Genomic binding and transcriptional regulation by the Drosophila Myc and Mnt transcription factors.

Deregulated expression of members of the myc oncogene family has been linked to the genesis of a wide range of cancers, whereas their normal expression is associated with growth, proliferation, differentiation, and apoptosis. Myc proteins are transcription factors that function within a network of transcriptional activators (Myc) and repressors (Mxd/Mad and Mnt), all of which heterodimerize with the bHLHZ protein Mad and bind E-box sequences in DNA. These transcription factors recruit coactivator or corepressor complexes that in turn modify histones. Myc, Mxd/Max, and Mnt proteins have been thought to act on a specific subset of genes. However, expression array studies and, most recently, genomic binding studies suggest that these proteins exhibit widespread binding across the genome. Here we demonstrate by immunostaining of Drosophila polytene chromosome that Drosophila Myc (dMyc) is associated with multiple euchromatic chromosomal regions. Furthermore, many dMyc-binding regions overlap with regions containing active RNA polymerase II, although dMyc can also be found in regions lacking active polymerase. We also demonstrate that the pattern of dMyc expression in nuclei overlaps with histone markers of active chromatin but not pericentric heterochromatin. dMyc binding is not detected on the X chromosome rDNA cluster (bobbed locus). This is consistent with recent evidence that in Drosophila cells dMyc regulates rRNA transcription indirectly, in contrast to mammalian cells where direct binding of c-Myc to rDNA has been observed. We further show that the dMyc antagonist dMnt inhibits rRNA transcription in the wing disc. Our results support the view that the Myc/Max/Mad network influences transcription on a global scale.

Drosophila C-terminal binding protein functions as a context-dependent transcriptional co-factor and interferes with both mad and groucho transcriptional repression.

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. We show here that dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. We find that mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and that dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, we propose that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function.

Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development.

Rho GTPases play an important role in diverse biological processes such as actin cytoskeleton organization, gene transcription, cell cycle progression and adhesion. They are required during early Drosophila development for proper execution of morphogenetic movements of individual cells and groups of cells important for the formation of the embryonic body plan. We isolated loss-of-function mutations in the Drosophila Rho1 (Rho1) gene during a genetic screen for maternal-effect mutations, allowing us to investigate the specific roles Rho1 plays in the context of the developing organism. Here we report that Rho1 is required for many early events: loss of Rho1 function results in both maternal and embryonic phenotypes. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Two phenotypes are associated with reduction of maternal Rho1 activity: the actin cytoskeleton is disrupted in egg chambers, especially in the ring canals and embryos display patterning defects as a result of improper maintenance of segmentation gene expression. Despite showing imperfect dorsal closure, Rho1 does not activate downstream genes or interact genetically with members of the JNK signaling pathway, used by its relatives dRac and dCdc42 for proper dorsal closure. Consistent with its roles in regulating actin cytoskeletal organization, we find that Rho1 interacts genetically and physically with the Drosophila formin homologue, cappuccino. We also show that Rho1 interacts both genetically and physically with concertina, a G(alpha) protein involved in cell shape changes during gastrulation.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system.

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

Myc and Max homologs in Drosophila.

The proteins encoded by the myc proto-oncogene family are involved in cell proliferation, apoptosis, differentiation, and neoplasia. Myc acts through dimerization with Max to bind DNA and activate transcription. Homologs of the myc and max genes were cloned from the fruit fly Drosophila melanogaster and their protein products (dMyc and dMax) were shown to heterodimerize, recognize the same DNA sequence as their vertebrate homologs, and activate transcription. The dMyc protein is likely encoded by the Drosophila gene diminutive (dm), a mutation in which results in small body size and female sterility caused by degeneration of the ovaries. These findings indicate a potential role for Myc in germ cell development and set the stage for genetic analysis of Myc and Max.

Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression.

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix (bHLH) proteins function as transcriptional repressors. We have examined the mechanisms of repression used by the Hairy and E(SPL) proteins by assaying the antagonism between wild-type or altered Hairy/E(SPL) and Scute bHLH proteins during sex determination in Drosophila melanogaster. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for their function: the bHLH, Orange, and WRPW domains. However, the suppression of Scute activity by Hairy does not require the WRPW domain. We show that the Orange domain is an important functional domain that confers specificity among members of the Hairy/E(SPL) family. In addition, we show that a Xenopus Hairy homology conserves not only Hairy's structure but also its biological activity in our assays. We propose that transcriptional repression by the Hairy/E(SPL) family of bHLH proteins involves two separable mechanisms: repression of specific transcriptional activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.

Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning.

Interaction between distinctly specified cells in adjacent compartments establishes organizing centres that control growth and specify cell fate in the developing limbs of Drosophila. Localized expression of the secreted Hedgehog protein (Hh) by cells in the posterior compartment induces expression of the secreted signalling molecules decapentaplegic (dpp) or wingless (wg) in nearby anterior cells. wg and dpp in turn organize spatial pattern in the wing and leg imaginal discs. The Hh signal is thought to act by antagonizing the ability of the patched (ptc) gene product to repress wg and dpp expression. Here we present evidence that removing activity of the gene encoding cyclic AMP-dependent protein kinase A (pka) is functionally equivalent to removing ptc activity or to providing cells with the Hh signal. These findings suggest that cyclic AMP-dependent protein kinase A is a component of the signal transduction pathway through which Hh and Ptc direct localized expression of dpp (or wg) and establish the compartment boundary organizer.

Sex determination and dosage compensation: lessons from flies and worms.

In both Drosophila melanogaster and Caenorhabditis elegans somatic sex determination, germline sex determination, and dosage compensation are controlled by means of a chromosomal signal known as the X:A ratio. A variety of mechanisms are used for establishing and implementing the chromosomal signal, and these do not appear to be similar in the two species. Instead, the study of sex determination and dosage compensation is providing more general lessons about different types of signaling pathways used to control alternative developmental states of cells and organisms.

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis.

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution.

Different genetic requirements for anterior RNA localization revealed by the distribution of Adducin-like transcripts during Drosophila oogenesis.

The proteins encoded by polar-localized mRNAs play an important role in cell fate specification along the anteroposterior axis of the Drosophila embryo. The only maternally synthesized mRNA known previously to be localized to the anterior cortex of both the oocyte and the early embryo is the bicoid mRNA whose localization is required to generate a homeodomain protein gradient that specifies position along the anteroposterior embryonic axis. We have identified and characterized a second mRNA that is localized to the anterior pole of the oocyte and early embryo. This mRNA encodes a Drosophila homolog of mammalian adducin, a membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. A comparison of the spatial distribution of bicoid and Adducin-like transcripts in the maternal-effect RNA-localization mutants exuperantia, swallow, and staufen indicates different genetic requirements for proper localization of these two mRNAs to the anterior pole of the oocyte and early embryo.

achaete-scute feminizing activities and Drosophila sex determination.

Sex determination in Drosophila depends on X-linked 'numerator' genes activating early Sex-lethal (Sxl) transcription in females. One numerator gene, sisterless-b (sis-b), corresponds to the achaete-scute (AS-C) T4 basic-helix-loop-helix (bHLH) gene. Two other closely related AS-C bHLH genes, T3 and T5, appear not to function as numerator elements. We analyzed endogenous AS-C expression and show that T4 is the major AS-C numerator gene because it is expressed earlier and more strongly than are T3 and T5. Only T4 expression is detectable during the early syncytial stages when Sxl state is being determined. Nevertheless, the effects of ectopic AS-C gene expression show that T3 and T5 proteins display weak but significant feminizing activities, enhancing male-lethality, and rescuing the female-lethality of sis mutations. Detailed examination of Sxl expression in rescued embryos suggests that female cells may be viable in the absence of detectable Sxl protein expression.

Mis-regulating segmentation gene expression in Drosophila.

We have used the hunchback (hb) gap-gene promoter to drive ectopic expression of the pair-rule genes fushi tarazu (ftz), even-skipped (eve) and hairy (h). Unexpectedly, flies transformed with such constructs are viable, despite spatial and temporal mis-regulation of pair-rule expression caused by the fusion genes. We show that fusion gene expression is transcriptionally regulated, such that ectopic expression is suppressed when pattern is established, and present evidence indicating that interstripe hb-ftz expression is repressed by eve. These results are considered in terms of redundant control of pair-rule gene striping. We also discuss the potential dangers of using mis-regulated gene expression to analyse normal function.

wimp, a dominant maternal-effect mutation, reduces transcription of a specific subset of segmentation genes in Drosophila.

wimp is a dominant maternal-effect mutation that interacts with a specific subset of early-acting maternal and zygotic Drosophila genes. We show that wimp is a change-of-function mutation, allelic to mutations of the 140-kD subunit of RNA polymerase, which causes reduced transcription of interacting genes. Loci that do not interact with wimp are expressed at normal levels. We discuss these results in terms of specific interactions between transcription factors and RNA polymerase. Embryos from wimp mothers show unaltered fate maps and develop normally, despite the reduction of transcript levels at least twofold. We suggest that spatial cues are determined by a balance of segmentation gene products rather than their absolute concentrations. We also demonstrate powerful genetic screens for otherwise undetected loci required for segmentation, sex determination, and other early functions.

X:A ratio, the primary sex-determining signal in Drosophila, is transduced by helix-loop-helix proteins.

Drosophila determines its sex by "counting" X chromosomes. We show that premature expression of the pair-rule segmentation gene hairy interferes with this process, resulting in female-specific lethality by inhibiting initiation of the master control gene Sex-lethal (Sxl). The female-specific lethality can be suppressed by a constitutive Sxl allele or by extra copies of X-linked "counting elements." These results are best explained by competition between hairy and other helix-loop-helix transcription factors that act in chromosome counting. We have confirmed this model by showing that misexpression of the achaete-scute T4 gene induces ectopic Sxl expression and male-specific lethality, confirming that achaete-scute T4 is the sisterless-b counting element. We propose that X chromosomes are counted through heterodimers of helix-loop-helix transcription factors that act synergistically to initiate Sxl expression.

Spatial control of hairy protein expression during embryogenesis.

We have used a polyclonal antiserum specific for the Drosophila segmentation gene, hairy (h), to analyse its expression during embryogenesis. The pattern of wild-type expression resembles that of h transcription, being expressed in stripes at the blastoderm stage. h is also expressed later in the stomodaeum, proctodaeum, tracheal pits and mesoderm. We demonstrate that h protein stripes show consistent phase relationships to those of the even-skipped (eve) pair-rule gene. We examine h protein patterns in embryos mutant for other segmentation genes, including h itself. We show that lack of h activity appears not to affect h striping, arguing that h expression is not under autoregulatory control. We also show that h activity is not needed for tracheal invagination. Mutations that are rearranged upstream of the h gene cause the loss of specific stripes, indicating that the h promoter includes activating elements that respond to specific spatial cues. Our observations suggest that pair-rule striping may be under redundant control, and we discuss possible implications for hierarchical models of pair-rule gene action.

The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein.

Homozygous mutations at the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of gypsy-induced alleles in a number of genes located throughout the Drosophila genome. To understand the molecular basis of this phenomenon, the su(Hw) locus was isolated by chromosomal walking from a cloned homeo-box-containing sequence. The exact location of the gene was determined by Southern analysis of the DNA alterations associated with several su(Hw) alleles. A 9.5-kb KpnI-SalI fragment, where all the DNA changes associated with su(Hw) mutations were mapped, was able to rescue the su(Hw) mutant phenotype after P-element-mediated germ-line transformation. This DNA fragment encodes a 3.3-kb RNA that is expressed in all stages of Drosophila development; the size or abundance of this RNA is affected in several su(Hw) alleles tested. This transcript encodes a protein that contains a highly acidic region and 12 repeats of the 'Zn finger' domain characteristic of some DNA-binding and transcription-activating proteins, supporting the hypothesis that the su(Hw) locus might encode a transcription factor that plays a role in the expression of the gypsy element.

Developmental expression of Drosophila melanogaster retrovirus-like transposable elements.

We have determined the pattern of temporal expression of several Drosophila retrovirus-like transposable elements. Some of these elements can be grouped into classes whose members show a similar profile of developmental transcription. The members of the 412 class, which includes 412, mdg1, 17.6 and 3S18, are transcribed mainly in the early larval and pupal stages of development, with small differences among the various members. HMS Beagle and Springer constitute another class where RNA accumulation in the larval stages is higher than in pupae and the adult flies accumulate more RNA than any other stage of development. Finally, the transcription of other elements such as copia, 297 and B104 follows a specific and individual pattern distinct from those described above. These results suggest the existence of evolutionary relationships among different transposable elements in Drosophila and the involvement of different cellular genes in the control of their expression.