PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of mbcLink to Publisher's site
 
Mol Biol Cell. 2010 July 1; 21(13): 2315–2326.
PMCID: PMC2893994

Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

William P. Tansey, Monitoring Editor

Abstract

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators.

INTRODUCTION

The Runx transcription factors comprise a family of vital developmental regulators that participate in multiple pathways extending from pattern formation and sex determination in Drosophila to blood, bone, neural, and stomach development in mammals (Duffy and Gergen, 1994 blue right-pointing triangle; Komori, 2002 blue right-pointing triangle, 2003 blue right-pointing triangle; Lian et al., 2003 blue right-pointing triangle; de Bruijn and Speck, 2004 blue right-pointing triangle; Enomoto et al., 2004 blue right-pointing triangle; Ito, 2004 blue right-pointing triangle). Runx genes have been identified in all animals, with single gene family members in basal metazoans such as sponges and sea anemones, as well as in nematodes, the spider Cupiennius salei and in sea urchins (Damen et al., 2000 blue right-pointing triangle; Robertson et al., 2002 blue right-pointing triangle; Sullivan et al., 2008 blue right-pointing triangle). Most vertebrates contain three Runx family members (Levanon and Groner, 2004 blue right-pointing triangle; Ito, 2008 blue right-pointing triangle), whereas four family members are conserved in the genomes of arthropod insects such as Drosophila, mosquitoes, beetles, bees, and wasps (Bao and Friedrich, 2008 blue right-pointing triangle).

The hallmark of the Runx proteins is the highly conserved DNA-binding Runt domain (Kagoshima et al., 1993 blue right-pointing triangle). In addition to interacting with DNA, this domain also mediates interaction with a conserved and unrelated partner protein, referred to as CBFβ/Bro. The CBFβ/Bro proteins do not bind DNA or make DNA contacts as a component of the resulting heterodimeric complex, but instead stabilize a conformation of the Runt domain that has enhanced DNA-binding affinity (Tang et al., 2000 blue right-pointing triangle; Bravo et al., 2001 blue right-pointing triangle; Zhang et al., 2003 blue right-pointing triangle). The Runt domain has also been shown to mediate functional interactions with a variety of factors that are involved in transcription regulation, including other sequence specific DNA-binding proteins as well as non-DNA–binding cofactors (for review see Ito, 2004 blue right-pointing triangle).

A second conserved characteristic shared among Runx proteins is a C-terminal pentapeptide VWRPY motif that mediates interactions with the TLE/Gro family of corepressor proteins (Aronson et al., 1997 blue right-pointing triangle; Levanon et al., 1998 blue right-pointing triangle; Javed et al., 2000 blue right-pointing triangle, 2001 blue right-pointing triangle) In Drosophila, the Runt VWRPY motif is required for repressing specific stripes of expression of the pair-rule genes hairy (h) and even-skipped (eve; Aronson et al., 1997 blue right-pointing triangle). The VWRPY motif and Gro both also participate in the maintenance, but are not required for the initial establishment of Runt-dependent repression of the en segment- polarity gene (Wheeler et al., 2002 blue right-pointing triangle). In mammals, the VWRPY-TLE interaction is similarly involved in a subset of Runx protein functions. Although a Runx1 derivative lacking the VWRPY motif supports the development of hematopoietic progenitors from either embryonic stem cells or from fetal liver cells, in both cases there are defects in CD4 silencing and abnormal thymocyte development (Nishimura et al., 2004 blue right-pointing triangle; Kawazu et al., 2005 blue right-pointing triangle). Likewise, although the VWRPY motif is not required for the Runx3-dependent development of either sensory neurons or dendritic cells, there is a failure to properly regulate dendritic cell maturation in Runx3 [ΔVWRPY] mice (Yarmus et al., 2006 blue right-pointing triangle). The C-terminal VWRPY motif is conserved in basal metazoans, including the sea anemone Nematostella vectensis and the freshwater hydra Hydra magnipapillata (Sullivan et al., 2008 blue right-pointing triangle), indicating this is an ancient aspect of Runx protein function.

Functional studies with all three mammalian Runx proteins have identified regions outside of the Runt domain and the VWRPY motif that contribute to the regulatory activities of these proteins. This includes regions that mediate interactions with a number of other transcription factors and different coactivators and corepressors (for review see Ito, 2004 blue right-pointing triangle). All three mammalian Runx proteins contain regions C-terminal to the Runt domain that contribute to transactivation in cell-based transcription assays (Kanno et al., 1998 blue right-pointing triangle; Thirunavukkarasu et al., 1998 blue right-pointing triangle; Ito, 1999 blue right-pointing triangle; Pande et al., 2008 blue right-pointing triangle). Two other notable properties shared by these C-terminal regions are the unique ability to interact with both BMP-responsive as well as TGFβ-responsive SMAD proteins (Miyazono et al., 2004 blue right-pointing triangle), and a conserved nuclear matrix–targeting signal (NMTS; Zeng et al., 1997 blue right-pointing triangle). Interestingly, the Runx1-dependent repression of CD4 in thymocytes requires the NMTS, but appears to be independent of interactions with the Sin3 or Groucho/TLE corepressors (Telfer et al., 2004 blue right-pointing triangle). The NMTS is also important for the activity of the Runx2 and Runx3 proteins (Zaidi et al., 2001 blue right-pointing triangle; Pande et al., 2008 blue right-pointing triangle).

The goal of this work is to identify regions that contribute to the regulatory properties of the Drosophila Runt protein, the founding Runx family member. The runt gene was initially identified based on the pair-rule segmentation defects in mutant embryos (Nusslein-Volhard and Wieschaus, 1980 blue right-pointing triangle) and was subsequently found to participate in other developmental pathways in the fly, including sex determination and neurogenesis (Duffy and Gergen, 1991 blue right-pointing triangle; Duffy et al., 1991 blue right-pointing triangle; Kaminker et al., 2002 blue right-pointing triangle). As found for other Runx proteins, Runt is capable of either activating or repressing gene transcription in a context-dependent manner. These dual regulatory properties are exemplified by the parasegment-specific regulation of the slp1 segmentation gene. Activation of slp1 in the two posterior-most cells of each odd-numbered parasegment in the late blastoderm embryo requires Runt in concert with the Zn-finger transcription factor encoded by the pair-rule gene odd-paired (opa). These same two transcription factors are also expressed in adjacent cells that comprise the anterior half of the even-numbered parasegments at this stage, but in these cells the presence of the homeodomain transcription factor Fushi-tarazu (Ftz) converts Runt from an activator to a repressor of slp1 transcription (Swantek and Gergen, 2004 blue right-pointing triangle). Although these combinatorial rules are well established and can indeed be used to manipulate slp1 expression in all cells in the late blastoderm embryo in a Runt-dependent manner, the specific molecular requirements for Runt-dependent activation versus repression have yet to be elucidated.

Regions outside of the conserved Runt domain clearly contribute to the specificity of Runt function. Neither the mammalian Runx1 protein, nor Lozenge, a second Drosophila Runx family member are effective in altering the expression of different Runt target genes in ectopic expression assays in the early Drosophila embryo (Pepling and Gergen, 1995 blue right-pointing triangle; Tracey et al., 2000 blue right-pointing triangle). However, a chimeric protein that contains the Runt domain of mammalian Runx1 in the context of the flanking N- and C-terminal regions of the Drosophila protein mimics Runt function in these assays (Pepling and Gergen, 1995 blue right-pointing triangle). Taken together, these observations indicate that regions of Runt outside of the Runt domain are distinct from those of these two other Runx family members and that these regions are functionally relevant.

A previous alignment of runt sequences from Drosophila melanogaster, D. pseudoobscura, and D. virilis identified eight regions of high sequence homology that are likely to contribute to the regulatory functions of Runt, including of course the Runt domain and the C-terminal VWRPY motif (Pepling and Gergen, 1995 blue right-pointing triangle). In this article, an extended phylogenetic analysis reveals that these eight homology regions are maintained in other drosophilid species, but that conservation in some regions dissipates when the comparison is widened to include other insects. We investigate the importance of four of the most well-conserved regions for Runt function during Drosophila development. We find that deletion derivatives lacking these different conserved regions all retain activity in vivo, but with differential effects on different activities of Runt. Indeed, the results indicate that Runt's conserved C-terminus contributes to the activation, rather than the repression of slp1 and identify a distinct conserved module that is required for repression of this target. These findings provide compelling evidence for the modular architecture of the Runt transcription factor and lay groundwork for identifying the molecular interactions that contribute to the context-dependent regulatory properties of this protein.

MATERIALS AND METHODS

Drosophila Mutations and Runt Deletion Transgene Lines

The maternally expressed P{GAL4-nos.NGT} Gal4-drivers have been described previously (Tracey et al., 2000 blue right-pointing triangle; Wheeler et al., 2002 blue right-pointing triangle), as have the P{UAS-runt.T}232, P{UAS-runt.T}15, P{UAS-runt[CK].L}77, P{UAS-runt[Δ8].S}4-3, P{UAS-opa.VZ}10,P{UAS-opa.VZ}12, and P{UAS-opa.VZ}14 transgenic lines (Li and Gergen, 1999 blue right-pointing triangle; Tracey et al., 2000 blue right-pointing triangle; Wheeler et al., 2002 blue right-pointing triangle; Swantek and Gergen, 2004 blue right-pointing triangle). P{UAS-ftz}263 is a third chromosome-linked transgene obtained from Leslie Pick (University of Maryland) that is comparable in activity to the previously described second chromosome-linked P{UAS-ftz}261 (Swantek and Gergen, 2004 blue right-pointing triangle; Lohr and Pick, 2005 blue right-pointing triangle). The GroBX22, GroE48, and Rpd304456 mutations are also as described previously (Wheeler et al., 2002 blue right-pointing triangle).

Excite PCR was performed with primers to generate in-frame deletions of different conserved regions of Runt essentially as described for the previously generated Runt[CK], Runt[FLAG] and Runt[FLAGΔ8] expression constructs (Kramer et al., 1999 blue right-pointing triangle; Wheeler et al., 2000 blue right-pointing triangle). pB:Runt[FLAG-Δ3] was created using the primers 5′-GCCAAGTCCTCGGCCTCC-3′ and 5′-TCTTGGCTCCCGTGGCCCGTC-3′. pB:Runt[FLAG-Δ6] was created using the primers 5′-GTGGCGGATTACAAGGATGACG-3′ and 5′-CTGGGTGGGCGAGGAGCTGG-3′. (Bases that transcribe the FLAG epitope-tag are underlined.) pB:Runt [FLAG-Δ7] was created using the primers 5′-GGTCCTGGAGCGGTAGCC-3′ and 5′-CTTATCGTCGTCATCCTTGTAATC-3′. The products lack amino acids S233-L284, H410-D453, and P456-S476 of the normal protein, respectively. The deletions were confirmed by sequencing the plasmid and a StyI/BstEII fragment (Runt [Δ3]) or a BbsI/ApaI fragment (Runt [Δ6], Runt [Δ7]) spanning the deletion was cloned into pB:ED(Bam-8ΔKS)Runt[FLAG] and digested accordingly. The p[UAS:Runt[FLAGΔ3]], p[UAS:Runt[FLAGΔ6]], and p[UAS:Runt[FLAGΔ7]] germline transformation constructs were generated by insertion of BamHI fragments from the appropriate pB:Runt[FLAG] construct into BglII-linearized pUAS:T vector and sequenced to confirm the orientation. Transformant lines were recovered by standard P-element germline transformation. The UAS-Runt[Δ8]79 and UAS-Runt[Δ8]49 lines were obtained by P-element–mediated mobilization of the previously described and weaker UAS-Runt[Δ8]4-3 line (Wheeler et al., 2000 blue right-pointing triangle).

Immunofluorescence Detection of Protein Expression and Nuclear Localization

Salivary gland expression of the different Runt deletions was obtained by mating the appropriate UAS constructs with the salivary gland GAL4 driver P{w[+mc] = Sgs3-GAL4.PD}TP1 (Tweedie et al., 2009 blue right-pointing triangle). Larvae were grown at 18°C. Flat-bottom wells were blocked with PBT (phosphate-buffered saline [PBS] and 10.1% Tween) and 2% BSA an hour before larval salivary gland dissections in PBS at 4°C. The glands were fixed in PBS, 2%BSA, and 3% formaldehyde for 5 min; washed in PBT and 2% BSA; incubated with anti-FLAG M2 antibody (Sigma, St. Louis, MO) for 30 min; washed in PBT and 2% BSA; incubated with FITC-conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA) for 30 min; washed in PBT; and mounted in PBS, 50% glycerol, and 2% n-propyl gallate.

Viability Assay

Females homozygous for NGT11 or NGT40 were crossed to males heterozygous for each transformed UAS-Runt, UAS-Runt[CK], UAS-Runt[FLAG], UAS-Runt[Δ3], UAS-Runt[Δ6], UAS-Runt[Δ7], and UAS-Runt[Δ8] line. Viability measurements for transgenes on chromosome II were determined using males heterozygous for the UAS transgene and the CyO balancer. Viability measurements with transgenes on chromosome III were determined using males heterozygous for the UAS transgene and a chromosome carrying the dominant Pr and Dr mutations due to the reduced fitness of flies heterozygous for either the TM3 or TM6 third chromosome balancers. The relative viability is the percent of progeny that inherit the UAS transgene relative to their CyO (or Pr Dr) sibs, rounded to the nearest decile, except for values between 0 and 10%, which are rounded to the nearest fifth percentile.

Axonal Redirection

The MT14-Gal4 driver was used to drive expression of different UAS-Runt constructs in third-instar larval photoreceptor neurons R2 and R5 as described previously (Kaminker et al., 2002 blue right-pointing triangle). Homozygous MT14-Gal4 virgin females were mated to males homozygous for the different UAS-Runt transgenes, and larvae were grown at 18°C before dissection of third-instar eye imaginal disk optic lobe preparations. Axonal projections within the optic lobes were detected using a 1:50 dilution of 24B10 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), an mAb that recognizes the photoreceptor membrane protein chaoptin (Van Vactor et al., 1988 blue right-pointing triangle). The biotinylated secondary antibody was blocked with an acetone-washed powder of ground larvae. The signal was amplified and visualized using the Vectastain DAB kit (Vector Laboratories, Burlingame, CA).

Embryo Manipulation and In Situ Hybridization

Ectopic expression of the different UAS-Runt deletion derivatives was obtained using the maternally expressed NGT GAL4 drivers. Experiments investigating en repression involved matings between homozygous NGT40 females and the different UAS-Runt males. Initial experiments to screen for the ability of the different Runt deletions to perturb slp1 expression were carried out using females homozygous for both NGT40 and NGTA that produce slightly higher levels of ectopic expression. Experiments investigating the maternal dose-dependent effects of Gro and Rpd3 mutations on slp1 repression and activation involved matings between females heterozygous for these different mutations that were also heterozygous for NGT40 and NGTA to homozygous UAS-Runt15; UAS-Ftz263 males or to homozygous UAS-Runt15 UAS-Opa14 males, respectively. In this case the increased potency of UAS-Runt15 compensates for the reduced levels of maternally provided GAL4, giving a level of ectopic Runt expression that gives clear effects on slp1 expression while remaining in a range that is sensitive to changes in the levels of runt activity (Swantek and Gergen, 2004 blue right-pointing triangle). Embryos were collected and processed for in situ hybridization with digoxigenin-labeled (Boehringer Mannheim, Indianapolis, IN) anti-sense RNA probes for en and slp1 as described previously (Swantek and Gergen, 2004 blue right-pointing triangle).

RESULTS

Evolutionary Conservation of Runt in Drosophila

A comparison of runt sequences of D. melanogaster, D. pseudoobscura, and D. virilis previously identified eight blocks of high sequence homology that were separated by nonconserved spacers (Pepling and Gergen, 1995 blue right-pointing triangle). This observation was interpreted to reflect a modular architecture. Alignment of Runt sequences from 12 different Drosophila species reveals that the eight previously identified regions are present and for the most part intact in all of the species except for a clear divergence of regions IV and V and the N-terminal half of region VI in D. willistoni (Figure 1). Region III is the largest conserved block and encompasses the entire 128-amino acid Runt domain with conserved N-terminal and C-terminal extensions of 14 and 54 amino acids, respectively. The Runt domain sequence is identical in all 12 species, with perfect identity extending contiguously for 12 residues to the N-terminus and 39 residues to the C-terminus (Figure 1). The little variation that is observed in region III is consistent with the generally accepted phylogenetic relationships of these species. The entire 196-amino acid region is identical in all six species within the melanogaster group. The two species within the obscura group, D. pseudoobscura and D. persimilis are nearly the same but share the deletion of a single glutamine residue in the C-terminal extension. This same deletion is shared by the more distantly related species D. mojavenesis, D. virilis, and D. grimshawi, all three of which also share a nearby alanine-to-glutamine substitution.

Figure 1.
Conservation of the Runt protein in Drosophila. The figure shows a ClustalW2-generated alignment of Runt protein sequences from 12 different Drosophila species. The D. melanogaster amino acid sequence (single -letter code) is given at the top of each ...

Three of the regions outside of the Runt domain that were identified in the initial three-way alignment are not intact in D. willistoni. The 12-way alignment also reveals that four of the regions outside of the Runt domain are subject to a sequence interruption (Table 1). The one region identified in the initial alignment that clearly does not survive as a discrete functional module in the 12-way alignment is region IV. This region corresponds to a 57-residue, alanine- and proline-rich region of the D. melanogaster protein that also contained a single site of sequence disruption in the initial three-way alignment. Even excluding D. willistoni from the alignment, this region is disrupted by sequence breaks at four different positions that identify five distinctive smaller regions of homology, the largest of which is an alanine-rich sequence that is identical at 16 of 17 positions (excluding D. willistoni). Based on this divergence, region IV does not correspond to a single functional module that is under high selective pressure. In contrast, the conservation observed across the Drosophilidae for other regions, especially the contiguous blocks of very high sequence homology identifies regions of the Runt protein that are under selective pressure and thus likely to be functionally important.

Table 1.
Conserved regions of Runt proteins in Drosophila and other insects

Conservation of Runt in Nondrosophilid Insects

Runt protein sequences are available for nondrosophilid insects including the mosquitoes Aedes aegypti and Anopheles gambia, the silkworm Bombyx mori, the flour beetle Tribolium castaneum, the bee Apis mellifera and the wasp Nasonia vitripennis. We compared these sequences to the D. melanogaster sequence in order to determine whether the conserved regions observed in the Drosophilidae extended to other insects. There are substitutions at a total of 42 different positions in the Runt domain relative to that in Drosophila and even an amino acid insertion in Tribolium (not shown). There are seven amino acids that are conserved within the Runt domain of these other insects that differ from a residue in Drosophila. Five of these seven substitutions involve a replacement also found in the vertebrate Runx proteins.

None of the regions N-terminal to the Runt domain appear to be conserved in these other insects. Indeed, the nondrosophilid proteins are smaller than those in Drosophila and contain only short (6–25 amino acid) regions N-terminal to the Runt domain. Except for A. aegypti, the N-termini of the insect Runt proteins start with MHLP (data not shown). It is interesting to note that a similar sequence, MRIP is found at the N-terminus of the vertebrate Runx proteins.

There is evidence of sequence conservation C-terminal to the Runt domain. The two most prominent regions of conservation are the C-terminal extension of the Runt domain and the hallmark VWRPY motif. The wasp sequence is unusual in that there are an additional two amino acids that follow the VWRPY sequence. In addition to the VWRPY motif there are several other residues from region VIII that are conserved in other insects, including a lysine and a somewhat further upstream SP-TK(I/L) sequence (Figure 2). The conservation of the C-terminal extension to the Runt domain is more extensive spanning a region of 37–44 amino acids. The nine-residue segment immediately C-terminal to the Runt domain includes two amino acids that are identical in all of these species with conservative substitutions at most of the other positions (Figure 2). Perhaps even more striking is a 15-amino acid region that begins 13 amino acids C-terminal to the Drosophila Runt domain. This segment is demarcated by conserved tyrosine and phenylalanine residues and includes four other absolutely conserved positions (Figure 2). A similar architecture, with two short conserved motifs located immediately adjacent and some 20 amino acids C-terminal to the Runt domain is found in several vertebrate Runx proteins and is thought to modulate DNA-binding activity (Ito, 1999 blue right-pointing triangle). However there is no apparent sequence homology between the C-terminal Runt domain extensions of the vertebrate and insect proteins. In vertebrates this region is rich in basic and acidic residues, whereas the corresponding conserved regions of the insect proteins are predominantly hydrophobic, including aromatic residues at six positions and include only single charged lysine and aspartic acid residues.

Figure 2.
Conservation of RUNT C-terminal modules in other insects. The figure shows an alignment of the regions of Runt proteins from six nondrosophilid insects with corresponding intervals from conserved regions III, VI, VII, and VIII of the Drosophila proteins. ...

All of the insect proteins have sizable regions (from 134 to 238 amino acids) located between the region III and region VIII homologies noted above. The three subregions of region IV that are conserved in several Drosophila species are not evident in these other insects, although all of the proteins are rich in proline in the region downstream of region III (data not shown). The homology to region V is evident in the more closely related mosquito sequences, but not in the bee and the wasp, although all of the proteins have serine- and proline-rich regions N-terminal to a block of homology with region VI (data not shown). The homology to region VI is comprised of several acidic residues followed by a hydrophobic patch and a basic lysine or arginine, a feature of the block of sequence identity shared by the Drosophila Runt proteins (Figure 2). Region VII is less well conserved, although there is homology centered on the 12-amino acid block that is identical in the Drosophila proteins (Figure 2). Although the sequence divergence is greater when the analysis is extended to these other insects, the conservation that is observed for the C-terminal extension of the Runt domain as well as for regions VI, VII, and VIII provides further evidence that these regions are functionally important.

In Vivo Activity of Runt Derivatives Lacking Conserved Modules Outside the Runt Domain

Deletion derivatives were generated to investigate the functional importance of the four regions that showed evidence of conservation in nondrosophilid insects. Not surprisingly, deletion of the Runt domain eliminates in vivo function (Kramer et al., 1999 blue right-pointing triangle). Indeed, a mutation in the Runt domain that abrogates the interaction with the Bro protein fully eliminates Runt activity in a number of different in vivo assays (Li and Gergen, 1999 blue right-pointing triangle). In contrast, the Runt[CK] derivative, which contains two point mutations in the Runt domain (C127S, K199A) that perturb DNA-binding (Kramer et al., 1999 blue right-pointing triangle), retains the ability to establish repression of the segment-polarity gene en (Vander Zwan et al., 2003 blue right-pointing triangle), indicating DNA-binding independent activities of Runt. The role of the conserved C-terminal extension of the Runt domain was not tested in these previous experiments. Therefore we generated a deletion derivative, termed Runt[Δ3] that retains the Runt domain but that lacks this conserved C-terminal extension, from amino acids S233 to L284 inclusive (Figure 3). To test the functional importance of regions VI, VII, and VIII, we also generated deletions that remove the full extent of each of these conserved regions. All of the different deletion derivatives were generated in the context of a UAS-Runt expression construct that also contains a FLAG epitope tag inserted between amino acids A455 and P456, i.e., in the linker region between conserved regions VI and VII (Figure 3). Therefore we also generated a construct containing the FLAG epitope tag inserted in this same position in the full-length Runt protein as a control. These several different Runt derivatives were all inserted into the pUAS-T germline transformation vector, and transgenic lines were generated for each of the UAS-Runt constructs. The activities of the different deletions were examined in a number of different in vivo assays and compared with results obtained with the full-length wild-type protein as well as the DNA-binding defective Runt[CK] derivative.

Figure 3.
Runt deletion constructs. Schematic diagram indicating the regions removed in different UAS-Runt deletion constructs and the location of the FLAG epitope tag between conserved regions VI and VII. The solid horizontal line represents the Runt protein, ...

We used a GAL4 driver that is expressed in larval salivary glands to examine the effects of the different deletions on protein expression and subcellular localization. The Runt-FLAG protein and all four of the different deletions show accumulation within nuclei, indicating that none of the deletions dramatically affect protein stability or nuclear localization (Figure 4). The Runt-FLAG, Runt[Δ6], and Runt[Δ8] derivatives all show a punctate pattern within the nucleus similar to that described for the nuclear matrix-associated mammalian Runx proteins (Zeng et al., 1997 blue right-pointing triangle; Zaidi et al., 2001 blue right-pointing triangle). In contrast, the Runt[Δ3] and Runt[Δ7] proteins show more uniform expression throughout the nucleus (Figure 4, C and E). This result suggests these two regions contribute to subnuclear localization, potentially mediating association with the nuclear matrix. We have not examined whether the punctate expression observed in these salivary gland preparations corresponds to bona fide association with the nuclear matrix and further note that there is no obvious homology of either region III or region VII with the conserved NMTS of the vertebrate Runx proteins.

Figure 4.
Expression and nuclear localization of the Runt deletion derivatives. Expression of different Runt deletion constructs in larval salivary glands as detected by the anti-FLAG M2 mAb. (A–F) Background antibody control of salivary glands that are ...

We used ectopic expression assays to investigate the in vivo functional activity of the different deletion derivatives. As an initial test that also provides information on the relative strength of different insertions of the same UAS transgene constructs, we measured the lethality produced by ectopic expression at the blastoderm stage in response to maternally expressed GAL4 (Tracey et al., 2000 blue right-pointing triangle). All of the different UAS-Runt transgene insertions for every construct show evidence of lethality using the strong NGT40 maternal GAL4 driver (Table 2), indicating that all of the different deletion derivatives retain activity in vivo. As expected, there is less lethality in crosses using the NGT11 driver, which has ~40% of the activity of NGT40 (Tracey et al., 2000 blue right-pointing triangle), and males are more sensitive to the lethality associated with ectopic Runt expression. The differences in lethality obtained with different insertions of the same construct provide an indication of the relative expression levels and allowed us to identify strong representative lines for each construct for use in subsequent studies (bold in Table 2). In each case these representative lines are completely lethal when expressed using the NGT40 driver but produce escapers in crosses with NGT11.

Table 2.
Lethality produced by blastoderm stage expression of Runt[Δ] derivatives

Distinct Requirements for Runt-dependent Axonal Pathfinding

Ectopic expression assays have been previously used to demonstrate a role for Runt in regulating the targeting of photoreceptor axonal projections during development of the Drosophila eye. Adult flies have ~800 ommatidiae in each eye, with each ommatidia containing eight photoreceptor neurons, R1–R8. During the third-instar larval stage these photoreceptors differentiate from epithelial cells of the eye imaginal disk and extend axons into the optic lobe. The axons of photoreceptors R1 through R6 terminate in the optic lobe within a layer referred to as the lamina plexus (Figure 5A), a structure that can be visualized based on expression of the membrane associated chaoptin (Figure 5B). In contrast, the axons of the Runt-expressing R7 and R8 photoreceptors project past the lamina plexus and terminate in the medulla. Interestingly, ectopic expression of Runt in photoreceptors R2 and R5 is sufficient to redirect all photoreceptor axons to extend beyond the lamina into the medulla via a process that does not involve changes in expression of cell-specific developmental markers (Kaminker et al., 2002 blue right-pointing triangle). We used this ectopic expression assay to investigate the functional requirements for Runt in this process. The phenotypic consequence of ectopic Runt is disappearance of the lamina plexus due to extension of all photoreceptor axons into the medulla (Figure 5C). A similar loss of the lamina plexus is produced by ectopic expression of Runt[CK] and Runt[Δ7] (Figure 5, D and G), but not by the Runt[Δ3], Runt[Δ6], and Runt[Δ8] derivatives (Figure 5, E, F, and H). The ability of the Runt[CK] protein to redirect axonal projections provides another example of a DNA-binding–independent activity of Runt. The observation that conserved region VII is not required for axonal redirection, whereas the flanking regions VI and VIII contribute to function in this assay provides a clear indication of the distinct functional activities of these different conserved regions.

Figure 5.
Functional specificity of Runt-dependent axonal pathfinding. (A) Schematic of eye disk and optic lobe in Drosophila third-instar larvae. Axons of wild-type photoreceptor neurons R1–R6 terminate between the lamina and medulla of the optic lobe ...

Distinct Contributions of the Conserved Modules to Transcription Regulation by Runt

The two most well-characterized targets for transcriptional regulation by Runt are the segmentation genes en and slp1. Expression of en in odd-numbered parasegments is extremely sensitive to the NGT-driven expression of Runt in blastoderm stage embryos and is repressed by ectopic expression levels that do not affect expression of other pair-rule and segment-polarity genes (Wheeler et al., 2002 blue right-pointing triangle). As described for wild-type Runt, NGT-driven expression of the epitope-tagged Runt[FLAG] protein is effective at repressing the odd-numbered en stripes in a gastrula stage embryo (Figure 6C) and maintains this repression during germband extension (Figure 6D). A similar result is obtained in embryos ectopically expressing the Runt[Δ6] (Figure 6, I and J) and Runt[Δ7] (Figure 6, K and L) proteins, indicating that these conserved regions are not required for en repression. Previous studies demonstrated that the Runt-dependent repression of en is separable into two distinct steps: establishment and maintenance (Wheeler et al., 2002 blue right-pointing triangle). As expected from this previous work, the Runt[CK] and Runt[Δ8] proteins are capable of establishing repression (Figure 6, E and M) but do not maintain this repression during germband extension (Figure 6, F and N). This same phenotype is observed in embryos expressing the Runt[Δ3] protein (Figure 6, G and H). The failure of Runt[CK] and Runt[Δ8] to maintain repression was interpreted to indicate the importance of DNA-binding and the recruitment of Gro in this process (Wheeler et al., 2002 blue right-pointing triangle). The observation that Runt[Δ3] is impaired in maintaining en repression indicates that this conserved region may contribute to one or both of these molecular interactions. It is notable that all of the Runt deletions tested in these experiments retained the ability to establish the initial repression of en. This initial Runt-dependent repression involves genetic interactions with the transcriptional repressor Tramtrack (Ttk; Wheeler et al., 2002 blue right-pointing triangle). The results presented above indicate that none of the conserved regions of Runt tested in these assays is critically involved in this interaction.

Figure 6.
Differential requirements in establishing and maintaining en repression. Expression of en mRNA as revealed by in situ hybridization in gastrula stage (left column) and germband extension stage (right column) Drosophila embryos. (A and B) Wild-type expression ...

Runt acts as both an activator and a repressor of slp1 (Swantek and Gergen, 2004 blue right-pointing triangle). A striking observation from this previous work was the ability of Runt to activate slp1 in the anterior head region when it is coexpressed with the Zn-finger transcription factor Opa. This anterior activation of slp1 is unique among the segmentation genes and occurs in cells that normally do not express Runt and the other pair-rule transcription factors. This anterior activation assay thus provides a means for investigating the ability of the different Runt deletion constructs to activate slp1 independent of activity from the endogenous Runt protein. The anterior activation of slp1 that is evident in embryos with NGT-driven expression of wild-type Runt and Opa (Figure 7A) is also observed with the Runt[Δ6] and Runt[Δ7] derivatives (Figure 7, D and E), indicating that these two conserved regions are not required for Runt-dependent slp1 activation. In contrast, activation of slp1 is reduced in embryos expressing the Runt[CK], Runt[Δ3], and Runt[Δ8] proteins (Figure 7, B, C, and F). It is interesting to note that the functional requirements for slp1 activation match the requirements for maintenance of en repression, i.e., regions VI and VII are not required whereas the DNA-binding activity of Runt, the region immediately C-terminal to the Runt domain and the VWRPY-containing C-terminus of the protein contribute to both the activation of slp1 at the gastrula stage and the maintenance of en repression during germband extension. This raises the possibility that maintenance of en repression during these later stages is due to the inappropriate prior activation of slp1 in anterior odd-numbered parasegments, i.e., in cells that would normally express the odd-numbered en stripes. However, the levels of ectopic Runt needed to establish and maintain en repression are below those needed to activate slp1, indicating that the maintenance of en repression and the activation of slp1 are two distinct regulatory targets of Runt.

Figure 7.
Differential requirements for slp1 activation and repression. Expression of slp1 mRNA as revealed by in situ hybridization. (A–F) gastrula stage expression of slp1 in response to NGT-driven coexpression of Opa and different Runt deletion derivatives. ...

Runt is converted from an activator to a repressor of slp1 by the homeodomain transcription factor Ftz (Swantek and Gergen, 2004 blue right-pointing triangle). Endogenous Runt and Ftz are expressed in the anterior half of the even-numbered parasegments where both are required to prevent slp1 expression. Ectopic expression of Ftz alone leads to repression of slp1 in the posterior half of the odd-parasegments where endogenous Runt is also present. More important for this work is the observation that NGT-driven coexpression of Runt and Ftz also leads to repression of slp1 in the posterior half of the even-numbered parasegments, resulting in the elimination of expression throughout the presegmental region of the embryo (Figure 7G). This coexpression assay thus provides an approach to investigating the ability of different Runt deletion constructs to repress slp1 independent of the activity of endogenous Runt protein. Two of the deletions, Runt[Δ6] and Runt[Δ8], retain activity as repressors (Figure 7, J and L), whereas the Runt[CK], Runt[Δ3], and Runt[Δ7] proteins fail to repress the even-numbered slp1 stripes in this assay (Figure 7, H, I, and K). The observation that these last three Runt derivatives fail to repress slp1, yet are competent in the initial establishment of en repression at this stage (Figure 6, E, G, and K) indicates a clear distinction in the molecular requirements for Runt-dependent repression of these two targets. It was noted above that the Runt[Δ8] protein is effective at establishing en repression at the gastrula stage, but this repression is not maintained during germband extension (Figure 6, M and N). In contrast, the Runt[Δ8]-dependent repression of slp1 is maintained during these later stages (Figure 7R). This observation provides a further indication for differences in the Runt-dependent repression of en and slp1. There are some differences in early versus late slp1 expression in embryos that coexpress Ftz and the Runt[CK], Runt[Δ3], and Runt[Δ7] proteins (Figure 7, N, O, and Q). However, it is difficult to interpret these changes without examining the response of other Runt targets in these embryos.

The Role of Groucho and Rpd3 in slp1 Regulation

The results presented above indicating that Runt's conserved C-terminal region VIII has no apparent role in slp1 repression, but instead contributes to Runt-dependent slp1 activation are somewhat surprising. The Runt[Δ8] protein lacks the C-terminal VWRPY motif that mediates interaction with the corepressor protein Gro (Aronson et al., 1997 blue right-pointing triangle). The maintenance of Runt-dependent en repression is sensitive to the maternal dosage of Gro and the Gro-interacting histone deacetylase Rpd3 (Wheeler et al., 2002 blue right-pointing triangle). We used assays similar to those used in this previous work to investigate whether Gro and Rpd3 have roles in Runt-dependent slp1 regulation. NGT-driven coexpression of Runt and Ftz is as effective at repressing slp1 in embryos from females heterozygous for either the GroBX22 or GroE48 mutations as in embryos with wild-type Gro dosage (Figure 8, A–C). This result is consistent with observations above indicating that the Gro-interacting C-terminus is not required for slp1 repression. The maternal dosage of Rpd3 also has no effect on slp1 repression (Figure 8D).

Figure 8.
Runt-dependent slp1 regulation is insensitive to Gro and Rpd3 dosage. Expression of slp1 mRNA in response to NGT-driven coexpression of UAS-Runt15 and UAS-Ftz263 (A–D) or UAS-Runt15 and UAS-Opa14 (E–H) transgenes. (A) Sixteen of 20 gastrula ...

The more interesting question is whether the requirement for region VIII in slp1 activation reflects a role for Gro in this process. We used the anterior activation assay described above to investigate to investigate whether the dosage of either Gro or Rpd3 influences Runt-dependent slp1 activation. In this case the activation of slp1 in anterior head regions in response to NGT-driven coexpression of Runt and Opa is not reduced in embryos from females that are heterozygous for mutations in either Gro or Rpd3 (Figure 8, E–H). The extent of anterior activation obtained with the specific combination of NGT drivers and UAS-Runt and UAS-Opa lines used for these experiments is strong, but not maximal. Importantly, slight reductions (less than twofold) in the level of ectopic Runt activity results in significantly weaker anterior activation (Swantek and Gergen, 2004 blue right-pointing triangle). These results thus provide strong evidence that the Runt-dependent activation of slp1 is not sensitive to Gro dosage. On the basis of these observations, we conclude that the requirement for region VIII in slp1 activation does not involve interactions with Gro but instead should involve interactions of Runt's conserved C-terminus with other factors that are involved in transcriptional activation.

DISCUSSION

Functionally Distinct Roles for Different Conserved Regions of Runt

The experiments presented above use several different assays to investigate the functional contributions of different conserved regions of Runt. Each of the four deletion derivatives affects a different set of properties (Table 3). The functional specificity demonstrated by the different deletions is consistent with the notion that different conserved regions correspond to functional modules that participate in distinct molecular interactions of the Runt protein. It is interesting to note that similar patterns of functional requirements for Runt are observed for two different pairs of assays. The observations that NGT-driven expression of all of the different Runt constructs is lethal and that all of the constructs also retain the ability to establish en repression are consistent with the idea that this initial repression of en is the principal basis for the lethality associated with ectopic Runt expression. Perhaps more interesting are the common functional deficits of the Runt[CK], Runt[Δ3], and Runt[Δ8] proteins in both slp1 activation and the maintenance of en repression (Table 3). As mentioned above, the levels of Runt needed to maintain en repression in anterior odd parasegments are less than those needed for activation of slp1 in these same cells, indicating that these are two distinct targets that have coincidental requirements for DNA-binding by Runt, the conserved region C-terminal to the Runt domain and the VWRPY-containing C-terminus.

Table 3.
Functional specificity of Runt's conserved regions

The inactivity of Runt[CK] in maintaining en repression and in both the activation and repression of slp1 strongly suggests that DNA binding by Runt is critical for these three aspects of Runt function. Runt[Δ3], which is also defective for these same three functions lacks a conserved region that is located just C-terminal of the DNA-interacting “tail region” loop of the Runt domain (Bravo et al., 2001 blue right-pointing triangle). Intramolecular interactions with regions C-terminal to the Runt domain of Runx1 modulate DNA binding in vitro (Kagoshima et al., 1996 blue right-pointing triangle; Kanno et al., 1998 blue right-pointing triangle) and can influence cooperative interactions with other DNA-binding factors (Gu et al., 2000 blue right-pointing triangle). Our results are consistent with the idea that the region C-terminal to the Runt domain makes important contributions to the in vivo DNA-binding activity of the Runt transcription factor.

Runt[Δ8] is similar to Runt[CK] and Runt[Δ3] in that it is defective in maintaining en repression and in activating slp1. A key difference between these three is the ability of the Runt[Δ8] to repress slp1. This result indicates that the C-terminal VWRPY motif is not required for slp1 repression, a finding also consistent with the results of our genetic experiments indicating that Runt-dependent slp1 repression is insensitive to Gro dosage. The observation that conserved region VIII contributes to slp1 activation is somewhat surprising, especially given the previously documented physical interaction between the C-terminal VWRPY motif and the Gro corepressor (Aronson et al., 1997 blue right-pointing triangle). On the basis of these results, we propose that conserved region VIII also mediates interactions with a separate factor that participates in transcription activation. One issue is whether this proposed interaction also involves the VWRPY motif. Conserved region VIII contains two contiguous blocks of sequence conservation separated by a variable linker, suggesting that the proposed interaction with an activator may involve these other conserved amino acids. In any event, the relatively compact size of region VIII makes it likely that interactions with Gro and the proposed cofactor involved in Runt-dependent activation will be mutually exclusive.

The unique activities of Runt[Δ6] and Runt[Δ7] underscore the modular architecture and context-dependent activities of Runt. These two conserved regions are separated by two amino acids in the five species that comprise the melanogaster subgroup (Figure 1). In the set of assays we used, the only activity that is disrupted by deletion of region VI is the redirection of axonal projections in the developing eye (Table 3). Region VII is not required for this activity of Runt, but appears to contribute to the punctate subnuclear localization of Runt and is required for slp1 repression. These last two properties are shared with Runt[Δ3]. Nuclear matrix association of Runx1 is important for CD4 repression (Telfer et al., 2004 blue right-pointing triangle), suggesting a potential parallel in the mechanisms of slp1 repression by Runt and the repression of CD4 by Runx1. The repression of slp1 by Runt also requires the activity of the homeodomain protein Ftz as well as the Ftz-interacting orphan nuclear receptor protein Ftz-F1 (Swantek and Gergen, 2004 blue right-pointing triangle; Hou et al., 2009 blue right-pointing triangle). It is reasonable to propose that regions III and VII are involved in molecular interactions with Ftz, Ftz-F1 and/or the nuclear matrix that are involved in converting Runt from an activator to a repressor of slp1 transcription. Taken all together, our results provide compelling evidence for the functional modularity of Runt and lay groundwork for identifying molecular interactions that contribute to the regulatory properties of this conserved family of transcriptional regulators.

ACKNOWLEDGMENTS

Xiangqun Ning played an important role in initiating functional studies on the different conserved regions of Runt. Comments from Xiaoling Wang and other members of the Gergen lab are appreciated. This research was supported by grants from the National Science Foundation (MCB 0344486 and MCB 0721430). We dedicate this work to the memory of Kevin King.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-11-0953) on May 12, 2010.

REFERENCES

  • Aronson B. D., Fisher A. L., Blechman K., Caudy M., Gergen J. P. Groucho-dependent and -independent repression activities of Runt domain proteins. Mol. Cell. Biol. 1997;17:5581–5587. [PMC free article] [PubMed]
  • Bao R., Friedrich M. Conserved cluster organization of insect Runx genes. Dev. Genes Evol. 2008;218:567–574. [PubMed]
  • Bravo J., Li Z., Speck N. A., Warren A. J. The leukemia-associated AML1 (Runx1)–CBF beta complex functions as a DNA-induced molecular clamp. Nat. Struct. Biol. 2001;8:371–378. [PubMed]
  • Damen W.G.M., Weller M., Tautz D. Expression patterns of hairy, even-skipped and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod. Proc. Natl. Acad. Sci. USA. 2000;97:4515–4519. [PubMed]
  • de Bruijn M. F., Speck N. A. Core-binding factors in hematopoiesis and immune function. Oncogene. 2004;23:4238–4248. [PubMed]
  • Duffy J. B., Gergen J. P. The Drosophila segmentation gene runt acts as a position-specific numerator element necessary for the uniform expression of the sex-determining gene Sex-lethal. Genes Dev. 1991;5:2176–2187. [PubMed]
  • Duffy J. B., Gergen J. P. Sex, segments, and the central nervous system: common genetic mechanisms of cell fate determination. Adv. Genet. 1994;31:1–28. [PubMed]
  • Duffy J. B., Kania M. A., Gergen J. P. Expression and function of the Drosophila gene runt in early stages of neural development. Development. 1991;113:1223–1230. [PubMed]
  • Enomoto H., Furuichi T., Zanma A., Yamana K., Yoshida C., Sumitani S., Yamamoto H., Enomoto-Iwamoto M., Iwamoto M., Komori T. Runx2 deficiency in chondrocytes causes adipogenic changes in vitro. J. Cell Sci. 2004;117:417–425. [PubMed]
  • Gu T. L., Goetz T. L., Graves B. J., Speck N. A. Auto-inhibition and partner proteins, core-binding factor beta (CBFbeta) and Ets-1, modulate DNA binding by CBFalpha2 (AML1) Mol. Cell. Biol. 2000;20:91–103. [PMC free article] [PubMed]
  • Hou H. Y., Heffer A., Anderson W. R., Liu J., Bowler T., Pick L. Stripy Ftz target genes are coordinately regulated by Ftz-F1. Dev. Biol. 2009;335:442–453. [PMC free article] [PubMed]
  • Ito Y. Molecular basis of tissue-specific gene expression mediated by the runt domain transcription factor PEBP2/CBF. Genes Cell. 1999;4:685–696. [PubMed]
  • Ito Y. Oncogenic potential of the RUNX gene family: “overview.” Oncogene. 2004;23:4198–4208. [PubMed]
  • Ito Y. RUNX genes in development and cancer: regulation of viral gene expression and the discovery of RUNX family genes. Adv. Cancer Res. 2008;99:33–76. [PubMed]
  • Javed A., Barnes G. L., Jasanya B. O., Stein J. L., Gerstenfeld L., Lian J. B., Stein G. S. runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins. Mol. Cell. Biol. 2001;21:2891–2905. [PMC free article] [PubMed]
  • Javed A., et al. Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription. J. Cell Sci. 2000;113:2221–2231. [PubMed]
  • Kagoshima H., Akamatsu Y., Ito Y., Shigesada K. Functional dissection of the alpha and beta subunits of transcription factor PEBP2 and the redox susceptibility of its DNA binding activity. J. Biol. Chem. 1996;271:33074–33082. [PubMed]
  • Kagoshima H., Shigesada K., Satake M., Ito Y., Miyoshi H., Ohki M., Pepling M., Gergen P. The Runt domain identifies a new family of heteromeric transcriptional regulators. Trends Genet. 1993;9:338–341. [PubMed]
  • Kaminker J. S., Canon J., Salecker I., Banerjee U. Control of photoreceptor axon target choice by transcriptional repression of Runt. Nat. Neurosci. 2002;5:746–750. [PubMed]
  • Kanno T., Kanno Y., Chen L. F., Ogawa E., Kim W. Y., Ito Y. Intrinsic transcriptional activation-inhibition domains of the polyomavirus enhancer binding protein 2/core binding factor alpha subunit revealed in the presence of the beta subunit. Mol. Cell. Biol. 1998;18:2444–2454. [PMC free article] [PubMed]
  • Kawazu M., et al. Functional domains of Runx1 are differentially required for CD4 repression, TCRbeta expression, and CD4/8 double-negative to CD4/8 double-positive transition in thymocyte development. J. Immunol. 2005;174:3526–3533. [PubMed]
  • Komori T. [Cbfa1/Runx2, an essential transcription factor for the regulation of osteoblast differentiation] Jpn. J. Clin. Med. [Nippon Rinsho] 2002;60(suppl 3):91–97. [PubMed]
  • Komori T. Requisite roles of Runx2 and Cbfb in skeletal development. J. Bone Mineral Metab. 2003;21:193–197. [PubMed]
  • Kramer S. G., Jinks T. M., Schedl P., Gergen J. P. Direct activation of Sex-lethal transcription by the Drosophila runt protein. Development. 1999;126:191–200. [PubMed]
  • Levanon D., Goldstein R. E., Bernstein Y., Tang H., Goldenberg D., Stifani S., Paroush Z., Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc. Natl. Acad. Sci. USA. 1998;95:11590–11595. [PubMed]
  • Levanon D., Groner Y. Structure and regulated expression of mammalian RUNX genes. Oncogene. 2004;23:4211–4219. [PubMed]
  • Li L. H., Gergen J. P. Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye. Development. 1999;126:3313–3322. [PubMed]
  • Lian J. B., et al. Runx1/AML1 hematopoietic transcription factor contributes to skeletal development in vivo. J. Cell. Physiol. 2003;196:301–311. [PubMed]
  • Lohr U., Pick L. Cofactor-interaction motifs and the cooption of a homeotic Hox protein into the segmentation pathway of Drosophila melanogaster. Curr. Biol. 2005;15:643–649. [PubMed]
  • Miyazono K., Maeda S., Imamura T. Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins. Oncogene. 2004;23:4232–4237. [PubMed]
  • Nishimura M., Fukushima-Nakase Y., Fujita Y., Nakao M., Toda S., Kitamura N., Abe T., Okuda T. VWRPY motif-dependent and -independent roles of AML1/Runx1 transcription factor in murine hematopoietic development. Blood. 2004;103:562–570. [PubMed]
  • Nusslein-Volhard C., Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature. 1980;287:795–801. [PubMed]
  • Pande S., et al. Subnuclear targeting of the Runx3 tumor suppressor and its epigenetic association with mitotic chromosomes. J. Cell. Physiol. 2008;218:473–479. [PMC free article] [PubMed]
  • Pepling M. E., Gergen J. P. Conservation and function of the transcriptional regulatory protein Runt. Proc. Natl. Acad. Sci. USA. 1995;92:9087–9091. [PubMed]
  • Robertson A. J., Dickey C. E., McCarthy J. J., Coffman J. A. The expression of SpRunt during sea urchin embryogenesis. Mech. Dev. 2002;117:327–330. [PubMed]
  • Sullivan J. C., Sher D., Eisenstein M., Shigesada K., Reitzel A. M., Marlow H., Levanon D., Groner Y., Finnerty J. R., Gat U. The evolutionary origin of the Runx/CBFbeta transcription factors–studies of the most basal metazoans. BMC Evol. Biol. 2008;8:228. [PMC free article] [PubMed]
  • Swantek D., Gergen J. P. Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription. Development. 2004;131:2281–2290. [PubMed]
  • Tang Y. Y., Crute B. E., Kelley J. J., Huang X., Yan J., Shi J., Hartman K. L., Laue T. M., Speck N. A., Bushweller J. H. Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA. FEBS Lett. 2000;470:167–172. [PubMed]
  • Telfer J. C., Hedblom E. E., Anderson M. K., Laurent M. N., Rothenberg E. V. Localization of the domains in Runx transcription factors required for the repression of CD4 in thymocytes. J. Immunol. 2004;172:4359–4370. [PubMed]
  • Thirunavukkarasu K., Mahajan M., McLarren K. W., Stifani S., Karsenty G. Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta. Mol. Cell. Biol. 1998;18:4197–4208. [PMC free article] [PubMed]
  • Tracey W. D., Jr, Ning X., Klingler M., Kramer S. G., Gergen J. P. Quantitative analysis of gene function in the Drosophila embryo. Genetics. 2000;154:273–284. [PubMed]
  • Tweedie S., et al. FlyBase: enhancing Drosophila Gene Ontology annotations. Nucleic Acids Res. 2009;37:D555–D559. [PMC free article] [PubMed]
  • Van Vactor D., Jr, Krantz D. E., Reinke R., Zipursky S. L. Analysis of mutants in chaoptin, a photoreceptor cell-specific glycoprotein in Drosophila, reveals its role in cellular morphogenesis. Cell. 1988;52:281–290. [PubMed]
  • Vander Zwan C. J., Wheeler J. C., Li L. H., Tracey W. D., Gergen J. P. A DNA-binding-independent pathway of repression by the Drosophila Runt protein. Blood Cells Mol. Dis. 2003;30:207–222. [PubMed]
  • Wheeler J. C., Shigesada K., Gergen J. P., Ito Y. Mechanisms of transcriptional regulation by Runt domain proteins. Semin. Cell Dev. Biol. 2000;11:369–375. [PubMed]
  • Wheeler J. C., VanderZwan C., Xu X., Swantek D., Tracey W. D., Gergen J. P. Distinct in vivo requirements for establishment versus maintenance of transcriptional repression. Nat. Genet. 2002;29:29. [PubMed]
  • Yarmus M., Woolf E., Bernstein Y., Fainaru O., Negreanu V., Levanon D., Groner Y. Groucho/transducin-like Enhancer-of-split (TLE)-dependent and -independent transcriptional regulation by Runx3. Proc. Natl. Acad. Sci. USA. 2006;103:7384–7389. [PubMed]
  • Zaidi S. K., Javed A., Choi J. Y., van Wijnen A. J., Stein J. L., Lian J. B., Stein G. S. A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene. J. Cell Sci. 2001;114:3093–3102. [PubMed]
  • Zeng C., et al. Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-alpha transcription factors. Proc. Natl. Acad. Sci. USA. 1997;94:6746–6751. [PubMed]
  • Zhang L., Li Z., Yan J., Pradhan P., Corpora T., Cheney M. D., Bravo J., Warren A. J., Bushweller J. H., Speck N. A. Mutagenesis of the Runt domain defines two energetic hot spots for heterodimerization with the core binding factor beta subunit. J. Biol. Chem. 2003;278:33097–33104. [PubMed]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology