A DNA-binding-independent pathway of repression by the Drosophila Runt protein

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A DNA-binding-independent pathway of repression by the Drosophila Runt protein Christine J. Vander Zwan,1 John C. Wheeler,3 Ling-Hui Li,2,4 William D. Tracey Jr.,1,5 and J. Peter Gergen* Department of Biochemistry and Cell Biology and the Center for Developmental Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794-5140, USA Submitted 6 January 2003 (Communicated by M. Lichtman, M.D., 7 January 2003)

Abstract DNA-binding proteins are important for regulating gene expression during development. It is widely assumed that this regulation involves sequence-specific DNA binding by these transcription factors to cognate cis-regulatory sequences of their downstream target genes. However, studies in both the Drosophila and the mouse model systems have provided examples in which the DNA-binding activity of a transcription factor is not essential for in vivo function. Using a system that allows for quantitative analysis of gene function in the Drosophila embryo, we have discovered a DNA-binding-independent activity of Runt, the founding member of the RUNX family of transcriptional regulators. Examination of the in vivo potency of a DNA-binding-defective form of Runt reveals differential requirements for DNA binding in the regulation of different downstream target genes. DNA binding is not required for establishing repression of the odd-numbered stripes of the segment polarity gene engrailed, but does contribute to Runt’s role as a regulator of sloppy-paired, another downstream target gene in the pathway of segmentation. We investigate this DNA-binding-independent pathway using a genetic screen for dose-dependent modifiers of runt activity. These studies reveal that DNA-binding proteins encoded by the tramtrack locus cooperate with Runt to repress engrailed. These results provide new insights into the context-dependent regulatory functions of Runt domain proteins and provide a paradigm for understanding DNA-binding-independent regulation by developmentally important transcription factors. © 2003 Elsevier Science (USA). All rights reserved. Keywords: RUNX; Tramtrack; Engrailed; Sloppy-paired; Segmentation

Introduction Gene expression during development is frequently regulated at the level of transcription initiation. It is generally accepted that interactions between sequence-specific DNAbinding transcription factors and their cognate cis-regula* Corresponding author. Fax: ⫹1-631-632-8575. E-mail address: [email protected] (J.P. Gergen). 1 Graduate Program in Genetics. 2 Graduate Program in Molecular and Cellular Biology. 3 Current address: The Rothberg Institute for Childhood Diseases, Guilford, CT 06437, USA. 4 Current address: Institute of Genetics, National Yang-Ming University, 112 Taipei, Taiwan. 5 Current address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

tory targets are central to the assembly of an active RNA polymerase complex at the initiation site in both prokaryotic and eukaryotic systems. However, more than one observation indicates that there may be exceptions to this simple view of transcriptional regulation. Krause and co-workers demonstrated that a version of the Drosophila Fushi-tarazu (Ftz) protein that was deleted for the conserved homeodomain retained the ability to regulate target genes in vivo [1]. More recently, work on mammalian transcription factors, including the glucocorticoid receptor and the bHLH protein SCL, indicates that the DNA-binding activities of these two important developmental regulators are not essential for function in vivo [2,3]. These results strongly suggest that these transcription factors can regulate transcription by mechanisms that do not involve DNA binding.

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RUNX proteins comprise a small family of developmental regulators that are identified by a highly conserved DNA binding domain, termed the Runt domain [4]. RUNX genes have been identified in all animal species examined, including runt and lozenge (lz) in Drosophila, three different RUNX genes in mammals, the RUNX1 orthologue Xaml in Xenopus, SpRunt in sea urchins, Cs-runt in the spider, Runxa and Runxb in zebrafish, and run in Caenorhabditis elegans [4 –10]. runt, the founding member of this family, has vital roles in several developmental pathways during Drosophila embryogenesis [11–13]. The lz gene was initially identified due to its role in eye development, but has additional functions including a role in blood development [6,14]. Mutations in Runx1 and Runx2 are associated with disease in humans [15–18], and targeted mutagenesis experiments in the mouse indicate that these genes have vital roles in the pathways of hematopoiesis and osteogenesis, respectively [19,20]. A unifying aspect of RUNX protein function in these many different developmental pathways is a role in cell fate specification, presumably by transcriptional regulation. Several lines of experimental evidence indicate that RUNX proteins directly participate in regulating transcription. The initial purification of the mammalian RUNX proteins was based on their biochemical interaction with cisregulatory DNA sequences in the Moloney murine leukemia virus and polyoma DNA tumor virus core enhancers [21,22]. Binding sites for RUNX proteins have now been identified in a large number of putative target genes, and in several cases these sites have been demonstrated to mediate RUNX-dependent transcriptional regulation [23–27]. In the pre-blastoderm Drosophila embryo, Runt works in cooperation with other sequence-specific DNA-binding proteins in a dose-dependent manner to trigger the sex-specific transcriptional activation of the Sex-lethal gene [28]. At a slightly later stage, runt regulates the transcription of numerous downstream target genes in the developmental pathway of segmentation [29 –34]. Recent work demonstrates a direct role for Lz in regulating the transcription of other genes during eye development [6,35]. An intriguing aspect of transcriptional regulation by Runt domain proteins is that they function both as activators or repressors, depending on the specific target gene and the developmental context. The mechanisms used to achieve this context-dependent specificity are not understood. The most significant conserved feature amongst RUNX proteins is the 128-amino-acid Runt domain. Runt domains between vertebrates and Drosophila share nearly 70% sequence identity, and almost all of the amino acid substitutions are conservative, suggesting that this conserved domain is central to the regulatory functions of these proteins [4]. Two different biochemical activities have been attributed to the Runt domain. In addition to sequence-specific DNA binding, the Runt domain also mediates interaction with an unrelated protein, CBF␤ in mammals, or the homologous Brother (Bro) and Big Brother (Bgb) proteins in

Drosophila. The CBF␤, Bro, and Bgb partner proteins do not bind to DNA themselves, but instead induce conformational changes that are associated with an increase in DNAbinding affinity by the Runt domain–partner protein complex [36 – 41]. The functional conservation of the Runt domain is further demonstrated by the observation that murine CBF␤ enhances in vitro DNA binding by the Drosophila Runt protein and by the finding that a chimeric Runt protein containing a mammalian Runt domain is functional in vivo [42]. Here we investigate the importance of the DNA-binding activity of the Runt domain for transcriptional regulation by Runt in the Drosophila embryo. Studies on the mammalian RUNX proteins have identified two conserved amino acids in the Runt domain that are specifically required for DNA binding. Mutation of either of these residues abolishes protein activity in reporter gene transactivation experiments in cell culture, indicating that DNA binding is critical in these assays [38,43]. Mutation of the analogous two residues in Runt interferes with DNA binding in vitro and abrogates Runt-mediated activation of Sxl [28]. We have further examined the activity of this DNA-binding-defective form of Runt, referred to here as Runt[CK], using a genetic system that allows for quantitative analysis of gene function in the Drosophila blastoderm embryo [30]. Surprisingly, we find that the Runt[CK] protein retains significant in vivo activity. Comparison of the relative potencies of the wild-type Runt[⫹] protein versus the Runt[CK] protein reveals differential requirements for DNA binding in the regulation of different downstream target genes. Both proteins are potent repressors of the odd-numbered stripes of the segment polarity gene engrailed (en). In contrast, the Runt[CK] protein is less effective than the Runt[⫹] protein in regulating expression of sloppy-paired (slp), another sensitive downstream target in the segmentation pathway. We present evidence that the DNA-binding-independent repression of en by Runt is a normal component of the segmentation gene network. Finally, we take advantage of the genetic tools available in the Drosophila system to design a screen for factors that cooperate with Runt to repress en. Using this approach, we find that maternal dosage of the tramtrack (ttk) locus contributes to the DNA-binding-independent repression of en by Runt. These results suggest a model whereby the DNA-binding activity of the Ttk proteins provides DNA-binding specificity and Runt’s role in establishing transcriptional repression of en is mediated through protein–protein interactions.

Materials and methods Drosophila stocks The pUAS-Runt[CK] plasmid for P-element-mediated germline transformation was generated by replacement of a BglII fragment of pUAS-Runt with an analogous fragment from pB:Runt[CK] that contains the C127S and K199A

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mutations [28]. Transformants carrying this transgene were established by standard protocols. Three different UASrunt[CK] lines (2, 43, and 77) and two different UASrunt[⫹] lines (14 and 232) are used in this report. The UAS-dpp/TM3 strain (P{UAS-dpp.S}) and UAS-lacZ strain (P{UAS-lacZ.B}4-1-2) are as previously described [30]. The control third chromosome strain used in the genetic screen, ss e ro, was obtained from the Bloomington Drosophila Stock Center and isogenized by standard genetic methods. Strains representing the “Deficiency Kits” and the ttk mutations ttkle11/TM3, ttk1/TM6B, and ttk02667/TM3 were obtained from the Bloomington Drosophila Stock Center. Stocks carrying the ttkD2-50, ttkB330, ttk10556, ttk3546, ttkrM730, and ttkosn mutations were obtained from C. Kla¨ mbt (U. Ko¨ ln). The runtLB5 and runtYP17 mutations and the runt⫹ Y chromosome duplication, y⫹ Y mal102, are as previously described [11,44]. NGT40 is a germline transformant strain, P{w[⫹mC] Scer⶿GAL4[nos.PG] ⫽ GAL4-nos.NGT}, containing the GAL4 coding region fused to the nanos promoter, followed by the 3⬘ UTR of an ␣-tubulin gene. Females homozygous for the NGT40 chromosome drive accumulation of approximately 125,000 molecules of ␤-galactosidase per cell in late blastoderm stage embryos that carry one copy of the UAS-lacZ4-1-2 transgene [30]. A related strain, NGTA, was generated by mobilization of the same transposon onto the third chromosome. The NGTA driver has 50% the activity of NGT40 based on enzyme activity measurements of extracts from embryos with the UAS-lacZ transgene. Thus, the combined NGT40; NGTA strain is 1.5-fold more active than the homozygous NGT40 strain and approximately 3-fold more active than NGT40 heterozygotes. Quantification of RNA levels The relative expression levels of different UAS-runt[⫹] and UAS-runt[CK] transgenes were determined by RNase protection. Two- to 4-h old embryos were collected at 25°C from crosses of homozygous NGT40 females and homozygous UAS-runt[⫹]14, UAS-runt[⫹]232, UAS-runt[CK]2, UAS-runt[CK]77, and UAS-runt[CK]43 males, respectively. Total RNA was isolated using a modification of a procedure for preparing adult fly RNA [45]. Preparation of RNA probes and the RNase protection assay followed established protocols. An anti-sense radiolabeled RNA probe corresponding to the 5⬘ region of the runt mRNA, which includes 71 nucleotides which are not contained in the UAS-runt transgenes, was synthesized from a pB:E25-5⬘(⌬NotI) template [46]. A total of 5 to 10 ␮g of total RNA was used in each hybridization reaction. After RNase treatment, the samples were loaded on a denaturing 6% polyacrylamide gel. For quantification, the autoradiograph of the gel was analyzed using the ImageQuant program (Molecular Dynamics). The RNA expression level of each transgenic fly line was determined relative to the endogenous runt gene and these ratios were normalized using the UAS-runt[⫹]232

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Table 1 Expression levels of UAS-runt transgenic lines Transgenic line runt runt runt runt runt

[⫹]14 [⫹]232 [CK]2 [CK]77 [CK]43

Relative RNA levela 0.52 ⫾ 0.01 1.00 ⫾ 0.03 0.66 ⫾ 0.01 1.48 ⫾ 0.33 2.58 ⫾ 0.28

a Embryos from homozygous NGT40 females carrying each of the indicated UAS trangenes were collected and total RNA was isolated for use in RNase protection assays as described previously [46]. The RNA expression level was determined relative to the endogenous runt gene and these ratios were normalized using UAS-runt[⫹]232 as a standard. The values given are the means of two to three independent experiments, each done in duplicate, ⫾ the standard deviation.

as a standard. Values shown are means of two to three independent experiments, each done in duplicate (Table 1). Genetics and phenotypic analysis Cuticle preparations were done on un-hatched embryos collected 24 h after egg-laying according to established procedures. To analyze gene expression patterns, embryos were collected for 2– 4 h and fixed, dehydrated, and stored in ethanol until they were subjected to standard in situ hybridization [47]. The probe used to detect slp expression was generated from a 600-base-pair PCR fragment cloned into the BamHI site of pBluescript(KS⫹) from Michael Weir (Wesleyan University). An anti-sense RNA probe was generated using T7 polymerase on this pB:slp1 plasmid that was linearized with EcoRI. Probes for en and other segmentation genes have been described previously [31]. To examine gene expression in embryos ectopically expressing Runt, NGT females were mated to homozygous UAS-runt males. Embryos were collected from such crosses at 25°C aged for 2– 4 h and then fixed and stored for in situ hybridization. To examine the effects of wild-type runt gene dosage on en expression, males with the genotype y w runtLB5/y⫹Ymal102 were mated to homozygous y w (runt⫹) females. All male embryos from this cross inherit the Y chromosome duplication and thus have twice the normal dosage of runt. In contrast, all female embryos from this cross are heterozygous for the runtLB5 null mutation and have only half the normal level of runt activity. The temperature-sensitive runtYP17 allele was used to investigate the temporal requirements for runt activity in segmentation. Heterozygous runtYP17/FM7 females were crossed to FM7/Y males. Cohorts of embryos were collected at 25°C for 60 min and were then shifted to the permissive temperature of 18°C and allowed to age for 4.5 h, at which point the embryos were shifted to 30°C. After 30 min of further development at this non-permissive temperature the embryos were immediately fixed and stored in ethanol until they were subjected to in situ hybridization. The screen for dose-dependent maternal suppressors of

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ectopic Runt lethality was done as follows. Homozygous NGT40 flies were crossed to individuals from balanced Drosophila stocks that carry different deficiency chromosomes. The 149 different deficiency chromosomes tested were obtained from the Bloomington Drosophila Stock Center. All together the chromosomal intervals spanned by this kit of deficiency chromosomes represent approximately 70% of the Drosophila genome. From this cross virgin females heterozygous for NGT40 and each particular deficiency chromosome were collected and crossed to UASrunt[⫹]232/CyO males and the viability of the UASrunt[⫹]232 progeny was determined relative to the number of CyO progeny. Previous experiments revealed that a single copy of the NGT40 driver reduced the viability of UAS-runt[⫹]232 progeny to fewer than 10% in this assay. Thirty-two of the deficiencies increased the viability of UAS-runt progeny from fewer than 10% to greater than 50%. In order to determine whether the suppression of UAS-runt lethality was specific, these deficiencies were tested for their effects on the lethality associated with NGTdriven expression of UAS-dpp and/or UAS-en transgenes as well as on the levels of ␤-galactosidase activity produced by NGT-driven expression of a UAS-lacZ transgene as described previously [30]. The suppression of UAS-runt lethality was categorized as non-specific if the deficiency allowed greater than 50% viability of UAS-dpp progeny (from a background of 1%), allowed for greater than 5% viability of UAS-en progeny (form a background of 0%), or led to greater than a twofold reduction in the levels of NGT-driven ␤-galactosidase activity. Based on these specificity tests, six intervals were selected for further analysis. P-element insertion lines and other mutations that mapped within these intervals were obtained from the Bloomington Stock Center and tested in a similar fashion. A tabulation of the results with all of the deficiency chromosomes and these additional mutations is available on request.

Results A DNA-binding-defective form of Runt is functional in vivo Kramer and colleagues demonstrated that two point mutations (C127S, K199A) in the Runt domain interfere with DNA binding but do not disrupt the interaction of Runt with the Bro partner protein. This mutant protein, which we refer to as Runt[CK], binds DNA in vitro with approximately 50-fold lower affinity than the wild-type protein and is incapable of activating Sxl transcription in vivo [28]. These previous in vivo experiments were done using an mRNA injection assay. We recently described a genetic system based on the use of the yeast transcription factor GAL4 that allows for quantitative manipulation of gene expression in the Drosophila embryo [30]. This approach involves mating females that express GAL4 in the germline to males that

carry inducible UAS transgenes. Reproducible genetic manipulation of expression levels is achieved in crosses between different NGT maternal GAL4 driver strains and different UAS transgenes. We established Drosophila strains that carry UAS-runt[CK] transgenes in order to examine the requirement for DNA binding by Runt in pathways other than Sxl activation. The relative expression levels obtained with three of these different strains and with two different UAS-runt[⫹] strains are indicated in Table 1. NGT-driven uniform expression of the wild-type Runt protein from the UAS-runt[⫹] transgenes at levels that are below the levels of endogenous Runt expression within the pair-rule stripes leads to segmentation defects and embryonic lethality [30,46]. NGT-driven expression of Runt[CK] at similar levels also causes embryonic lethality. This unexpected observation indicates that this DNA-binding-defective form of the Runt protein retains activity in vivo. In order to compare further the activity of the Runt[⫹] and Runt[CK] proteins, we examined cuticle preparations from non-viable embryos. The expression levels of the Runt protein obtained in crosses between females homozygous for the strong maternal GAL4 driver NGT40 and our benchmark UAS-runt[⫹]232 line produce strong pair-rule segmentation defects as well as severe defects in the head skeleton (Fig. 1A and B). In contrast, expression of the Runt[CK] protein at levels 1.5-fold higher than this causes only minor defects in segmentation but is still associated with severe defects in the head skeleton (Fig. 1C and D). The embryos shown in Fig. 1 are typical examples from these crosses. The difference in the in vivo activity of these two proteins is confirmed by scoring the severity of the patterning defects (Fig. 2). The reduced potency of the Runt[CK] protein in affecting segmentation is observed at both lower and higher expression levels. The UAS-runt[⫹]14 and UAS-runt[CK]2 lines are both expressed at approximately 50% of the level of UAS-runt[⫹]232. This lower level of expression of wildtype Runt protein still produces clear segmentation defects in over 80% of the embryos, whereas fewer than 20% of the UAS-runt[CK]2-carrying embryos have any apparent defects in segmentation. In contrast, a similar range of head defects is observed in these two sets of embryos. The differential effects of the Runt[⫹] and Runt[CK] proteins on patterning in the head and on segmentation are also observed at higher expression levels. The UAS-runt[CK]43 line gives 2.6-fold more expression than the UASrunt[⫹]232 line. This higher level of Runt[CK] expression has severe effects on patterning in the head, yet is markedly weaker than UAS-runt[⫹]232 in generating segmentation defects (Fig. 2). Taken together, these results indicate that the Runt[⫹] and Runt[CK] proteins have similar potencies at disrupting patterning in the head, but that the Runt[CK] protein is less effective at interfering with segmentation. Assuming that the patterning defects in these embryos are due to alterations in transcriptional regulation by the ectopically expressed Runt[⫹] and Runt[CK] proteins, these results strongly suggest that high-affinity DNA binding is

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Fig. 1. Ectopic expression of Runt[CK] produces embryonic lethality. Homozygous NGT40 females were crossed to UAS-runt[⫹]232 or UAS-runt[CK]77 males. In both cases this level of ectopic expression results in fully penetrant embryonic lethality. (A) Phase contrast photomicrograph showing the defects in the head region that result from NGT-driven expression of the wild-type Runt protein. The most sensitive regions are the posterior wall of the pharynx and the ventral arms of the head skeleton which derive from the intercalary and mandibular segments, respectively. (B) Dark field photomicrograph showing the pair-rule fusion of denticle belts in these UAS-runt[⫹]232 embryos. NGT-driven expression of Runt[CK] results in similar defects in head development (C), but leads to only minor defects in segmentation (D).

required for some, but not all of Runt’s regulatory properties in vivo. Differential requirements for DNA binding in the regulation of Runt target genes To investigate more directly the transcriptional regulatory properties of the Runt[⫹] and Runt[CK] proteins we examined the response of various target genes to ectopic expression of these proteins. The most sensitive target of Runt in the segmentation pathway is en, specifically the stripes of expression that arise in the odd-numbered parasegments. These stripes normally emerge in the region between the seven runt stripes and fail to become expressed in the presence of low, uniform levels of ectopic Runt expression. Two lines of evidence indicate that this is direct repression of en transcription by Runt. First, the expression of other pair-rule and segment polarity genes remains unaffected at this level of NGT-driven Runt expression [30]. Second, the rapid kinetics of repression following induction of a hs-runt transgene strongly suggest that the odd-numbered en stripes are directly repressed by Runt [29]. The level of Runt expression obtained with one copy of NGT40

and UAS-runt[⫹]232 is sufficient for repression of the oddnumbered en stripes in approximately 90% of early gastrula stage embryos. Surprisingly, NGT-driven expression of Runt[CK] at a similar level also interferes with the initial activation of these en stripes (Fig. 3B and C). The implication of this result is that this early repression of en occurs via a mechanism that does not require high-affinity DNA binding by the Runt protein. There is an important difference between the effects of Runt[⫹] and Runt[CK] on en expression. In embryos expressing the wild-type Runt protein the repression of en is maintained through germband extension and is still apparent in stage 9 embryos (Fig. 3F). In contrast, in embryos expressing Runt[CK] the initial repression of the odd-numbered stripes is not maintained and en expression recovers in the odd parasegments during germband extension (Fig. 3G). This recovery of en expression helps to explain why Runt[CK] was less effective than the wild-type protein in causing segmentation defects in terminally differentiated embryos. At higher levels of Runt[CK] expression the repression of the odd-numbered en stripes is maintained (Fig. 3D and H), which correlates with the segmentation defects shown in Fig. 2. At this higher level of expression there are

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Fig. 2. Differential sensitivity of the head and body to the Runt[CK] protein. The cuticle phenotypes produced by expression of different UAS-runt and UAS-runt[CK] transgenes in embryos from homozygous NGT40 females were scored using a system based on five phenotypic classes. The identity of the different UAS-runt and UAS-runt[CK] lines and the expression level (EL ⫽) of each line relative to the UAS-runt[⫹]232 line are indicated on the left. The relative expression levels were determined using RNase protection (see Materials and methods). Also given in parentheses is the number of embryos (n ⫽) scored for each line. The charts on the left show the severity of defects in the anterior head region. The phenotypic classes are (1) wild-type; (2) slightly truncated head skeleton; (3) severely truncated head skeleton; (4) only trace remnants of head skeleton, usually two small fragments; and (5) no head skeleton apparent. The embryos in Fig. 1A and C are examples of category (5) and category (4) head defects, respectively. The charts on the right show the severity of segmentation defects associated with differing levels of Runt and Runt[CK] expression. The phenotypic classes for segmentation defects are as follows: (1) wild-type; (2) partial pair-rule fusion of denticle belts in one segment; (3) partial to complete fusion of two or more denticle belts; (4) partial to complete pair-rule fusion of denticle belts in each double segment repeat; and (5) defects more extreme than pair-rule. The embryo in Fig. 1B is an example of a category (3), the embryo in Fig. 1D is an example of category (1).

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Fig. 3. Runt[CK] is a potent repressor of en. Females carrying one copy of the NGT40 driver were crossed to control y w/Y males (A, E), homozygous UAS-runt[⫹]232 males (B, F), or homozygous UAS-runt[CK]77 males (C, G) and the resultant embryos were analyzed for en expression. Expression of en in stage 6 (gastrula stage) is shown in the embryos on the left, and expression at stage 9 (germ band extended) is shown on the right. The black circles indicate the positions of en stripes 3 and 5. The level of ectopic Runt expression in these crosses is approximately half of that obtained with the homozygous NGT40 females used for the experiments in Figs. 1 and 2 and is at the threshold for repression of en. At this level, wild-type Runt interferes with activation of en in the odd parasegments of 90% of stage 6 embryos (B) and maintains this repression through stage 9 (F). Runt[CK] also interferes with activation of en in 90% of the embryos at stage 6 (C), but cannot maintain repression through stage 9 (G). To examine the effects of expressing Runt[CK] at higher levels, homozygous NGT40; NGTA females were crossed to UAS-runt[CK]43 and the resultant embryos were analyzed for en expression (D, H). NGT40; NGTA females produce approximately 3-fold more GAL4 activity than heterozygous NGT40 females, and the UAS-runt[CK]43 transgene is 2.6-fold more active than UAS-runt[⫹]232. Thus, the embryos in D and H express approximately 8-fold higher levels of Runt[CK] than the levels of ectopic Runt[⫹] produced in the embryos in (B) and (F). This higher level of ectopic Runt[CK] allows for maintained repression of en and is also associated with changes in the expression of other segmentation genes.

also changes in the expression patterns of other segmentation genes. Thus, it is not known whether this later effect on en is direct or indirect. Another sensitive target of Runt is slp, which shows an interesting dual response. Normally, slp is expressed as a two-cell-wide stripe in the posterior half of each of the 14

parasegments [48]. Ectopic Runt expression represses slp in the even-numbered parasegments and has the opposite effect of activating slp in the odd-numbered parasegments. This is apparent in embryos from a cross between homozygous NGT40 females and UAS-runt[⫹]232 males in which 7 broadened stripes of slp expression are obtained (Fig. 4B).

Fig. 4. Runt[CK] is less effective at regulating slp expression. Homozygous NGT40 females were crossed to UAS-runt[⫹]232 (B) or UAS-runt[CK]77 (C) males and the resultant embryos were analyzed for slp expression. This is the same combination of lines used for the embryos shown in Fig. 1 and is at a level of NGT-driven expression that is approximately twice that obtained with the heterozygous NGT40 females used in Fig. 3. (A) Wild-type pattern of slp expression at stage 6. (B) Ectopic wild-type Runt expression causes a shift in the slp pattern, such that expression is repressed in even parasegments and activated in odd parasegments. (C) Ectopic expression of Runt[CK] at similar levels has no effect on the slp expression pattern. (D) Females homozygous for NGT40; NGTA were crossed to UAS-runt[CK]43 and the resultant embryos were analyzed for slp expression. Expression of Runt[CK] at approximately four-fold higher levels than the level of ectopic Runt obtained in (B) results in intermediate effects on slp expression.

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Runt[CK] is less effective than Runt[⫹] at regulating slp and normal expression is observed in embryos from a cross of homozygous NGT40 females with UAS-runt[CK]77 males (Fig. 4C). Effects on slp are obtained in crosses that involve our strongest maternal GAL4 driver and the strongest UAS-runt[CK] line (Fig. 4D). However, these effects are less pronounced than those observed with significantly lower levels of the Runt[⫹] protein (Fig. 4B). These data suggest that DNA binding by Runt is important for the regulation of slp expression. More importantly, the contrasting effects of the Runt[⫹] and Runt[CK] proteins on en and slp demonstrate that there is a differential requirement for DNA binding in the regulation of these two target genes by the Runt protein. Is en repression a normal function of Runt? The above results are based on ectopic expression assays. Thus, it is important to consider whether the repression of en by Runt has physiological relevance in the segmentation pathway. To begin to address this concern it is useful to compare the dynamics and phasing of en and runt expression during early embryogenesis. Expression of runt commences prior to completion of the final syncitial nuclear division cycle in a broad band that encompasses nearly the entire pre-segmental region of the embryo. This gap genelike pattern resolves into seven four-cell-wide stripes during the process of cellularization. The 14-striped en pattern is established in two steps. The even-numbered stripes appear first during the cellular blastoderm stage. Expression of the odd-numbered stripes first emerges in late blastoderm stage embryos, and expression in the even- and odd-numbered parasegments is not equivalent until after gastrulation and the onset of germband extension [49,50]. The even-numbered en stripes arise in Ftz-expressing cells that lie in the center of the Runt stripes, whereas the odd-numbered stripes arise in cells located between the Runt stripes. The sensitivity of the odd-numbered en stripes to low levels of ectopic Runt expression suggests that the clearance of Runt protein from the inter-stripe regions may be critical for allowing activation of en in odd parasegments. Segmentation is sensitive to runt dosage. Female embryos heterozygous for runt mutations show partially penetrant runt-like segmentation defects, whereas male embryos that carry a Y chromosome duplication for runt have complementary, anti-runt-like defects [11]. We examined the early evolution of the en pattern in embryos from a cross where 50% of the progeny are males with increased runt dosage and the other 50% of the progeny are heterozygous females with reduced runt dosage. In half of the embryos from this cross, the presumptive duplication males, the odd-numbered en stripes are somewhat delayed in their emergence compared to wild-type (Fig. 5A and B). Conversely, in the other half of the embryos, the presumptive females with reduced runt dosage, the odd-numbered en stripes emerge precociously and become as intense as the

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even-numbered stripes prior to the onset of germband extension (Fig. 5C). These observations provide strong evidence that the temporal activation of en in odd parasegments is sensitive to runt dosage. The characterization of runt as a direct regulator of en has been obscured by runt’s earlier role as a primary pairrule gene. We used the temperature-sensitive runtYP17 mutation to try and separate runt’s role in regulating the expression of other pair-rule genes from its role as a direct regulator of en. Embryos were collected from a cross that produces runtYP17/Y mutant males and allowed to develop at the permissive temperature through the early blastoderm stages when the seven stripe patterns of the pair-rule genes are established. These embryos were then shifted to a nonpermissive temperature of 30°C for 30 min and then immediately fixed and processed for in situ hybridization. As expected, 25% of the embryos from this cross show abnormal en expression. Consistent with the hypothesis that runt plays a role in repressing the odd-numbered en stripes, we find that elimination of runt activity causes these stripes to broaden (Fig. 5D). Interestingly, the transient elimination of runt activity is also associated with a loss of the evennumbered en stripes in these embryos. Thus Runt has a dual role in en regulation—activation in even parasegments and repression in odd parasegments. Based on these several observations we conclude that the repression of en in odd parasegments is a physiologically relevant function of the Runt protein. Maternal Ttk cooperates with Runt to repress en The level of NGT-driven Runt expression that is associated with repression of en in the odd-numbered parasegments coincides with the threshold level that is associated with embryonic lethality. The progeny from crosses where one copy of the maternal NGT40 driver is used to drive expression of UAS-runt[⫹]232 show nearly fully penetrant early repression of the odd-numbered en stripes. At this level of ectopic Runt expression approximately 10% of the female progeny survive to adulthood and there are no surviving UAS-runt[⫹]232 males. Experiments with other combinations of lines indicate that there is a substantial increase in the viability of embryos that have half this level of NGT-driven Runt expression [30]. We took advantage of this sensitivity to conduct a genetic screen for suppressors of ectopic Runt lethality (see Materials and methods). If a particular gene product contributes in a dose-dependent manner to the potency of Runt in the early embryo, then a 50% reduction in this gene product should reduce Runt function by 50%, thus increasing the viability of UAS-runt flies. We screened a collection of deficiency chromosomes altogether uncovering approximately 70% of the genome for their ability to act as dose-dependent maternal suppressors of NGT-driven Runt lethality (see Materials and methods). Several of these deficiencies showed reproducible

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C.J. Vander Zwan et al. / Blood Cells, Molecules, and Diseases 30 (2003) 207–222 Table 2 Ttk mutations suppress lethality of NGT-driven runt expression Maternal genotypea

UAS-runt viability (n ⫽)b

Comments (Ref.)c

ss e ro Df(3R)faf-BP l(3)02667 ttk[1] ttk[1e11] ttk[D2-50] ttk[B330] ttk[10556] ttk[5346] ttk[rM730] ttk[osn]

3% (421) 40% (250) 17% (544) 19% (221) 18% (321) 8% (199) 32% (157) 27% (181) 56% (129) 4% (188) 0% (323)

Control chromosome Deficiency 100D1-2; 100E-F (1) P-insertion (2) P-insertion; blocks Ttk-p88 (3) Imprecise excision of ttk[1] (3) EMS point mutant (4) EMS point mutant (5) P-insertion (4) P-insertion (4) P-insertion; upstream (6) P-insertion (7)

a Experimental females carrying one copy of the strong NGT40 driver were generated by crosses between homozygous NGT40 females and males carrying different mutant third chromosomes. The different mutant chromosomes that are indicated in the genotype column are also present in a single copy in the experimental females. b The relative adult viability of UAS-runt progeny was determined by mating males heterozygous for the UAS-runt[⫹]232 transgene, which is located on the second chromosome insertion, and the CyO balancer chromosome. The numbers given represent the percentage of viability of UAS progeny relative to their siblings. The total number of sibs recovered in each series of experimental crosses is given in parentheses. c References on the different mutant chromosomes are as follows: (1) [64]; (2) [65]; (3) [52]; (4) [53]; (5) [66]; (6) [51]; (7) [67].

effects on increasing the viability of UAS-runt progeny. One possible explanation for suppression of ectopic Runt lethality is that the deficiency affects the efficiency of the NGT system. To identify such non-specific effects we analyzed the effects of these deficiencies on other NGT-driven phenotypes, including the lethality of UAS-dpp and UAS-en transgenes and the enzyme activity levels of a UAS-lacZ transgene. For those deficiency intervals that were judged to have the strongest specific effect on runt we then tested the P-element insertion lines that were available from the Bloomington Drosophila Stock Center. Among the 43 mutations tested in this manner, the strongest specific effect was obtained with l(3)02667, a P-element insertion in the tramtrack (ttk) locus [51]. The suppression of UAS-runt lethality associated with this P-element insertion is less than that obtained with the deficiency chromosome for this interval, Df(3R)faf-BP (Table 2). However, the ttk02667 mutation also appears to be more specific and has no effect on the viability of UAS-dpp progeny (data not shown).

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In order to confirm that the effects on runt were due to ttk dosage and not to other mutations on the l(3)02667 chromosome we obtained several other alleles of ttk and tested them in similar assays. The most well-characterized mutations were ttk1, an independently generated P-element insertion in the ttk locus, and ttkle11, which was derived from ttk1 by imprecise excision [52]. Both of these mutations show a dose-dependent maternal effect on UAS-runt viability (Table 2). This suppression is specific as there is no effect on the lethality associated with NGT-driven UAS-dpp expression and UAS-lacZ expression is not significantly different from that obtained from females heterozygous for the control ss e ro chromosome (data not shown). Several other ttk mutations, including two independently generated EMS mutations, also suppressed the lethality associated with NGT-driven runt expression (Table 2). The two exceptional cases, ttkosn and ttkrM730, are both P-element-induced hypomorphic alleles [53]. Based on these genetic results we conclude that maternal ttk dosage contributes to the potency of Runt in this ectopic expression assay system. We examined the effects of ttk dosage on the repression of the odd-numbered en stripes by Runt. For these experiments we used a slightly stronger NGT driver in order to increase the penetrance of en repression. The level of Runt expression produced by single copies of the NGT40 and NGTA drivers in combination with UAS-runt[⫹]232 blocks normal activation of en in greater than 90% of gastrula stage embryos and 60% of these embryos show no en expression in the odd-numbered parasegments. Mutations in ttk relieve this Runt-dependent repression of en (Fig. 6). The ttk locus encodes two different Zn-finger DNA-binding proteins that both have been characterized for their roles as transcriptional repressors. These observations suggest an obvious model for repression by the DNA-binding-defective Runt[CK] protein whereby the Ttk protein(s) provide DNAbinding specificity and Runt’s role depends on protein– protein interactions. A prediction of this model is that reductions in maternal ttk dosage should also suppress the potency of the Runt[CK] protein. Consistent with this model, we found that ttk mutations relieved repression of en by the Runt[CK] protein (Fig. 6). These results confirm the importance of maternal ttk dosage for repression by Runt and strongly suggest that the DNA-binding-independent repression of en by Runt involves interactions with one or more of the DNA-binding proteins encoded by the ttk locus.

Fig. 5. A normal role for runt in regulating en. (A) Wild-type expression of en at gastrulation (stage 6). The black circles indicate the positions of en stripes 3 and 5. In wild-type embryos, expression in odd parasegments is weaker than in even parasegments at this stage. (B and C) Embryos were collected from homozygous y w females crossed to runtLB5/y⫹Ymal102 males and analyzed for en expression. In 50% of the embryos from this cross the odd-numbered en stripes are weak and incomplete relative to the wild-type pattern (B). In the other 50% of the embryos from this cross, the odd and even stripes are expressed at equivalent levels at this stage. We interpret these two classes of embryos to represent males that are duplicated for runt and females that are heterozygous mutant for runt, respectively. (D) Embryos collected from runtYP17/FM7 females crossed to FM7/Y males were allowed to develop for 4.5 h at 18°C and then shifted to the non-permissive temperature (30°C) for 30 min prior to fixation for in situ hybridization. Transient elimination of runt in this manner leads to broadened en stripes in odd parasegments and also is associated with a loss of expression in the even-numbered parasegments.

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Fig. 6. Ttk cooperates with Runt to repress en. Control embryos derived from NGT40/⫹; NGTA/ss e ro females crossed to UAS-runt[⫹]232 (A) or UAS-runt[CK]77 (B) males were analyzed for en expression. Activation of the odd-numbered en stripes is defective in more than 90% of stage 6 embryos that express either the Runt or the Runt[CK] proteins at this level. Complete repression, as shown in these two embryos, is obtained in 60% of UAS-runt[⫹]232 embryos (n ⫽ 60) and in 50% of RuntUAS-runt[CK]77 embryos (n ⫽ 50), respectively. Embryos derived from NGT40/⫹; NGTA/ttk1 females crossed to UAS-runt[⫹]232 or UAS-runt[CK]77 males are shown in (C) and (D), respectively. Reduction of the maternal ttk dose leads to nearly wild-type en expression in 29% (n ⫽ 134) of Runt-expressing and in 47% (n ⫽ 51) of Runt[CK]-expressing embryos. Similar dominant maternal suppression of Runt[⫹]- and Runt[CK]-mediated repression is obtained with the ttk1e11 and ttk02667 mutations.

Discussion Regulation of en by Runt The results presented here confirm and extend previous observations on runt’s role in regulating expression of the segment polarity gene en. A new finding is the evidence that runt may directly participate in activating en in even-numbered parasegments. The even-numbered stripes arise in Ftz-expressing cells that lack Odd-skipped (Odd) [29,54]. These cells are in the center of the runt stripes. The elimination of the even-numbered en stripes in embryos that have suffered a transient loss of runt could reflect a loss of ftz expression and/or a failure to repress odd. However, the kinetics of the response to the temperature shift are consistent with a more direct effect. Although runt is necessary for the even-numbered en stripes, it is not sufficient, as these stripes are not significantly broadened in response to ectopic runt expression. It is interesting to note that activation of the even-numbered en stripes requires the maternally provided FTZ-F1 orphan nuclear receptor [55,56]. Indeed, mutagenesis of a FTZ-F1-binding site within a cis-element that drives the even-numbered en stripes reduces expression of an en-lacZ reporter gene to background levels [57]. Binding sites for FTZ-F1 in the ftz zebra element contribute to the activation of ftz by runt [32]. These observations suggest that interactions between Runt and FTZ-F1 may also contribute to en activation. The odd-numbered en stripes have been interpreted to be direct targets of repression by Runt based both on the

kinetics and on the sensitivity of the response to ectopic runt expression [29,30]. The surprising result from this study is that this repression does not require high-affinity DNA binding by the Runt protein. A solution to this apparent paradox is provided by the finding that repression by Runt involves dose-dependent interactions with Zn-finger DNAbinding proteins encoded by the ttk locus. The ttk locus produces two protein isoforms, p69 and p88, via alternative splicing. Both proteins are expressed maternally and are degraded during early embryogenesis, such that they are nearly undetectable in mid-stage 7 embryos [58]. Runt is first zygotically expressed in a broad central domain of the embryo; during stages 5–7 this pattern refines to seven pair-rule stripes of expression, such that Runt is no longer detectable in the anterior cells of the odd parasegments (Fig. 7). We propose that Runt and Ttk work together to prevent activation of the odd-numbered en stripes and that expression of these stripes awaits removal of both repressors from these cells. An interesting question is which Ttk isoform works with Runt to repress en? Mutations in ttk have been classified previously based on their effects on the embryonic central nervous system (CNS), which is thought to primarily reflect the activity of Ttk-p69 [53]. Comparison of these previous observations with our results strongly suggests that Ttk-p88 is more relevant for en repression. For example, the ttkD2-50 and ttkosn mutations both have strong effects on CNS development but show no evidence of suppressing the lethality associated with NGT-driven Runt expression. Conversely, the ttk1 mutation suppresses Runt-dependent en repression,

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of en repression by Runt [61]. Further characterization of single locus suppressor mutations that affect establishment should provide further insight into the molecular mechanism of en repression by Runt and Ttk. Other DNA-binding-independent targets of Runt

Fig. 7. Regulation of en by Runt and Ttk. Relationship of en expression to the expression of Runt and Ttk in blastoderm stage Drosophila embryos. In cycle 13, Runt (black) is expressed in a broad central domain throughout the pre-segmental region of the embryo and maternally provided Ttk (cross-hatched) is expressed uniformly. Expression of en in the oddnumbered parasegments in late cellular blastoderm stage embryos correlates with the elimination of Runt in the inter-stripe regions and an overall reduction of Ttk protein levels.

but has no effect on the embryonic CNS [53]. The ttk1 allele consists of a P-element insert in the intron of the p88 isoform (the 3⬘UTR of the p69 isoform) and specifically affects expression of Ttk-p88 in the eye [52]. Ttk-p69 and Ttk-p88 differ in their Zn-finger domains and have distinct DNA-binding specificity [58,59]. The hypothesis that Ttkp88 is directly involved in en repression is consistent with the finding that Ttk-p88 binds to sequences in the en cisregulatory region [58]. Further analysis of the molecular basis of the different ttk mutations and their effects on expression of Ttk-p69 and Ttk-p88 during early embryogenesis should help to clarify which isoform is critical for the DNA-binding-independent repression activity of Runt. Our working model is that the DNA-binding activity of Ttk-p88 is used to target Runt to the en cis-regulatory region. This proposed mechanism is analogous to transrepression by the glucocorticoid receptor, which involves tethering to DNA via the AP-1 transcription factor [60]. In the simplest version of this model, repression would be mediated by binding sites for Ttk-p88 in the en cis-regulatory region. Unfortunately, the cis-elements necessary for expression of the odd-numbered en stripes that would allow this model to be tested have not yet been identified. We also imagine that protein–protein interactions account for Runt’s ability to repress transcription via a DNA-binding-independent mechanism. Preliminary yeast two hybrid assays have not uncovered direct interactions between Runt and either of the Ttk proteins (J.C. Wheeler, unpublished). However, the interactions between Runt and Ttk need not be direct, as other interacting proteins (e.g., Bro) may act as a bridge. Indeed, our genetic screen uncovered five other chromosomal deficiencies that specifically suppress the lethality associated with NGT-driven runt expression. Interestingly, identification and characterization of the interacting genes within three of these deficiencies demonstrate that there are distinct requirements for establishment versus maintenance

Our results indicate that other targets of runt are also regulated by DNA-binding-independent mechanisms. The Runt[CK] and Runt[⫹] proteins are equally effective in disrupting pattern formation in the anterior region of the embryo. We have not yet identified a specific target gene that can account for these head defects, although previous studies with hs-runt embryos demonstrated that Runt interferes with transcriptional activiation by the Bicoid (Bcd) homeodomain protein [31]. This interference was observed on minimal reporter gene constructs containing multimerized copies of a Bcd binding site, suggesting that sequencespecific DNA binding by Runt is not essential for preventing Bcd-dependent activation. There is an interesting difference in the effects of ectopic Runt[CK] expression in the anterior versus pre-segmental regions of the embryo. Although en is efficiently repressed by Runt[CK], the expression recovers during the germband extension stages and there are no apparent defects in the cuticular segmentation pattern in embryos that have severe head defects. The stable maintenance of en repression in the pre-segmental region also requires Runt’s conserved Cterminal VWRPY motif and involves interactions with the Groucho co-repressor and the Rpd3 histone deacetylase [61]. These results suggest that stable repression involves recruitment of the Gro:Rpd3 complex by a DNA-bound form of Runt. Once recruited, the histone deacetylase activity of Rpd3 would then prevent subsequent activation by reducing accessibility to transcriptional activators and/or the transcriptional machinery. Consistent with this model, the transcription factor Paired (Prd), which is responsible for activation of en in odd-numbered parasegments [62], continues to be expressed in the appropriate cells during these stages in the presence of ectopic Runt activity (D. Swantek and J.P. Gergen, unpublished observation). We presume that the failure of the head to recover from the effects of transient Runt[CK] expression reflects developmental differences in the regulatory information against which runt activity is being measured in these different regions. In contrast to Prd, maternally provided Bcd protein decays soon after gastrulation [63] and would not be available for subsequent activation of target gene expression during germband extension. Conserved functions of the DNA-binding Runt domain The Runt domain is the largest and by far the most conserved feature of the Runt protein. This domain is responsible for sequence-specific DNA binding as well as for interaction with the Bro and Bgb partner proteins. Given

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this conservation, the discovery that the DNA-binding activity of the Runt domain is not absolutely required for all functions is somewhat surprising. This result is also in contrast to the previous observation that a point mutation within the Runt domain that specifically disrupts the interaction with the Bro/Bgb partners renders the protein nonfunctional in vivo by all criteria [46]. Thus, even the DNAbinding-independent repression by Runt presumably involves a Runt/Bro or Runt/Bgb protein complex. These results parallel results obtained with the bHLH transcription factor SCL. An SCL mutant protein that is defective in DNA binding retains the ability to rescue hematopoiesis in SCL⫺/⫺ embryonic stem cells, whereas a mutant that is defective in heterodimerization with other E-box proteins is inactive in the same assay [2]. Hence, while DNA binding is important for regulation of some targets, it appears that interactions with other proteins are more critical for the in vivo function of these transcription factors. Our results also demonstrate that the relative importance of DNA binding by a transcription factor depends on the target gene and developmental context. High-affinity DNA binding is absolutely required for activation of Sxl by Runt, but is not required for repression of en. The intermediate response of slp to the Runt[CK] protein suggests that DNA binding contributes to Runt’s potency as both an activator and a repressor of this target gene. Thus, we anticipate that the cis-regulatory elements for slp will contain binding sites for Runt, but that interaction with other proteins will also be critical for the regulation of slp transcription by Runt. Depending on the target gene and the concentrations of other interacting factors, one can imagine that the threshold of a response to Runt will be a function of the relative affinities for DNA and/or other protein factors in a cell-specific manner. The network of protein–protein and protein–DNA interactions that account for the reproducible and high-fidelity regulation of gene transcription during development presents a formidable challenge for the future. The genetic approaches available in the Drosophila embryo will be invaluable for further unraveling the molecular mechanisms of transcriptional activation and repression by Runt domain proteins.

Acknowledgments Many aspects of this work benefited from the expert technical assistance of Deborah Swantek. The manuscript was improved by constructive comments from Sean Boykevisch, Maurice Kernan, Joe Landry, Pegine Walrad, and Kathy Wojtas. We thank Michael Weir (Wesleyan University) for providing the plasmid used to make probes for slp expression. Several of the ttk mutations used in this work were generously provided by Christian Kla¨ mbt. The invaluable services provided by the Drosophila Stock Center in Bloomington, Indiana, are also greatly appreciated. This work was supported by grants from NIH (CA77221) and

NSF (MCB-0090956) to J.P.G. This paper is based on a presentation given at a Focused Workshop on Transcriptional Regulation of Runx Proteins in Development and Leukemia sponsored by the Leukemia & Lymphoma Society in Charlottesville, Virginia, in August 2002.

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