Mechanisms of transcriptional regulation by Runt domain proteins.

Runt domain proteins have vital roles in regulating transcription in developmental pathways extending from sex determination and segmentation in fruit fly embryos to the development of blood and bone in mammals. Many of the insights into the mechanisms by which these proteins act to regulate transcription originate either from studies on the Drosophila runt gene, the founding member of this family, or from work on the mammalian PEBP2/CBF transcription factor. Genetic experiments in the Drosophila system reveal that runt functions both to activate and to repress transcription of different downstream target genes and indicate that different mechanisms are used in the regulation of different specific downstream target genes. These studies have also identified other nuclear factors that work with Runt in some of these pathways. Studies in mammalian systems have provided additional evidence for the complexity of transcriptional regulation by Runt domain proteins and have identified other transcription factors that cooperate with Runt domain proteins to regulate the activity of different specific cis-regulatory enhancers. The emerging view from studies in both systems is that these proteins act as context-dependent regulators of transcription, activating or repressing gene expression dependent upon the constititution of a particular promoter/enhancer in a particular cell type. These results have yielded new insights into the molecular mechanisms that control animal development and provide a framework for investigating fundamental issues in eukaryotic transcriptional regulation.

Quantitative analysis of gene function in the Drosophila embryo.

The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye.

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBF(beta), which interacts with the mammalian CBF(alpha) Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. In both mouse and human there is genetic evidence that the CBF(alpha) and CBF(beta) proteins function together in hematopoiesis and leukemogenesis. Here we demonstrate functional interactions between Brother proteins and Runt domain proteins in Drosophila. First, we show that a specific point mutation in Runt that disrupts interaction with Brother proteins but does not affect DNA binding activity is dysfunctional in several in vivo assays. Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sxl-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, we examine the effects of expression of a Brother fusion protein homologous to the dominant negative CBF(beta)::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. Moreover, we find that the effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. Our results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding.

Direct activation of Sex-lethal transcription by the Drosophila runt protein.

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

Pair-rule gene runt restricts orthodenticle expression to the presumptive head of the Drosophila embryo.

Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizes transcriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid. Here we investigate the relevance of runt's activity as a repressor of otd in normal Drosophila embryogenesis otd expression is activated in the posterior region of embryos that are mutant for runt. This posterior expression domain of otd depends on the activity of the orphan nuclear receptor protein Tailless. Repression of otd by runt does not require the conserved VVVRPY motif that mediates interaction between Runt and the co-repressor protein Groucho. The observed functional interactions between runt and tailless on otd expression may indicate there are other contexts where members of these two families of transcriptional regulators interact to regulate gene expression during development.

A Xenopus homologue of aml-1 reveals unexpected patterning mechanisms leading to the formation of embryonic blood.

The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventral blood island in a pattern that anticipates that of later globin. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventral blood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, we propose that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos.

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.

Groucho-dependent and -independent repression activities of Runt domain proteins.

Runt domain proteins are transcriptional regulators that specify cell fates for processes extending from pattern formation in insects to leukemogenesis in humans. Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. We demonstrate an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. The interaction, however, is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins. Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. This suggests that the Runt domain protein-Groucho interaction may be regulated.

Disperse versus compact elements for the regulation of runt stripes in Drosophila.

The segmented body pattern of the Drosophila embryo is established through a hierarchical network of interacting genes. At each successive step in this pathway, transcriptional regulation is used to convert coarse positional information into finer patterns of gene expression. Central to this process are the cis-regulatory regions that drive the dynamic spatial expression of the different segmentation genes. Here we describe the cis-regulatory region of the runt gene. As found for both other primary pair-rule genes, hairy and even-skipped, there are stripe-specific elements which mediate the initial regulation of runt stripes by gap genes. We did not find autoregulatory elements as described for even-skipped and fushi tarazu. The regulation of runt by other pair-rule genes is mediated by a large region, extending over 5 kb upstream and downstream of the transcription start site. This "disperse" element cannot be subdivided into functionally independent subelements or minimal elements. Such disperse elements mediating pair-rule gene interactions may have escaped detection in other segmentation genes and may involve molecular mechanisms different from those mediating regulation by gap genes.

Dosage-sensitive maternal modifiers of the drosophila segmentation gene runt.

The protein encoded by the pair-rule gene runt functions as a transcriptional regulator during anterior-posterior patterning of the Drosophila embryo. Results of over-expression experiments as well as parallels drawn from the recent characterization of vertebrate homologues indicate that interactions with other proteins are likely to be central to the function of the Runt protein. To identify factors important for runt activity, we took advantage of an adult visible phenotype observed in animals heterozygous for runt mutations. Using a set of 126 different deficiency chromosomes we screened approximately 65% of the genome for genes that act as dose-sensitive maternal modifiers of runt. Eighteen deficiencies representing 12 putative loci were identified as maternally acting enhancers of runt haplo-insufficiency. Further characterization of two of these regions led to the identification of the interacting loci. Both of these loci affect the spatial regulation of runt transcription and appear genetically complex. Furthermore, the effects of one of these loci, M(1)1B, is indirect and mediated through effects on the transcriptional regulation of posterior gap genes.

Drosophila homologs of the proto-oncogene product PEBP2/CBF beta regulate the DNA-binding properties of Runt.

The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt.

Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development.

Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, we have examined a collection of Drosophila female-sterile mutants for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of bicoid and Toll mRNAs. Cortex embryos contain a bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly(A)) tail. Injection of polyadenylated bicoid mRNA into cortex embryos allows translation demonstrating that insufficient polyadenylation prevents endogenous bicoid mRNA translation. In contrast nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. Cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development.

Conservation and function of the transcriptional regulatory protein Runt.

A phylogenetic approach was used to identify conserved regions of the transcriptional regulator Runt. Alignment of the deduced protein sequences from Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virilis revealed eight blocks of high sequence homology separated by regions with little or no homology. The largest conserved block contains the Runt domain, a DNA and protein binding domain conserved in a small family of mammalian transcription factors. The functional properties of the Runt domain from the D. melanogaster gene and the human AML1 (acute myeloid leukemia 1) gene were compared in vitro and in vivo. Electrophoretic mobility-shift assays with Runt/AML1 chimeras demonstrated that the different DNA binding properties of Runt and AML1 are due to differences within their respective Runt domains. Ectopic expression experiments indicated that proteins containing the AML1 Runt domain function in Drosophila embryos and that sequences outside of this domain are important in vivo.

Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs.

Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs (mRNAs) whose protein products determine cell fate. Three mRNAs that dictate anterior, dorsoventral, and terminal specification--bicoid, Toll, and torso, respectively--showed increases in polyadenylate [poly(A)] tail length concomitant with translation. In contrast, posteriorly localized nanos mRNA, although also translationally activated, was not regulated by poly(A) status. These results implicate at least two mechanisms of mRNA activation in flies. Studies with bicoid mRNA showed that cytoplasmic polyadenylation is necessary for translation, establishing this pathway as essential for embryogenesis. Combined, these experiments identify a regulatory pathway that can coordinate initiation of maternal pattern formation systems in Drosophila.

Gap gene properties of the pair-rule gene runt during Drosophila segmentation.

The Drosophila Runt protein is a member of a new family of transcriptional regulators that have important roles in processes extending from pattern formation in insect embryos to leukemogenesis in humans. We used ectopic expression to investigate runt's function in the pathway of Drosophila segmentation. Transient over-expression of runt under the control of a Drosophila heat-shock promoter caused stripe-specific defects in the expression patterns of the pair-rule genes hairy and even-skipped but had a more uniform effect on the secondary pair-rule gene fushi tarazu. Surprisingly, the expression of the gap segmentation genes, which are upstream of runt in the segmentation hierarchy was also altered in hs/runt embryos. A subset of these effects were interpreted as due to an antagonistic effect of runt on transcriptional activation by the maternal morphogen bicoid. In support of this, expression of synthetic reporter gene constructs containing oligomerized binding sites for the Bicoid protein was reduced in hs/runt embryos. Finally, genetic experiments demonstrated that regulation of gap gene expression by runt is a normal component of the regulatory program that generates the segmented body pattern of the Drosophila embryo.

Regulation of runt transcription by Drosophila segmentation genes.

The runt gene plays an important role in the genetic hierarchy that generates the segmented body pattern during the early stages of Drosophila embryogenesis. We studied mRNA expression in mutant embryos in order to investigate the regulation of runt transcription during these stages. We used sensitive whole-mount in situ hybridization procedures to identify the earliest, and therefore most likely direct regulatory effects. There are several distinct phases of runt expression in the early embryo. We find that each phase depends on a different set of regulators. The first phase of expression is a broad-field of mRNA accumulation in the central regions of syncytial blastoderm stage embryos. This pattern is due to terminal repression by the anterior and terminal maternal systems. The effect of the terminal system, even at this early stage, is mediated by two zygotic gap genes, tailless and huckebein. A 7 stripe pattern of runt mRNA accumulation emerges during the process of cellularization. The initial formation of this pattern depends on position-specific repression by zygotic gap genes. Examination of the early RNA patterns of the pair-rule genes even-skipped, hairy, and fushi tarazu indicate that they are also regulated in a similar manner. Three pair-rule genes, hairy, even-skipped, and runt itself, also affect runt's 7 stripe pattern. The effects of runt are stripe specific; the effects of hairy are more uniform; and the patterns obtained in even-skipped mutant embryos show a combination of both stripe specific and uniform regulatory effects. A third distinct phase of expression occurs at the onset of gastrulation when runt becomes expressed in 14 stripes. fushi tarazu plays a negative regulatory role in generating this pattern, whereas the pair-rule genes paired and odd-paired are required for activating or maintaining runt expression during these stages.

The Drosophila segmentation gene runt has an extended cis-regulatory region that is required for vital expression at other stages of development.

The Drosophila runt gene functions in several developmental pathways during embryogenesis. This gene was initially characterized due to the pivotal role that it plays in the genetic regulatory network that establishes the segmented body pattern. Recently it was found that this X-chromosome-linked gene is one of several dosage-sensitive, X-linked components that is involved in activating the Sex-lethal gene in blastoderm stage female embryos. Finally, this gene is also extensively re-expressed in later stages of embryogenesis in the developing nervous system where it plays an important role in the development of specific neural lineages. We have initiated an analysis of the runt cis-regulatory region in order to investigate runt's roles in these (and other) developmental pathways. Analysis of both the function and the expression patterns of runt genes with truncated cis-regulatory regions indicates that there are multiple elements that make quantitative contributions to runt regulation during segmentation. We find that sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis. Genetic experiments indicate that the post-blastoderm expression of runt is vital to the organism.

Expression and function of the Drosophila gene runt in early stages of neural development.

The Drosophila gene runt was initially identified on the basis of its role during segmentation. Recent molecular and genetic studies have demonstrated that the runt gene encodes a novel nuclear protein whose developmental importance is not exclusive to segmentation. This report addresses the functional relevance of runt expression in the developmental pathway of neurogenesis. Antibodies against the runt protein reveal that it is expressed in a subset of neuroblasts, ganglion-mother cells and neurons. A subset of these neurons also co-express the segmentation gene even-skipped (eve). Using eve as a marker, we show that runt is required for the normal development of these neurons. A runt P-transposon that lacks neural cis-regulatory elements is used to show that these neurons require runt activity independent of its activity during segmentation. These results are confirmed using a temperature-sensitive runt allele. Further temperature-shift experiments indicate that the requirement for runt is during an early stage of neurogenesis. Based on its pattern of expression and its temporal requirements, runt is distinguished as one of the earliest acting genes involved in the generation of diverse cell fates in the developing Drosophila nervous system.