase RING domain and is distinct from its antiapoptotic functions. The results predict that small molecules targeting the RING domain of XIAP rather than its BIR domains would represent more selective inhibitors of Wnt signaling. Alternatively, drugs targeting both antiapoptotic and pro-Wnt functions of XIAP may be particularly effective against Wnt-driven cancers. Recent findings indicate that inducing apoptosis results in 'compensatory proliferation' of surrounding surviving cells due to release of mitogenic signals (e.g., Wnt) from dying cells, suggesting that drugs targeting both aspects of XIAP function may be particularly useful anticancer therapies even in non-Wnt-driven tumors (Hanson, 2012).

Chick Groucho homologs

Alar plate of chick mesencephalon differentiates into the optic tectum. It has been shown that factors expressed in the mes-metencephalic boundary induce the tectum and give positional specificity. Chick Grg4 is expressed at first in the anterior neural fold. The expression localizes from the posterior diencephalon to the mesencephalon by stage 10. To investigate the function of Grg4 in mesencephalic development, Grg4 overexpression was carried out by in ovo electroporation. After Grg4 overexpression, expression of En-2, Pax5, Fgf8, and EphrinA2 is repressed, and Pax6 is upregulated in the mesencephalic region. Grg4 overexpression causes the morphological change; mesencephalic swelling becomes smaller and the di-mesencephalic boundary shifts posteriorly, that is, the anterior limit of tectum shifts posteriorly. Importantly, cotransfection of Grg4 with Pax5 cancels the tectum-inducing activity of Pax5. These results suggest that Grg4 works as an antagonist against tectum-organizing activity. Transfected N-terminal domains of Grg4 induce En-2 expression. Since N-terminal domains are transported to the nucleus in the neuroepithelium, they could act in a dominant negative fashion for endogenous Grg4 production. These results indicate that Grg4 has repressing activity against the organizing proteins and suggest that Grg4 plays important roles in formation of anterior tectal boundary and polarity (Sugiyama, 2000).

Identification and expression of mammalian Groucho homologs

Mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophila enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins exhibit approximately 50% identity to the amino-terminal region of Drosophila groucho, mouse ESG and human transducin-like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb are abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb are predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb are found only in testis, while human TLE 1 transcripts of 4.5 kb is more abundant in muscle and placenta, as compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines (Miyasaka, 1993).

Mammalian Groucho homologs TLE 1 and TLE 3 are expressed during a number of cell-determination events, including embryonic segmentation, central and peripheral neurogenesis, and epithelial differentiation. This expression pattern is in agreement with the involvement of Groucho in similar fate choices in Drosophila and suggests that Groucho and TLE proteins perform similar developmental roles. TLE genes are co-expressed during a variety of cell-fate choices with several vertebrate homologs of genes implicated in the Drosophila Notch cascade, suggesting a role for the TLE proteins in mammalian Notch signaling (Dehni, 1995).

Grg4, a murine groucho-related gene, is detected in tissues adjacent to other murine neurogenic gene homologs during embryonic development. Grg4 is detected in proliferating epithelial tissues undergoing mesenchymal induction, overlapping with Grg3, Notch1 and Hairy enhancer of split 1 expression. Grg4 is also expressed in the central nervous system and somites, but in cells adjacent to Grg3, Notch1 and Hes1 expressing cells. This distinct pattern of expression suggests a role for Grg4 in later stages of cell differentiation than for the other mouse neurogenic gene homologs (Koop, 1996).

Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development, since overexpression of Mash-2 reduces giant cell formation. The role that the Notch signaling pathway may play in trophoblast development has been investigated. Mash-2 is a homolog of Drosophila achaete/scute complex genes. In the developing mouse placenta, all elements of the Notch pathway are expressed. In particular, the Notch-2, HES-2, and HES-3 genes are coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family are expressed in trophoblasts; TLE3 is expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 is limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development (Nakayama, 1997).

Protein interactions and post-translational modification of mammalian Groucho homologs

Groucho forms transcription complexes with the basic helix-loop-helix proteins encoded by the hairy/Enhancer of split ("hairy-like") gene family. These interactions are mediated by the carboxyl-terminal WRPW motif of Hairy-like proteins. TLE1 interacts with HES-1, a murine homologue of Drosophila Hairy-like proteins, both in the yeast two-hybrid assay and in an interaction assay based on glutathione S-transferase fusion proteins. Groucho/TLE proteins and Hairy-like/HES proteins are involved in similar interactions in Drosophila and mammals; these proteins appear to perform conserved cellular functions (Grbavec, 1996).

Two populations of phosphorylated mammalian Groucho homologs, TLE (transducin-like Enhancer of split), can be identified based on their interaction with the nuclear compartment. More slowly migrating proteins with an apparent molecular mass of roughly 110 kDa interact strongly with the nuclei, while faster migrating proteins displaying molecular masses roughly 84-85 kDa show lower affinity for the nuclear compartment. Similarly, the proteins with an apparent molecular mass of roughly 118 kDa, exhibit higher affinity for the nuclear compartment than do faster migrating forms with apparent molecular masses of 90-93 kDa. The nuclear, more slowly migrating, TLE1 proteins are induced during neural determination of P19 embryonic carcinoma cells. These results implicate phosphorylation in the activity of Groucho/TLE1 proteins and suggest that phosphorylated forms of higher molecular mass are involved in nuclear functions. Different TLE proteins respond in different ways to the neural commitment of P19 cells, suggesting that individual members of this protein family may have non-redundant functions (Husain, 1996).

Groucho is a Drosophila transcriptional repressor involved in neurogenesis, segmentation, and sex determination, together with basic helix-loop-helix proteins of the Hairy/Enhancer of split (HES) family. Several mammalian Groucho homologs, the Transducin-like Enhancer of split (TLE) 1 through 4 proteins, share similar properties with their Drosophila counterpart, suggesting that TLE proteins perform functions analogous to the roles of Groucho in Drosophila. The aim of this study was to examine this possibility by characterizing the properties of TLE2 and extending the analysis of TLE1. TLE2 and TLE1 are transcriptional repressors that contain two separate repression domains, located either within a Gln-rich amino terminal region or within an internal domain characterized by an abundance of Ser, Thr, and Pro residues. In addition, both TLE2 and TLE1 can homo- and heterodimerize through a short region that is part of their amino-terminal transcription repression domains. Finally, TLE2 interacts and is co-expressed with mammalian HES proteins in both neural and non-neural tissues. Taken together, these findings implicate TLE2 in transcriptional repression and define the structural elements that mediate transcriptional and protein-protein interaction functions of Groucho/TLE proteins (Grbavec, 1998).

The yeast proteins TUP1 and SSN6 form a transcription repressor complex that is recruited to different promoters via pathway-specific DNA-binding proteins and regulates the expression of a variety of genes. TUP1 is functionally related to invertebrate and vertebrate transcriptional repressors of the Groucho/transducin-like Enhancer of split (TLE) family. The aim was to examine whether similar mechanisms underlie the transcription repression functions of TUP1 and Groucho/TLEs by determining whether TLE family members can interact with yeast SSN6 and mammalian SSN6-like proteins. SSN6 has been shown to bind to TLE1 and mediates transcriptional repression when expressed in mammalian cells. Moreover, TLE1 and TLE2 interact with two mammalian proteins related to SSN6, designated as the products of the ubiquitously transcribed tetratricopeptide-repeat genes on the Y (or X) chromosomes (UTY/X). These findings suggest that mammalian TLE and UTY/X proteins may mediate repression mechanisms similar to those performed by TUP1-SSN6 in yeast (Grbavec, 1999).

The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, it has been demonstrated that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which, in transfected Jurkat T cells, contain functional AML binding sites. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling (Levanon, 1998).

The AML1 gene encodes DNA-binding proteins that contain the Runt domain. The gene is found at the breakpoints of some translocations associated with leukemias. It has been reported that AML1 plays pivotal roles in myeloid differentiation, probably through the transcriptional regulation of various hematopoietic genes. This study demonstrates the physical and functional interaction between AML1 and TLE1 (transducin-like Enhancer of split), the human homolog of Groucho that is known to be a corepressor of Hairy-related proteins. TLE1 binds to AML1 through the Runt domain and the C terminus of AML1, which includes the VWRPY motif. The interaction is mainly mediated by the SP domain of TLE1. Moreover, TLE1 inhibits AML1-induced transactivation of the target promoters through the C terminus of AML1. These results suggest that TLE1 acts as a repressor of AML1 and provide important insights into the mechanism of the negative regulation of the AML1 functions in hematopoiesis and leukemogenesis (Imai, 1998).

The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of beta-interferon (IFN-beta) and c-myc gene expression. PRDI-BF1 represses transcription of the IFN-beta promoter and of an artificial promoter through an active repression mechanism. A minimal repression domain has been identified in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein. Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors. In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain. The amino-terminal glutamine-rich domains of hGrg and TLE1 are found to be sufficient to mediate dimerization of the two Groucho family proteins. Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-beta promoter activity after virus induction. It is concluded that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and it is suggested that such corepressor complexes are required for the postinduction repression of the IFN-beta promoter (Ren, 1999).

The Groucho family includes three types of proteins. The larger proteins such as Groucho and its mammalian homologs [transducin-like enhancer of split (TLE) 1 through 3] share five domain structures. These proteins exhibit a common feature including an amino-terminal glutamine-rich region (Q domain), a glycine/proline-rich region (GP domain), a CcN domain containing a casein kinase II site and nuclear localization sequence, a serine/proline-rich region (SP domain), and COOH-terminal WD40 repeats. Three of these domains, the Q, CcN, WD40 domains, are most highly conserved. A shorter protein, the human TLE4, contains all the domains except for the amino-terminal Q domain. Shortest proteins in the Groucho family, which contain only the Q domain and the GP domain, are designated as amino-terminal enhancer of split (AES) or the Groucho-related gene (Grg). Significant homology is observed in the Q domain between AES and other Groucho proteins, except for TLE4. AES encodes a 197-amino acid protein that is homologous to the NH(2)-terminal domain of the Drosophila Groucho protein but lacks COOH-terminal WD40 repeats. Although the Drosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast two-hybrid system, a protein-protein interaction has been identified between AES and the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed by in vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES represses p65-driven gene expression. AES also inhibits NF-kappaB-dependent gene expression induced by tumor necrosis factor alpha, interleukin-1beta, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-kappaB activation. These data indicate that AES acts as a corepressor for NF-kappaB and suggest that AES may play a pivotal role in the regulation of NF-kappaB target genes (Tetsuka, 2000).

These findings suggest that the transcriptional activity of NF-kappaB may be regulated by a balance between the counteracting effects of Groucho corepressors and coactivators, because it was previously found that NF-kappaB binds to coactivator proteins p300/CREB-binding protein. It is also speculated, although not yet proven, that the interactions of NF-kappaB with coactivators or corepressors may be modulated by signal-induced modification of these proteins. These findings suggest a putative role for vertebrate NF-kappaB in transcriptional repression. Inhibition of NF-kappaB activity by localized expression of transdominant-negative IkappaBalpha in chick embryos results in the arrest of limb growth. In this phenotype, inhibition of NF-kappaB leads to inhibition of twist expression but derepression of a vertebrate decapentaplegic homolog, the bone morphogenic protein-4. Taken together, these data indicate that the vertebrate NF-kappaB may also act as a transcriptional repressor by recruiting Groucho family corepressors, such as AES and TLEs (Tetsuka, 2000 and references therein).

In the present study, it was noted that AES and TLE1 inhibit NF-kappaB-mediated gene expression but do not actively repress NF-kappaB-driven gene expression to less than basal level (the level of gene expression without NF-kappaB and AES/TLE1). Lack of active repression may be because NF-kappaB-dependent reporter constructs, which do not contain additional repression elements, were used. In Drosophila, recruitment of Groucho is obligatory but is not sufficient for Dorsal-mediated repression. Dorsal-mediated repression requires additional repression elements in the proximity of the Dorsal binding sites and the binding of other DNA-binding repressor proteins to these elements. Dorsal-mediated repression has been shown to require the formation of a multiprotein DNA-bound complex that includes Groucho, Dorsal, and additional DNA-binding proteins, such as Cut and Dead ringer. NTF-1/Grainyhead is known to bind to the repression elements in decapentaplegic promoter, and it may play a role in decapentaplegic repression that is analogous to the role of cut and/or dead ringer in zerknüllt repression. Thus, if NF-kappaB acts as an active repressor in vertebrates, it is likely that there are additional repressor binding sites and their respective DNA-binding proteins (Tetsuka, 2000 and references therein).

Members of the hepatic nuclear factor 3 (HNF3) family, including HNF3alpha, beta and gamma, play important roles in embryonic development, the establishment of tissue-specific gene expression and the regulation of gene expression in differentiated tissues. The transducin-like Enhancer of split (TLE) proteins, the human homologs of Drosophila Groucho, directly associate with HNF3beta. The CRII region of HNF3beta (a.a. 361-388) is responsible for the interaction with TLE1. The CRII region is conserved between all three mammalian HNF3 proteins and the Drosophila Forkhead protein. The CRII region of HNF3beta does not contain the WRPW motif that is required for the interaction between TLE/Groucho and the Hairy, Hairy/Enhancer of split-like (HES). The CRII region also does not resemble the eh1/GEH motif that is required for the interaction between TLE/Groucho and Engrailed or Goosecoid. The CRII region, however, does have an FNHPF sequence that may serve as a TLE binding site, since it has two aromatic residues separated by a proline. A mammalian two hybrid assay was used to confirm that this interaction occurs in vivo. Overexpression of TLE1 in HepG2 and HeLa cells decreases transactivation mediated through the C-terminal domain of HNF3beta, and Grg5, a naturally occurring dominant negative form of Groucho/TLE, also increases the transcriptional activity of this region of HNF3. These results suggest that TLE proteins could influence the expression of mammalian genes regulated by HNF3 (Wang, 2000).

Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family (Eberhard, 2000).

Three groucho-related genes coding for full-length Grg proteins (Grg1, 3a and 4) have been identified to date in the mouse genome. Using transient transfection assays, all three murine Grg proteins have been shown to be phosphorylated in a Pax5-dependent manner and can repress the transcriptional activity of Pax5 efficiently. Even the distantly related Groucho protein of Drosophila is able to interact with Pax5 and to down-modulate the activity of this transcription factor in heterologous mammalian cells. Furthermore, GST pull-down assays have demonstrated that the mouse Pax8 and Drosophila Pax2/5/8 proteins can bind full-length Grg4 with an affinity similar to that of human Pax5. Moreover, the transcriptional activity of the mouse Pax8, zebrafish Pax2.1 and Drosophila Pax2/5/8 proteins could be repressed efficiently by Grg4 in transfected plasmacytoma cells. These different Pax proteins are also able to promote additional phosphorylation of Grg4 in transfected COP-8 fibroblasts. Collectively, these data demonstrate, therefore, that the interaction between distantly related members of the Pax2/5/8 and Groucho protein families has been conserved in evolution (Eberhard, 2000).

Inspired by the high evolutionary conservation of the Groucho-Pax2/5/8 protein interaction, an investigation was carried out to see whether the octapeptide motif can function in vivo as a repression domain during Drosophila development. Based on the transcriptional regulation of the Sex lethal (Sxl) gene in Drosophila embryos, a repression assay was employed. Sxl is a key regulator of sex determination and dosage compensation: Sxl transcription is initiated only in female blastoderm embryos. In male embryos, Sxl expression is prevented by the transcriptional repressor Deadpan (Dpn), which is a member of the Hairy-related basic helix-loop-helix (bHLH) protein family. The negative effect of Dpn can be mimicked in female embryos by ectopic expression of the related Hairy protein at the time of sex determination. Premature Hairy expression under the control of the hunchback (hb) promoter represses Sxl transcription in the anterior part of female embryos, which leads to female-specific lethality. Repression of Sxl by Hairy depends on the interaction of its C-terminal WRPW motif with Groucho and, consequently, does not occur in embryos deprived of maternal Groucho function. Moreover, substitution of the C-terminal Hairy sequences by a heterologous repression domain still leads to down-regulation of Sxl expression, thus providing a convenient assay for the study of Groucho-dependent repression domains in vivo (Eberhard, 2000 and references therein).

This assay was used to examine the in vivo function of the octapeptide motif by replacing the C-terminal region of Hairy with a sequence encompassing the 90 amino acids located between the paired domain and partial homeodomain of zfPax2.1. The octapeptide motif is the only conserved element that is shared between this zebrafish Pax2.1 sequence and the corresponding region of the Drosophila Pax2/5/8 protein. Expression of the chimeric HairyPax2.1 protein under the control of the hb promoter results in significant reduction of Sxl expression in the anterior half of transgenic female embryos, as compared with the uniform Sxl staining of wild-type embryos. Moreover, the repression of Sxl by HairyPax2.1 is dependent on Groucho, as it is not observed in embryos lacking maternal gro function. However, the HairyPax2.1 protein is clearly less active in repressing the Sxl gene than a HairyGsc protein containing the GEH motif of Goosecoid (Gsc) as a potent repression domain. This difference in repression activity is also reflected by the fact that ectopic expression of HairyGsc caused female lethality, whereas HairyPax2.1 doesnot significantly affect female viability. These data indicate that the octapeptide motif of the zebrafish Pax2.1 protein can function as a weak Groucho-dependent repression domain in Drosophila embryos (Eberhard, 2000).

Six3 is a vertebrate homeobox gene that is expressed in the anterior neural plate and eye anlage. Dominant transcriptional activator or repressor forms of Six3 were overexpressed in zebrafish embryos to analyze their effect on eye and forebrain formation. RNA injection of the activator form of Six3 into zebrafish embryos causes reduction of the expression domains for rx2, pax2, and emx1 in the anterior neural plate, resulting in eye and forebrain hypoplasia. However, overexpression of the repressor form of Six3 or wild-type Six3 shows phenotypes the opposite of those of the activator form. Six3 has eh1-related motifs, motifs crucial for transcriptional repression function of Drosophila Engrailed which plays a role in tethering the Groucho corepressor to the promoters. One of the zebrafish Groucho family genes, grg3, has been isolated and an interaction between Six3 and Grg3 has been demonstrated using yeast two-hybrid analysis. Point-mutations in the eh1-related motifs in Six3 reduce both its eye and forebrain enlarging activities and its interaction with Grg3. These results strongly argue that Six3 functions as a Groucho-dependent repressor in eye and forebrain formation. Furthermore, zebrafish Six2 and Six4 also interact with Grg3, implying a conserved function among the Six family proteins as transcriptional repressors (Kobayashi, 2001b).

Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family (Drosophila homolog: Optix), plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the Eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6 (Drosophila homolog: Sine Oculis), which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. The interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, a specific DNA motif has been identified that is bound by Six3 and it has been demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. This interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae leads to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos results in failure of lens placode invagination and production of delta-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development (Zhu, 2002).

Brain factor 1 (BF-1: Drosophila homologs Slp1 and Slp2) is a winged-helix transcriptional repressor that plays important roles in both progenitor cell differentiation and regional patterning in the mammalian telencephalon. The aim of this study was to elucidate the molecular mechanisms underlying BF-1 functions. BF-1 is shown to interact in vivo with global transcriptional corepressors of the Groucho family and also to associate with the histone deacetylase 1 protein. The ability of BF-1 to mediate transcriptional repression is promoted by Groucho and inhibited by the histone deacetylase inhibitor trichostatin A, suggesting that BF-1 recruits Groucho and histone deacetylase activities to repress transcription. These studies also provide the first demonstration that Groucho mediates a specific interaction between BF-1 and the basic helix-loop-helix protein Hes1 and that BF-1 potentiates transcriptional repression by Hes1 in a Groucho-dependent manner. These findings suggest that Groucho participates in the transcriptional functions of BF-1 by acting as both a corepressor and an adapter between BF-1 and Hes1. Taken together with the demonstration that these proteins are coexpressed in telencephalic neural progenitor cells, these results also suggest that complexes of BF-1, Groucho, and Hes factors may be involved in the regulation of progenitor cell differentiation in the telencephalon (Yao, 2001).

These studies have provided the first demonstration that BF-1 and Groucho/TLE proteins can physically interact with each other in vivo. Their interaction appears to be direct, since it was also observed in binding assays using bacterially purified TLE proteins and in vitro-translated BF-1 preparations. Two separate TLE domains, the amino-terminal Q domain and the carboxy-terminal WDR region, are involved in BF-1 binding. This finding is in agreement with previous investigations showing that these two domains mediate protein-protein interactions with a number of other factors, including RUNX, NK-3, and UTY proteins. These observations suggest that the use of multiple protein-protein interaction domains is a strategy regularly utilized by Groucho/TLEs, perhaps to achieve a specificity that may not be provided by each interaction domain alone. A short region of BF-1, located immediately after the winged-helix domain, is involved in TLE binding. This region contains a sequence, YWPMSPFLSLH, that is conserved among all BF-1 family members and is characterized by two adjacent aromatic residues followed by the motif PFLSL. This arrangement of aromatic residues separated by one or two proline residues is reminiscent of the bona fide Groucho/TLE-binding motif WRP(W/Y) found in Hes and RUNX family members. Importantly, a similar sequence, YAFNHPFSINN, is present in the CRII region that mediates the interaction of TLEs with the winged-helix protein hepatic nuclear factor 3ß. Thus, it is possible that these short sequences may perform the common task of mediating the interaction of these winged-helix proteins with TLEs (Yao, 2001).

BF-1 also associates with HDAC1 in mammalian cells. This interaction is not direct and may be mediated by TLE proteins, which can bind to both BF-1 and HDAC1. Importantly, BF-1-mediated transcriptional repression is reduced by an inhibitor of histone deacetylase activities. Thus, it is proposed that BF-1 can recruit TLEs and histone deacetylases to repress transcription, a possibility consistent with previous studies showing that histone deacetylases are involved in transcriptional repression mediated by Groucho/TLE proteins. It remains to be determined, however, whether the recruitment of histone deacetylase activity represents the general mechanism normally utilized by BF-1 to repress transcription or whether other mechanisms may also be utilized. For instance, it will be important to determine whether Groucho/TLE proteins are always involved in repression by BF-1 or whether the latter can also repress transcription independently of the former. Moreover, Groucho/TLEs may contribute to BF-1 mediated repression in ways that may not always involve the recruitment of histone deacetylases (Yao, 2001).

BF-1 is an important regulator of the progenitor-to-neuron transition in the mammalian telencephalon. In the absence of BF-1, telencephalic progenitor cells differentiate prematurely, leading to early depletion of the progenitor population. These findings suggest that BF-1 promotes cell proliferation and/or inhibits cell differentiation in the telencephalon. BF-1 does not appear to have a direct growth-promoting activity, however, since disruption of BF-1 function in BF-1 minus mice has a demonstrable effect on the proliferation of neuroepithelial cells only after embryonic day 10.5, even though BF-1 is expressed in these cells at earlier stages. It is possible that BF-1 may act as a regulator of the activities of growth-regulatory signals. Support for this hypothesis derives from the finding that the loss of BF-1 leads to ectopic expression of BMP4 in the telencephalic neuroepithelium. This observation suggests that BF-1 may, at least in part, facilitate proliferation by inhibiting BMP4 expression, since BMP4 inhibits telencephalic progenitor cell proliferation. In addition, Xenopus XBF-1 may be a direct regulator of the p27Xic1 gene, the amphibian counterpart of the mammalian cell cycle inhibitor p27Kip1 (Yao, 2001 and references therein).

It is proposed that BF-1 may control telencephalon development by coordinating the control of cell proliferation with the timing of differentiation in the neuroepithelium. In both invertebrates and vertebrates, Hes and Groucho/TLE proteins act as negative regulators of neuronal differentiation by preventing progenitor cells from differentiating prematurely. The finding that BF-1 interacts with and enhances the transcription repression activity of Hes1 suggests that BF-1 may contribute to the regulation of the timing of neuronal differentiation together with Hes1 and TLE proteins. This possibility is consistent with the demonstration that Hes1 minus mice display a forebrain phenotype very similar to that of BF-1 minus mice, namely, premature differentiation of precursor cells with consequent depletion of the progenitor cell population. In the future, it will be important to determine whether BF-1 is involved in the regulation of the expression of genes that are thought to be targets of the transcriptional inhibitory functions of Hes proteins, like the proneural gene Mash1 (Yao, 2001).

A functional interaction between BF-1 and Hes1 may also help explain the results of studies of Xenopus embryos showing that ectopic expression of high doses of XBF-1 causes suppression of neuronal differentiation in the injected area in a cell autonomous way. It is conceivable that the BF-1-Hes1 interaction may be favored in cells expressing high doses of BF-1. As a result, the inhibitory function of Hes1 during neuronal differentiation may be promoted due to the potentiation of its transcription repression activity, leading to suppression of neuronal differentiation within the areas of high BF-1 expression. It remains to be determined, however, whether a similar situation may occur at lower BF-1 concentrations. Studies in Xenopus have shown that microinjection of low doses of XBF-1 does not cause suppression of neuronal differentiation but instead leads to the formation of supernumerary neurons within the injected area. This observation suggests that at low concentrations, BF-1 may not be able to enhance Hes1 activity but may still be able to suppress the growth-inhibitory function of p27Kip1 and/or other antiproliferation factors. This would lead to increased proliferation without an antineurogenic effect, eventually resulting in supernumerary neurons when the progenitor cells differentiate. These observations suggest that changes in BF-1 protein levels may have important repercussions during the progenitor-to-neuron transition and underscore the importance of the mechanisms that regulate BF-1 expression and function (Yao, 2001).

Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos, suggesting that they might be the functional counterparts of the Drosophila genes sine oculis (so) and/or optix. Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, yeast two-hybrid screens were performed, using either Six3 or Six6 as a bait. The results of the screen using Six6 is described that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domains of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish (Oryzias latipes) embryos, overlapping with the distribution of both Six3 and Six6. Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1, which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that controls eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes. Interestingly, Drosophila Optix shows similar interactions with Groucho as well as with TLE1 and AES (López-Ríos, 2003).

Regulation of gene expression by tissue-specific transcription factors involves both turning on and turning off transcription of target genes. Runx3, a runt-domain transcription factor, regulates cell-intrinsic functions by activating and repressing gene expression in sensory neurons, dendritic cells (DC), and T cells. To investigate the mechanism of Runx3-mediated repression in an in vivo context, mice were generated expressing a mutant Runx3 lacking the C-terminal VWRPY, a motif required for Runx3 interaction with the corepressor Groucho/transducin-like Enhancer-of-split (TLE). In contrast with Runx3–/– mice, which displayed ataxia due to the death of dorsal root ganglia TrkC neurons, Runx3VWRPY–/– mice are not ataxic and have intact dorsal root ganglia neurons, indicating that ability of Runx3 to tether Groucho/TLE is not essential for neurogenesis. In the DC compartment, the mutant protein Runx3VWRPY– promoted normally developed skin Langerhans cells but failed to restrain DC spontaneous maturation, indicating that this latter process involves Runx3-mediated repression through recruitment of Groucho/TLE. Moreover, in CD8+ thymocytes, Runx3VWRPY– up-regulates alphaE/CD103-like WT Runx3, whereas unlike wild type, it fails to repress alphaE/CD103 in CD8+ splenocytes. Thus, in CD8-lineage T cells, Runx3 regulates alphaE/CD103 in opposing regulatory modes and recruits Groucho/TLE to facilitate the transition from activation to repression. Runx3VWRPY– also failed to mediate the epigenetic silencing of CD4 gene in CD8+ T cells, but normally regulated other pan-CD8+ T cell genes. These data provide evidence for the requirement of Groucho/TLE for Runx3-mediated epigenetic silencing of CD4 and pertain to the mechanism through which other Runx3-regulated genes are epigenetically silenced (Yarmus, 2006).

The homeodomain protein Nkx2.2 (Nkx2-2) is a key regulator of pancreatic islet cell specification in mice; Nkx2.2 is essential for the differentiation of all insulin-producing β-cells and of the majority of glucagon-producing alpha-cells, and, in its absence, these cell types are converted to a ghrelin cell fate. To understand the molecular functions of Nkx2.2 that regulate these early cell-fate decisions during pancreatic islet development, Nkx2.2-dominant-derivative transgenic mice were created. In the absence of endogenous Nkx2.2, the Nkx2.2-Engrailed-repressor derivative is sufficient to fully rescue glucagon-producing alpha-cells and to partially rescue insulin-producing β-cells. Interestingly, the insulin-positive cells that do form in the rescued mice do not express the mature β-cell markers MafA or Glut2 (Slc2a2), suggesting that additional activator functions of Nkx2.2 are required for β-cell maturation. To explore the mechanism by which Nkx2.2 functions as a repressor in the islet, the pancreatic expression was assessed of the Groucho co-repressors, Grg1, Grg2, Grg3 and Grg4 (Tle1-Tle4), which have been shown to interact with and modulate Nkx2.2 function. Grg3 is highly expressed in the embryonic pancreas in a pattern similar to Nkx2.2. Furthermore, Grg3 physically interacts with Nkx2.2 through its TN domain. These studies suggest that Nkx2.2 functions predominantly as a transcriptional repressor during specification of endocrine cell types in the pancreas (Doyle, 2007).

Activating the PARP-1 sensor component of the Groucho/ TLE1 corepressor complex mediates a CaMKinase II-dependent neurogenic gene activation pathway

Switching specific patterns of gene repression and activation in response to precise temporal/spatial signals is critical for normal development. This study reports a pathway in which induction of CaMKII triggers an unexpected switch in the function of the HES1 transcription factor from a TLE-dependent repressor to an activator required for neuronal differentiation. These events are based on activation of the poly(ADP-ribose) polymerase1 (PARP-1) sensor component of the groucho/TLE-corepressor complex mediating dismissal of the corepressor complex from HES1-regulated promoters. In parallel, CaMKII mediates a required phosphorylation of HES1 to permit neurogenic gene activation, revealing the ability of a specific signaling pathway to modulate both the derepression and the subsequent coactivator recruitment events required for transcriptional activation of a neurogenic program. The identification of PARP-1 as a regulated promoter-specific exchange factor required for activation of specific neurogenic gene programs is likely to be prototypic of similar molecular mechanisms (Ju, 2004).

A covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins mediated by the poly(ADP-ribose) polymerase1 (PARP-1) enzyme. PARP-1 catalyzes the transfer of ADP-ribose chains onto glutamic acid residues of acceptor proteins, including itself (automodification), histones, transcription factors, and DNA repair proteins using NAD+ as a substrate involved in chromatin decondensation, DNA replication, and DNA repair. Therefore, poly(ADP-ribosyl)ation by PARP-1 affects cellular processes such as apoptosis, necrosis, cellular differentiation, malignant transformation, and modulations activities of transcription factors. While it has been recently reported that PARP influences both the expression and silencing at diverse times during Drosophila development (Tulin, 2002), it has been demonstrated that high PARP enzymatic activity is observed in areas of high transcriptional activity and chromatin decondensation on the polytene chromatin (Tulin, 2003). Together these observations suggest that PARP-1 may exert its function in transcription through direct binding to the gene-regulating sequences and through modification of transcription factors by poly(ADP-ribosyl)ation (Ju, 2004).

This study finds that HES1-dependent repression of MASH1 is dependent upon the actions of the TLE1 corepressor complex. Not only are additional insights provided into the molecular mechanisms of TLE1-mediated repression but also the molecular mechanism of the switch to activation function has been uncovered. The composition of this TLE1 complex is distinct from those of other reported corepressor complexes such as N-CoR/SMRT and CtBP. Interestingly, roles for transcriptional regulation and chromatin remodeling activities have been described for most of the components of the TLE1 complex identified, but no component alone is indispensable for at least some level of TLE1-mediated repression (Ju, 2004).

Consistent with the observation that the enzymatic activity of PARP-1 is not required for HES1-mediated MASH1 repression, these data favors a model predicting that in the TLE1 holorepressor complex, the enzymatic activity of PARP-1 is inhibited. Exposure to a signal inducing neuronal differentiation causes activation of CaMKIIdelta, which is proven to be required for activation of neurogenic genes. Once sufficient levels of CaMKIIdelta are achieved (5-7 hr), it will, directly or indirectly, mediate phosphorylation and activation of PARP-1, which then catalyzes poly(ADP-ribosyl)ation of TLE1 and most of the other components to the corepressor complex. This is consistent with the observation that calcium signaling evoked by extrinsic and intrinsic cues can induce auto-poly(ADP-ribosyl)ation of PARP-1; however, CaMKIIdelta may also be activated in a calcium-independent fashion. This covalent modification is suggested to result in their dismissal from the biochemical complex and derepression of the MASH1 gene. The role of PARP-1 in derepression of MASH1 and its retention on the activated MASH1 promoter is quite consistent with reports that poly(ADP-ribosyl)ation of chromatin-associated proteins induce major changes in chromosomal architecture. However, in the case of MASH1, it was found that derepression alone is insufficient for induction. This is in accord with the findings for other regulated transcription factors. For example, the loss of the N-CoR corepressor is not alone sufficient to activate most AP-1-regulated genes, and only in a subset of RAR target genes does derepression result in a signal-independent 'default' activation of gene targets.

These data also indicate that, in addition to Ca2+-CaMKII-dependent dissociation of the TLE1 corepressor from HES1, a covalent modification of HES1 itself is required to permit activation of MASH1. Thus, activation of MASH1 is linked to sequential CaMKIIdelta-dependent activation of PARP-1 enzymatic activity, which was previously inhibited in the TLE1 holocorepressor complex, permitting dismissal of the TLE1 complex, derepression and phosphorylation of HES1, and recruitment of specific coactivators and thus causing and maintaining derepression of genes mediating neuronal differentiation. The actions of PARP-1 in the TLE1-mediated events are thus analogous to the effects of covalent modifications by phosphorylation and acetylation, as mediators of switches from repressor to activator function (Ju, 2004).

While HES1 is recognized to regulate tissue morphogenesis by maintaining undifferentiated cells and preventing differentiation, continued occupancy of the MASH1 promoter by HES1 in the differentiating neural stem cells has surprisingly proven to be required to initiate MASH1 activation events. Indeed, previous, seemingly contradictory reports of transient transfection assays in which HES1 can inhibit acid beta-glucosidase genes in HepG2 cells but cause activation in fibroblasts are likely to be explained by findings of signal-dependent HES1 switching events (Ju, 2004).

This cortical progenitor culture system has permitted identification of a regulatory pathway that may be, at least in part, partially compensated in vivo because many nestin-positive neural stem cells in the subventricular zone proliferate without losing the multipotentiality to differentiate into neurons in HES1 mutant or even HES1/HES5 double mutant mice. It is suggested that there may be additional HES1-like repressors or unidentified protein partners, including HERP and other E box binding proteins, that are also potentially involved in MASH1 gene activation. The identification of this unexpected mechanism of HES1 action in cortical progenitor cell cultures suggest that, in this system, the other molecules that could assume a similar function were either not expressed or required. The finding of a requirement of HES1 in activation of neurogenic genes is consistent with suggestions that HES1 might promote differentiation, in addition to its role in maintenance of the undifferentiated state, at multiple steps of neural stem cell development (Ju, 2004).

In summary, a pathway is suggested in which a PARP-1-containing TLE1 complex is recruited by the Notch-induced bHLH factor, HES1, initially mediating repression of MASH1 in the proliferating neural stem cells. The data suggest that signals that induce neuronal differentiation, such as PDGF in neural stem cells, act to induce the CaMKIIdelta isoform, which, in turn, is required for HES1-mediated MASH1 activation . The temporal aspects of CaMKIIdelta induction appear to account for the delay in derepression and activation of MASH1 expression following critical PDGF signaling. CaMKIIdelta-induced phosphorylation of a specific serine residue in the orange domain of HES1 permits it to recruit coactivators, including CBP, and has proven to be required for activation of the MASH1. The conserved relationship of the HES1 orange domain with the HLH domain raises the possibility of an additional role in protein interactions that include dimerization (Ju, 2004).

In a sense, the observation that a component of the TLE1-mediated repression complex, PARP-1, is also required for derepression events and maintenance of activation, parallels the requirement of TBL1/TBLR1 complex for ligand-dependent exchange of N-CoR corepressor complexes for coactivators in the switch of nuclear receptor function from repression to activation. TBLR1 is required for recruitment of the ubiquitylation/19S proteosome complex to prevent N-CoR/SMRT-dependent maintenance of a repression checkpoint. It is suggested that PARP-1 may achieve the same effects by a distinct modification strategy and serves as a regulated sensor of neuron-inducing signals based on the actions of CaMKIIdelta induced by the initial stimulus to neuronal differentiation. Therefore, it is tempting to speculate that PARP-1 and TBLR1 may be critical for the exchange events required to overcome the repression checkpoint for TLE- and N-CoR-regulated repressors, respectively (Ju, 2004).

The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway indicate that while the initial Notch signal causes repression of neurogenic genes by induction of HES1, it simultaneously arms the response to subsequent Ca2+-CaMKII signals that permit MASH1 gene activation events. The data illustrate how a single signaling pathway can mediate a sequential, two-step derepression/activation process required for development in gene activation. Induction of a Ca2+/CaMKII-dependent program initiates both PARP-1 activation, which is required for dismissal of the TLE1 corepressor complex, and a second event, covalent modification of HES1, which is required for target gene activation. The requirement for both a derepression and independently mediated activation event is likely to be prototypic of many similar functions of PARP-1 factors in development. The sequential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic gene expression to signals and to permit temporally precise patterns of cellular response (Ju, 2004).

Mammalian Groucho homologs: Effects of mutation

The murine Grg gene encodes a 197 amino acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. Grg is a 25 kd nuclear protein that can participate in specific protein-protein interactions. Mice homozygous for a null mutation complete embryogenesis and are born, but exhibit varying degrees of post-natal growth deficiency. No dosage-sensitive genetic interaction is detected between the Notch1 and Grg genes in mice heterozygous for a Notch1 mutant allele and homozygous for the Grg null mutation (Mallo, 1995).

EGFR signaling via the MAPK pathway attenuates Groucho-dependent repression to antagonize Notch transcriptional output

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway, whether Gro is at a crossroad between this and other pathways was tested. This study shows that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways (Hasson, 2005).

Tcf HMG box transcription factors interact with Groucho-related co-repressors

Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, beta-catenin translocates to the nucleus, interacts with Tcf and thus activates transcription of target genes. Tcf factors (see Drosophila Pangolin) also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors. All known mammalian Groucho family members have been tested for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, the expression of all Tcf and Grg/TLE family members were determined in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The 'long' Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for Drosophila Groucho, this study shows that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription (Brantjes, 2001).

Mammalian Groucho homologs: Developmental roles

Transducin-like Enhancer of split (TLE) 1 is a mammalian transcriptional corepressor homologous to Drosophila Groucho. In Drosophila, Groucho acts together with bHLH proteins of the Hairy/Enhancer of split (HES) family to negatively regulate neuronal differentiation. Loss of the functions of Groucho or HES proteins results in supernumerary central and peripheral neurons. This suggests that mammalian TLE/Groucho family members may also be involved in the regulation of neuronal differentiation. Consistent with this possibility, TLE1 is expressed in proliferating neural progenitor cells of the central nervous system, but its expression is transiently down-regulated in newly generated postmitotic neurons. Based on these observations, an investigation was carried out to see whether persistent TLE1 expression in postmitotic neurons would perturb the normal course of neuronal development. Transgenic mice were derived in which the human TLE1 gene is regulated by the promoter of the Talpha1 alpha-tubulin gene, which is exclusively expressed in postmitotic neurons. In these mice, constitutive expression of TLE1 inhibits neuronal development in the embryonic forebrain leading to increased apoptosis and neuronal loss in the ventral and dorsal telencephalon. These results provide the first direct evidence that TLE1 is an important negative regulator of postmitotic neuronal differentiation in the mammalian central nervous system (Yao, 2000).

The pattern of neuronal specification in the ventral neural tube is controlled by homeodomain transcription factors expressed by neural progenitor cells, but no general logic has emerged to explain how these proteins determine neuronal fate. Most of these homeodomain proteins possess a conserved eh1 motif that mediates the recruitment of Gro/TLE corepressors. The eh1 motif underlies the function of these proteins as repressors during neural patterning in vivo. Inhibition of Gro/TLE-mediated repression in vivo results in a deregulation of cell pattern in the neural tube. These results imply that the pattern of neurogenesis in the neural tube is achieved through the spatially controlled repression of transcriptional repressors -- a derepression strategy of neuronal fate specification (Muhr, 2001).

Graded inductive signals specify cell fates in a position-dependent manner in the neural tube. Within the ventral neural tube, the identities of neural progenitor cells are assigned initially by the actions of Sonic hedgehog (Shh). Graded Shh signaling establishes distinct ventral progenitor domains by regulating the spatial pattern of expression of a set of homeodomain (HD) proteins that comprise members of the Pax, Nkx, Dbx, and Irx families. These HD proteins can be subdivided into class I and class II proteins based on their differential regulation by Shh signaling. The class I proteins are expressed by neural progenitor cells in the absence of Shh signaling, and their expression is repressed by Shh. In contrast, the expression of the class II proteins depends on exposure to Shh (Muhr, 2001 and references therein).

How do these HD proteins specify neuronal fate. The establishment of progenitor cell identity appears to involve cross-regulatory interactions between complementary pairs of class I and class II HD proteins that share a common boundary. These interactions define the spatial extent of individual progenitor domains and establish sharp boundaries between adjacent domains, thus ensuring that cells within individual domains express distinct combinations of HD proteins. The profile of class I and class II HD protein expression within a progenitor cell appears to direct neuronal fate. Most strikingly, several of these progenitor HD proteins have the ability to induce the ectopic generation of neuronal subtypes when misexpressed outside the confines of their normal progenitor domains. The inductive activities of these progenitor HD proteins involve the activation of expression of downstream transcription factors that serve intermediary roles in the determination of neuronal fate. In addition, gene targeting studies in mice have established the essential role of many of these class I and class II proteins in the specification of ventral neuronal identity (Muhr, 2001 and references therein).

Eight of the ten progenitor HD proteins implicated in ventral neural patterning share a motif related to the core eh1 region of the Engrailed repressor (EnR) domain. This motif mediates in vitro interactions of class I and class II HD proteins with Groucho-TLE (Gro/TLE) corepressors, and underlies the function of these proteins as repressors in neural patterning in vivo. Disruption of Gro/TLE function in neural cells in vivo leads to an impairment of ventral patterning. Three conclusions have been reached: (1) there is a common mechanism of action of the class I and class II progenitor HD proteins involved in ventral patterning; (2) Gro/TLE corepressors play a role in patterning the ventral neural tube; (3) the spatial pattern of neurogenesis in the ventral neural tube is achieved through the repression of repressors (Muhr, 2001).

To identify functional domains that mediate the neural patterning activity of the Nkx proteins, a focus was placed on a conserved ~10 amino acid motif, termed the TN, or NK decapeptide, domain. Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2 and Drosophila Ventral nervous system defective (Vnd) each possess a TN domain. This domain shows sequence similarity to the core region of the engrailed homology-1 (eh1) domain present in Engrailed (En), a transcriptional repressor. The eh1 motif interacts with Gro/TLE corepressors, and Gro/TLE proteins can bind to certain Nkx class proteins (Muhr, 2001).

The idea that Gro/TLE corepressors mediate the neural patterning activity of progenitor HD proteins currently rests on three lines of evidence: (1) the presence of an eh1 domain in class I and class II proteins underlies their Gro/TLE binding activity in vitro, and is required for their repressor functions in vivo; (2) Gro/TLE genes are expressed in the ventral neural tube at the time that neural pattern is established; (3) Grg5, a protein that inhibits Gro/TLE repressor function, deregulates the pattern of progenitor HD protein expression and blocks ectopic neuronal specification in vivo (Muhr, 2001).

The dorsal expansion in the domains of expression of the class II proteins Nkx6.1 and Nkx2.2 observed after Grg5 expression provides evidence that Gro/TLE function is required normally to establish the p1/p2 and pMN/p3 progenitor domain boundaries (MN refering to motor neuron). Expression of Grg5 also disrupted the normal mutual exclusion in the domains of expression of the class I/class II protein pairs Dbx2/Nkx6.1 and Pax6/Nkx2.2. Thus, a reduction in Gro/TLE activity blocks the ability of class II proteins to repress class I protein expression. However, there is not a ventral expansion in the domains of expression of the class I proteins Dbx2 and Pax6. One possible explanation for this asymmetry in HD protein deregulation is that a higher level of Gro/TLE activity is required for the repressor activity of the class I proteins than for the class II proteins. In addition, the detection of higher levels of ventral Gro/TLE gene expression than dorsal implies that expression of Grg5 will be more effective in reducing the net level of Gro/TLE activity in dorsal than ventral regions of the neural tube, favoring a dorsal expansions in progenitor HD protein expression. The early onset of Nkx6.1 expression, together with a higher level of ventral Gro/TLE gene expression, may also explain why Grg5 expression blocks the Nkx6.1-mediated induction of ectopic MNs, but is not able to inhibit the generation of MNs within the pMN domain. The findings with Grg5 support an essential role for Gro/TLE proteins in neural patterning, but there is still a need to define changes in neuronal fate that occur after elimination of the Gro/TLE proteins themselves (Muhr, 2001).

Pax6, in contrast to most other class I proteins, lacks an eh1 domain and functions as an activator. Nevertheless, Pax6 represses Nkx2.2 expression in vivo, implying that its function at the pMN-p3 boundary is achieved through an intermediary repressor. The finding that the domain of Nkx2.2 expression expands dorsally upon Grg5 overexpression implies that this intermediary repressor itself functions in a Gro/TLE-dependent manner. Taken together, these observations suggest that the establishment and maintenance of ventral progenitor domains -- whether achieved by direct repression or by activation of intermediary repressors -- depend on the activity of Gro/TLE corepressors (Muhr, 2001).

The finding that the activity of progenitor HD proteins depends on Gro/TLE-mediated repression provides several insights into the strategies used to establish neuronal diversity in the central nervous system. Focus is placed here on how transcriptional repression mediates the functions of the class II repressor proteins Nkx6.1 and Nkx2.2, although similar arguments apply for many of the class I proteins. The class II proteins Nkx6.1 and Nkx2.2 are required for the generation of MNs and V3 neurons, respectively. These activities appear to be achieved through the expression of downstream determinants of neuronal subtype identity. For example, within the pMN domain, Nkx6.1 promotes the expression of MNR2, a dedicated MN determinant. Nkx6.1 functions as a repressor during the specification of MNs in dorsal regions of the neural tube, favoring the idea that Nkx6.1 controls the expression of MNR2 within the pMN domain itself through its role as a repressor of class I proteins, although this remains to be established. In this view, the loss of MNs in Nkx6.1 mutant mice results from the ectopic ventral expression of class I proteins rather than from the loss of an Nkx6.1 activator function (Muhr, 2001).

How do Nkx6.1 and Nkx2.2 induce MNs and V3 neurons along the entire dorsoventral axis of the neural tube? In ventral progenitor cells, the inductive activities of Nkx6.1 and Nkx2.2 appear to depend on their ability to act as repressors of their complementary class I proteins, Dbx2 and Pax6. But in the dorsal neural tube, progenitor cells lack expression of many of the ventral class I repressor proteins. Thus, dorsal neural progenitors must also express repressors of MN and V3 neuronal differentiation -- repressors that are themselves subject to repression by Nkx6.1 or Nkx2.2. The identity of the dorsal repressors of MN and V3 neuron generation is not known, but the Gsh1/2 HD proteins are plausible candidates as suppressors of MN specification. Both Gsh proteins possess an eh1 motif (see Supplemental table) and are normally restricted to the dorsal neural tube, but are ectopically expressed ventrally in mouse Nkx6.1 mutants (Muhr, 2001).

The class II proteins also inhibit alternative neuronal fates within their normal domains of expression. Within the pMN and p2 domains, the expression of Nkx6.1 prevents V1 interneuron generation, and within the p3 domain, Nkx2.2 expression prevents MN generation. Thus, the Nkx proteins promote certain neuronal fates and block others, even though both activities are mediated primarily through repression. This reliance on repression distinguishes the function of Nkx proteins in neural fate specification from that of many other transcription factors whose roles in the selection of cell fates appears to reflect a combination of activator and repressor functions. The expression of Nkx2.2 and Nkx6.1 persists in certain post-mitotic neurons, and thus it remains possible that putative activator functions of these proteins are relevant for aspects of neuronal differentiation other than those examined in this study. Indeed, in other regions of the developing nervous system, the Phox2 HD proteins have been shown to function as activators of neuronal differentiation genes (Muhr, 2001).

How is neuronal fate decided when two repressor HD proteins are coexpressed within individual neural progenitor cells? Within the p3 domain, cells coexpress Nkx6.1 and Nkx2.2, yet the activity of Nkx2.2 is dominant, and progenitors generate V3 neurons rather than MNs. One conceivable reason for this is that Nkx2.2 has a higher affinity than Nkx6.1 for Gro/TLE proteins and thus sequesters available Gro/TLE corepressor activity, preventing Nkx6.1 function. Against this idea, Dbx2 is ectopically expressed in p3 domain progenitors in Nkx6.1 mutants, indicating that Nkx6.1 still functions as a repressor in this domain. A second and more plausible explanation is that Nkx2.2 blocks MN generation in p3 progenitors at a step downstream of progenitor HD proteins by repressing the expression of MN subtype determinants. Thus, instances of coexpression of class I and/or class II repressor proteins within progenitor cells may reflect the selection of neuronal fate through repression at the level of downstream neuronal subtype determinants rather than at the level of progenitor HD proteins (Muhr, 2001).

Taken together, these findings favor a model in which the pattern of neuronal specification is achieved primarily through the selectivity of repressor interactions with cis-acting DNA sequences present in the regulatory regions of different progenitor HD proteins and neuronal subtype determinants. This model requires that repressor HD proteins with distinct activities in neuronal specification recognize distinct DNA target sequences. In support of this idea, the class II proteins Nkx2.2 and Nkx6.1 have different patterning activities in the neural tube, possess divergent HDs, and recognize distinct target DNA sequences. In addition, the finding that hybrid class I and class II proteins consisting solely of the HD fused to the EnR or TN domain mimic the activity of the full-length proteins indicates that the distinct activities of class II and class I repressor proteins in neural patterning are likely to reside in the specificity of DNA recognition encoded in the HD (Muhr, 2001).

The finding that class II proteins and most class I proteins function as repressors leaves unresolved the issue of the role of transcription factors that activate the expression of neuronal subtype determinants. The results imply that progenitor cells arrayed along the entire dorsoventral axis of the neural tube possess a latent potential for activation of expression of all neuronal subtype determinants. In an extreme view, these subtype determinants may be activated by a single common activator protein that is expressed in a uniform manner along the entire dorsoventral axis of the neural tube. The ability of such an activator to induce different subtype determinant genes would then be constrained by the repertoire of cis-acting binding sites for class I and class II HD protein repressors present in their regulatory regions. This view argues that the specificity of neuronal subtype generation emerges largely from the patterned expression of repressors (Muhr, 2001).

In principle, it is possible to consider an alternative view in which distinct activator proteins are expressed within individual progenitor domains, with these activators operating upstream of but in a linear pathway with neuronal subtype determinants such as MNR2. In this view, the patterns of expression of these upstream activators would themselves need to be defined by the repressor activities of the class I and class II HD proteins. But the question of what activates the domain-restricted expression of these upstream activators immediately resurfaces. Thus, at its root, the activation of subtype determinants along the dorsoventral axis of the neural tube is likely to be a spatially unrestricted process. Clarification of this issue will require the identification of proteins that activate the expression of neuronal subtype determinants (Muhr, 2001).

In this context, it is intriguing that several basic helix-loop-helix (bHLH) transcriptional factors are expressed in discrete domains along the dorsoventral axis of the neural tube. Some of these genes transgress progenitor domain boundaries, whereas others are restricted to individual progenitor domains. Studies of bHLH protein function in vertebrates have begun to suggest that these proteins can influence neuronal subtype identity, in addition to their more general roles in neurogenesis. Determining whether and how the activity of bHLH proteins is integrated with progenitor HD protein-mediated repression during the specification of neuronal fate may help in the further dissection of mechanisms of ventral neuronal patterning (Muhr, 2001).

This analysis of the function of progenitor HD proteins has focused on neuronal specification along the dorsoventral axis of the neural tube. There are also clear restrictions in the potential for neuronal generation along the rostrocaudal axis of the neural tube. It is noteworthy that many HD proteins implicated in rostrocaudal neural patterning—including other Pax and Nkx proteins, and the Gsh, Msx, Gbx, and Tlx proteins—also possess eh1-like domains. Indeed, in a sample of 165 vertebrate HD proteins, many expressed by neural cells, ~36% were found to possess an eh1 domain (see Supplemental table). Gro/TLE-dependent repression may, therefore, have a more pervasive role in establishing precise spatial patterns of neuronal generation along both major axes of neural tube development. In addition, since homologs of the Nkx, Msx, and Gsh proteins control neuronal patterning along the dorsoventral axis of the Drosophila CNS, these results suggest that Gro/TLE-mediated corepression may be an evolutionarily conserved step in CNS patterning (Muhr, 2001).

During the development of the pituitary gland, two highly related paired-like homeodomain factors, a repressor (Hesx1/Rpx) and an activator (Prop-1) are expressed in sequential, overlapping temporal patterns (note: there are no known close Drosophila homologs). While the repressive actions of Hesx1/Rpx may be required for initial pituitary organ commitment, progression beyond the appearance of the first pituitary (POMC) lineage requires both loss of Hesx1 expression and the actions of Prop-1. Although Hesx1 recruits both the Groucho-related corepressor TLE1 and the N-CoR/Sin3/HDAC complex on distinct domains, the repressor functions of Hesx1 require the specific recruitment of TLE1, which exhibits a spatial and temporal pattern of coexpression during pituitary organogenesis. Furthermore, Hesx1-mediated repression coordinates a negative feedback loop with FGF8/FGF10 signaling in the ventral diencephalon, required to prevent induction of multiple pituitary glands from oral ectoderm. These data suggest that the opposing actions of two structurally-related DNA-binding paired-like homeodomain transcription factors, binding to similar cognate elements, coordinate pituitary organogenesis by reciprocally repressing and activating target genes in a temporally specific fashion, on the basis of the actions of a critical, coexpressed TLE corepressor (Dasen, 2001).

The sequential actions of transcriptional repressors and activators on overlapping sets of gene targets, in concert with requisite coregulatory machinery, is likely to be a central strategy in mammalian organogenesis. This study has explored the opposing roles of two highly-related paired-like homeodomain factors, Hesx1/Rpx and Prop-1, that exhibit temporally distinct, but overlapping patterns of expression over the entire period of pituitary organ commitment, patterning, and cell-type determination. These data suggest that Hesx1, although clearly modified by the actions of linked modifier genes on the basis of genetic background, is required for early organ commitment and cell determination events. These actions extend temporally to include the appearance of the dorsal POMC lineage, which then, with a specific coexpressed corepressor, Hesx1, serves to prevent Prop-1 from initiating the program required for asymmetric division and proliferation of the Pit-1 and gonadotrope lineages. Premature expression of Prop-1 can block pituitary organogenesis, phenocopying the effects of Hesx1-gene deletion, suggesting that the switch of binding of a paired-homeodomain repressor for a paired-homeodomain activator, with resultant alteration in the expression of key target genes, now prevents organogenesis. Conversely, expression of Hesx1 with the obligate corepressor TLE1 can block the activation of Prop-1-dependent genes required for the appearance of four anterior pituitary cell types. This provides a striking example of a potent strategy in mammalian organogenesis, in which opposing actions of related repressors and activators, putatively binding to overlapping sets of gene targets, provide critical temporal control of organ development. Interestingly, later persistent expression of Prop-1 under control of the alphaGSU promoter causes decreased gonadotrope differentiation and causes increased adenomatous hyperplasia (Dasen, 2001).

In addition to its early and later roles in pituitary organogenesis and cell type determination, analysis of Hesx1-/- mice has also revealed an intriguing regulatory loop. Early in development, Hesx1 is expressed in a broad region of the anterior neural plate that will later give rise to the ventral diencephalon and pituitary. Deletion of the Hesx1 gene causes a rostral extension of FGF8 and FGF10 expression in the ventral diencephalon, into an area that transiently expresses Hesx1, leading to ectopic Lhx3 induction and formation of supernumerary pituitary glands, confirming that FGF8/FGF10 signaling is required and sufficient to signal pituitary commitment from oral ectoderm. Further, the data showing that FGF8 suppresses Hesx1 gene expression indicate a negative regulatory loop with Hesx1 acting early to repress FGF8/FGF10, which in turn, directly or indirectly, represses Hesx1 gene expression at the time of the emergence of pituitary cell types from Rathke's pouch. Thus, a paired-like homeodomain repressor serves to establish boundaries of FGF8/10 gene expression in the ventral diencephalon and thus restricts the spatial domains at which pituitary organogenesis can occur (Dasen, 2001).

Together, these data suggest that Hesx1 can exert both cell-autonomous and noncell-autonomous roles in pituitary development. Early in development, Hesx1 is required for restricting and maintaining the proper expression domains FGF8 and FGF10, consistent with its putative role as a repressor in the anterior neural plate, which establishes boundaries of morphogen expression. Later in development, after its expression becomes restricted to Rathke's pouch between E9 and E12, Hesx1 is required for regulating the appropriate ventral proliferation patterns of pituitary progenitor lineages. These observations are based on the analysis of Hesx1 mutants, in which the pituitary did not exhibit defects in the ventral diencephalon, but continued to proliferate, and are further supported by the in vivo effects of maintained TLE1 and Hesx1 expression (Dasen, 2001).

Mature chick optic tecta consist of 16 laminae: SO (stratum opticum), SGFS (stratum griseum et fibrosum superficiale, a-j), SGC (stratum griseum centrale), SAC (stratum album centrale), SGP (stratum griseum periventriculare), SFP (stratum fibrosum periventriculare) and ependyma. The optic tecta receive retinal fiber projections in a precise retinotopic manner. Retinal axons arborize in laminae a-f of the SGFS, but do not cross the border between lamina f and g. In order to elucidate molecular mechanisms of tectal laminar formation, the migration of tectal postmitotic cells was examined. The migration pattern of postmitotic cells changes around E5 and late migratory cells form intervening laminae between those formed by early migratory cells. The coincident appearance of expression of Groucho family member Grg4 in the tectal ventricular layer and the change in migration pattern suggests an important role for Grg4. Clonal misexpression of Grg4 results in cells migrating to laminae h-j of the SGFS. Massive misexpression of Grg4 results in disruption of laminae that are formed by early migratory cells, in particular lamina g of the SGFS. Application of Grg4 morpholino antisense oligonucleotide or the misexpression of a dominant-negative form of Grg4 exerts the opposite effect. It is concluded that Grg4 may direct tectal postmitotic cells to follow a late migratory pathway (Sugiyama, 2003).

Repression by Groucho/TLE/Grg proteins: genomic site recruitment generates compacted chromatin in vitro and impairs activator binding in vivo

Groucho-related (Gro/TLE/Grg) corepressors meditate embryonic segmentation, dorsal-ventral patterning, neurogenesis, and Notch and Wnt signaling. Although Gro/TLE/Grgs disrupt activator complexes and recruit histone deacetylases (HDAC), activator complexes can be disrupted in various ways, HDAC recruitment does not account for full corepressor activity, and a direct role for Gro/TLE/Grg binding and altering chromatin structure has not been explored. Using diverse chromatin substrates in vitro, this study shows that Grg3 creates higher order, condensed complexes of polynucleosome arrays. Surprisingly, such complexes are in an open, exposed configuration. Chromatin binding enables Grg3 recruitment by a transcription factor and the creation of a closed, poorly accessible domain spanning 3-4 nucleosomes. Targeted recruitment of Grg3 blankets a similar sized region in vivo, impairing activator recruitment and repressing transcription. These activities of a Groucho protein represent a newly discovered mechanism which differs from that of other classes of corepressors (Sekiya, 2007).

In the absence of recruitment by a transcription factor, Grg3 readily binds mononucleosome particles, at least in part via the interaction with the tails of histones H3 and H4. However, another level of Grg3 interaction with chromatin has been discovered: with a dinucleosome subunit, helping to generate a condensed structure. In vivo studies suggest that the yeast Tup1 homolog bridges across adjacent nucleosomes (Ducker, 2000). Since it was found that N-terminally deleted Grg3, deficient in tetramerization, also elicits chromatin condensation, it is unlikely that condensation is mediated by bridging across nucleosomes by different Grg3 subunits in a native tetramer. It is suggested that chromatin condensation may be generated by an individual Grg3 subunit interacting simultaneously with H3 tails of adjacent nucleosomes or with a new structure generated by the interface of adjacent nucleosomes. The ability of different domains of Tup1 to interact independently with core histones is consistent with the former possibility (Sekiya, 2007).

By contrast, aggregation of separate nucleosome arrays requires an intact tetramerization domain of Grg3, as does the generation of recruitment-based, nuclease-resistant chromatin in vitro and transcriptional repression in vivo. Deletion of the tetramerization domain diminished, but did not eliminate, FoxA1 interactions, making it difficult to assess the extent to which the ability to aggregate arrays is necessary for the repressed chromatin structure upon FoxA1-based recruitment. Regardless, the middle region of Grg3 becomes exposed to V8 protease when full-length Grg3 binds to nucleosome arrays. Thus, chromatin binding induces a conformational change in the tetramer so that the subunits become exposed. This could facilitate the bridging of Grg3 between arrays to generate the aggregates. Secondarily, the conformational change in Grg3 induced by chromatin binding may indirectly facilitate stable interactions with FoxA, since it was found that the middle region of free Grg3 impairs the protein from binding to FoxA factors (Sekiya, 2007).

Significantly, chromatin binding by Grg3 is indispensable for FoxA1- and Hes1-based genomic site recruitment; neither transcription factor could recruit Grg3 to free DNA. The existence of positioned nucleosomes at the alb1 enhancer in liver cells may facilitate the ability of FoxA factors to recruit Grg3. In summary, it is suggested that Gro/TLE/Grg proteins scan chromatin domains for being able to transiently bind nucleosomes, which stabilizes an open tetramer and consequently allows the tetramer to bind a recruiting transcription factor. Since Groucho/TLE/Grg proteins are thought to be refractory to binding highly acetylated chromatin, associated HDAC activity could enable stable transcription factor-based recruitment (Sekiya, 2007).

Furthermore, this study found that Grg3 generates a compact chromatin structure that is resistant to nucleases only when binding both chromatin and a recruiting transcription factor. FoxA1-Grg3-array complexes are resistant to three different nucleases over the distance of approximately 3-4 nucleosomes in vitro. Strikingly, this corresponds to the same region over which Grg3 was recruited to the alb1 enhancer by FoxA1 in vivo, causing repression. Three to four nucleosomes spans the size of many enhancer and promoter elements. It is therefore suggested that the recruitment-based blanketing of Grg3 over such a chromatin domain is part of the mechanism for transcriptional repression by the Gro/TLE/Grg corepressors (Sekiya, 2007).

Functionally, this study found that recruitment-based 'blanketing' of local chromatin by Grg3 results in impaired recruitment of a transcriptional activator, TBP, and RNA polymerase II. This distinguishes the mechanism of repression by Grg with that of Sir complexes. Strikingly, Grg3 impaired the enhanced recruitment of NF-1, TBP, and pol II that normally occurs when the H2.35 liver cell line is shifted to differentiating culture conditions. Interestingly, local levels of linker histone were not significantly affected by Grg3, indicating that the Grg3-bound chromatin neither excludes linker histone nor is excluded by it (Sekiya, 2007).

When Grg recruitment sites are separated in chromatin, as for the afp enhancers, Grgs can apparently span a larger chromatin domain; though this terminates distal to recruitment sites. Although Gro/TLE/Grg proteins can repress genes distal to their site of recruitment, it is suggested that by blanketing an enhancer, or an extended enhancer domain (as for afp), Gro/TLE/Grg proteins indirectly suppress distal enhancer-promoter interactions. This is opposed to long-range spreading of a repressive and resistant chromatin structure from a recruitment site, as seen with Sir proteins. The yeast homolog Tup1 was found to spread along an entire STE6 gene on an episome (Ducker, 2000). Interestingly, the linker regions of the episomal Tup1-STE6 chromatin remained sensitive to MNase, similar to the linker region sensitivity to nucleases of the condensed chromatin generated by Grg3 alone on nucleosome arrays (Sekiya, 2007).

In conclusion, these studies emphasize the importance of reconstituting regulatory complexes on chromatin templates of different complexity and assessing the concordance with endogenous genes, to unveil mechanisms of genetic control. The mechanisms and consequences of chromatin binding that were found for Grg are different than those of other corepressor classes. Understanding how the condensed chromatin structure generated by Grg alone, in vitro, is converted to a resistant chromatin domain by transcription factor recruitment could unveil new molecular targets for antagonizing corepressor activity (Sekiya, 2007).

Meis2 competes with the Groucho co-repressor Tle4 for binding to Otx2 and specifies tectal fate without induction of a secondary midbrain-hindbrain boundary organizer

The transcription factor Otx2 is expressed throughout the anterior neuroectoderm and is required for the formation of all forebrain- and midbrain-derived structures. The molecular determinants that cooperate with Otx2 to subdivide its expression domain into distinct functional units are, however, poorly understood at present. This study shows that the TALE-homeodomain protein Meis2 is expressed in the chick tectal anlage and is both necessary and sufficient for tectal development. Unlike known tectum-inducing genes, the ability of Meis2 to initiate tectal development does not involve the formation of a secondary midbrain-hindbrain boundary organizer, but instead requires direct interaction with Otx2. Using an Otx2-dependent reporter assay it was demonstrated that Meis2 competes with the Groucho co-repressor Tle4 (Grg4) for binding to Otx2 and thereby restores Otx2 transcriptional activator function. Together, these data suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over tectal development. Controlled formation of Meis2-containing higher order protein complexes might thus serve as a general mechanism to achieve subdivision of the anterior neuroectoderm into distinct functional units during embryogenesis (Agoston, 2009).

Meis2 is a key regulator of tectal development. In contrast to other known genes involved in tectal development, Meis2 initiates tectal fate specification without inducing a secondary MHB organizer. Instead, Meis2 binds to Otx2 in the absence of DNA, competes with the co-repressor Tle4 for binding to Otx2 and thereby restores Otx2 transcriptional activator function. As discussed below, these results suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over the onset of tectal development. These data thus argue for a novel, potentially DNA-independent function of TALE-homeodomain proteins: the controlled assembly and disassembly of transcription regulator complexes (Agoston, 2009).

Tectum development is induced when an ectopic Fgf8 source is generated in the prosencephalon through transplantation of an ectopic MHB organizer or implantation of Fgf8-releasing beads into the lateral wall of the diencephalon. In addition to Fgf8, several transcription factors can trigger tectal development upon misexpression, including Otx2, Pax2/5, En1/2 and Pax3/7. Unlike Meis2, expression of these proteins is not specific for the tectal anlage. Moreover, each of these proteins participates in the interdependent, positive maintenance loop at the MHB organizer and, consequently, induces ectopic expression of MHB marker genes, including Fgf8, when misexpressed. These molecules therefore evoke tectal development indirectly through formation of an ectopic MHB organizer. Meis2, by contrast, is unique as it can initiate tectal development without participating in MHB organizer function or maintenance. Endogenous Meis2 expression is repressed when metencephalic development is experimentally induced through activation of the Ras-MAP kinase pathway in the mesencephalon and is upregulated concomitantly to the metencephalic-to-mesencephalic fate change that occurs when Ras-MAP kinase signaling is blocked in rhombomere 1 (Vennemann, 2008; Agoston, 2009).

Meis2 expression must therefore be directly or indirectly under control of the MHB organizer. Notably, a single, transient transfection of Meis2 in the diencephalic alar plate at the 10- to 11-somite stage was sufficient to initiate long-term expression of endogenous Meis2 in transfected cells. Meis2, once induced, can therefore stabilize its own expression. Together, these results suggest a model in which regulation of tectal development by signals from the MHB is mediated via induction and subsequent auto-maintenance of Meis2 expression (Agoston, 2009).

Meis family proteins act as co-factors of other transcriptional regulators. To date, Meis-interacting proteins have been isolated from non-neuronal tissue and the posterior hindbrain, yet Meis co-factors in the developing anterior brain have remained elusive. GST pull-down experiments from tectal extracts or with in vitro translated proteins as well as co-immunoprecipitation experiments were performed with native tectal proteins to demonstrate direct interaction of Meis2 and Otx2 during early midbrain development. Using deletion constructs of Otx2, it was found that complex formation requires a short motif N-terminal of the Otx2 homeodomain. The region of the Otx2 polypeptide chain that contacts Meis2 thus differs from the tryptophan-containing hexapeptide that mediates cooperative DNA binding of Hox or myogenic bHLH proteins with TALE-homeodomain proteins. Meis family proteins can therefore interact with different protein motifs present in a variety of transcription factors (Agoston, 2009).

Employing an Otx2-dependent reporter assay, evidence was provided that Meis2 competes with the co-repressor Tle4 for binding to Otx2. Tle4 expression begins in the anterior primitive streak (thus preceding that of Meis2), is later strong in the anterior neural tube and decreases after the 20- to 25-somite stage. Tle4 binding to Otx2 was previously shown be required for the ability of Otx2 to repress Fgf8 anterior to the MHB, an important step in the formation and stabilization of the MHB organizer. Overexpression of Tle4 in the mesencephalic vesicle, in turn, disrupts normal development and lamination of the optic tecta. Together with the results presented in this study, these data might allow reconstruction of the probable temporal sequence of tectal fate specification in the embryo. In the anterior neural plate and anterior neural tube at early somite stages, Tle4 is co-expressed with Otx2 but Meis2 is missing. In the absence of Meis2, Otx2 and Tle4 can interact, prevent precocious tectal differentiation and inhibit Fgf8 expression anterior to the organizer, which stabilizes the MHB signaling center (Agoston, 2009).

Meis2 expression in the mesencephalic alar plate begins at HH11-12 (13-16 somites) and is strong from the 20- to 22-somite stage onwards, at which time the MHB organizer is established. Meis2 competes with Tle4 for binding to Otx2 in the tectal anlage, releases Otx2 from Groucho-mediated repression and thereby allows tectal development to commence (Agoston, 2009).

If correct, two predictions can be drawn from this model. First, loss of Tle4 from the diencephalic vesicle (where Tle4 is co-expressed with Otx2 at early somite stages) should lead to derepression of tectal genes. Second, precocious and ectopic expression of Meis2 in the MHB territory may destabilize the Fgf8 expression domain through premature restoration of Otx2 transcriptional activator function. Indeed, as previously demonstrated, transfection of a putative dominant-negative form of Tle4 - a truncation comprising only the first 203 amino acids of the protein - into the diencephalic vesicle causes widespread ectopic induction of En2 transcripts. In addition, when Meis2 was ectopically introduced into the MHB region at the 4- to 6-somite stage, small ectopic patches of Fgf8 transcripts anterior to the normal Fgf8 expression domain at the MHB were visible. These ectopic patches of Fgf8 expression might correspond to cells that have escaped Fgf8 downregulation by Otx2-Tle4 during the period of MHB organizer formation owing to the precocious inactivation of the Otx2-Tle4 complex by Meis2HA (Agoston, 2009).

Meis2 had to be transfected in excess to Tle4 in order to restore Otx2 transactivation in the Otx2-dependent reporter assays. This observation is consistent with the fact that between the 24- and 44-somite stages, Meis2 transcripts are abundant in the dorsal midbrain, whereas Tle4 expression is barely detectable. Tle4 thus appears to bind to Otx2 with higher affinity than does Meis2, which might allow for tight control over the tectum-inducing activity of Otx2. Recently, the spatial-temporal windows of Otx2 control over head, brain and body development were defined by Tamoxifen-induced deletion of Otx2. Interestingly, Otx2 deletion at E10.5-12.5 resulted in a mesencephalic-to-metencephalic fate change without shifting the molecular MHB. Hence, the interaction of Otx2 with Meis2 in the tectal anlage reported in this study occurs at a similar developmental stage to that at which Otx2 is required for mesencephalic fate determination but not MHB organizer positioning (Agoston, 2009).

Possible targets of Otx2/Meis2 include ephrin B1 (Efnb1) and Dbx1, both of which carry several potential consensus Bicoid- and Meis-binding sites upstream of their transcriptional start sites. Direct regulation of a midbrain-specific regulatory element of the EphA8 gene by Meis2 has also been demonstrated in mice. However, because Meis2 binding to Otx2 does not require either protein to be bound to DNA, Meis2-Otx2 interaction and restoration of Otx2 transcriptional activator function might in fact take place before both proteins have contacted the regulatory elements of downstream genes. Regulation of gene expression by putative transcription factors independent of DNA binding is not unprecedented. For instance, several Hox proteins can modulate gene expression by inhibiting the activity of CBP histone acetyltransferases (HATs) without forming DNA-binding complexes with CBP HAT. Meis family members might therefore affect gene expression by multiple, DNA-dependent and -independent mechanisms. This view is supported by the fact that despite the identification of Meis proteins as transcriptional co-factors, few direct target genes of these proteins have been reported to date (Agoston, 2009).

In summary, the results reported in this study strongly suggest that Tle4 and Meis2 compete for binding to Otx2 in the mesencephalic vesicle and that the balance between these proteins provides spatial and temporal control over the onset of tectal differentiation. Formation of spatially and temporally distinct higher order protein complexes involving Meis proteins and known regulators of neural patterning or fate determination might serve as a simple, yet versatile, mechanism to subdivide broad territories into smaller functional units during brain development (Agoston, 2009).

O-GlcNAc transferase is critical for TLE-mediated repression of canonical Wnt signaling

The Drosophila Groucho protein and its mammalian orthologues the transducin-like enhancers of split (TLEs) are critical transcriptional corepressors that repress Wnt and other signaling pathways. Although it is known that Groucho/TLEs are recruited to target genes by pathway-specific transcription factors, molecular events after the corepressor recruitment are largely unclear. This study, carried out in mammalian cultured cells, reports that association of TLEs with O-GlcNAc transferase, an enzyme that catalyzes posttranslational modification of proteins by O-linked N-acetylglucosamine, is essential for TLE-mediated transcriptional repression. Removal of O-GlcNAc from Wnt-responsive gene promoters is critical for gene activation from Wnt-responsive promoters. Thus, these studies identify a molecular mechanism by which Groucho/TLEs repress gene transcription and provide a model whereby O-GlcNAc may control distinct intracellular signaling pathways (Yang, 2014).

Molecular functions of the TLE tetramerization domain in Wnt target gene repression

Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of beta-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. This paper describes the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. Differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between β-catenin and TLE for TCFs as part of an activation-repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression (Chodaparambil, 2014).

groucho: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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