The interaction of DIAP1 with dOmi/HtrA2 regulates cell death in Drosophila.

Mitochondrial proteins such as cytochrome c, Smac/DIABLO and Omi/HtrA2 play important roles in the cell death pathways of mammalian cells. In Drosophila, the role of mitochondria in cell death is less clear. Here, we report the identification and characterization of the Drosophila ortholog of human Omi/HtrA2. We show that Drosophila Omi/HtrA2 is imported into the mitochondria where it undergoes proteolytic maturation to yield two isoforms, dOmi-L and dOmi-S. dOmi-L contains a canonical N-terminal IAP-binding motif (AVVS), whereas dOmi-S contains a distinct N-terminal motif (SKMT). DIAP1 was able to bind to both isoforms via its BIR1 and BIR2 domains. This resulted in cleavage of the linker region of DIAP1 between the BIR1 and BIR2 domains and further degradation of the BIR1 domain by the proteolytic activity of dOmi. The binding of DIAP1 to dOmi also resulted in DIAP1-mediated polyubiquitination of dOmi, suggesting that DIAP1 could target dOmi for proteasomal degradation. Consistent with this, expression of DIAP1 in Drosophila eye discs protected them from dOmi-induced eye ablation, indicating that DIAP1 plays an important role in protecting cells from the potentially lethal effects of dOmi. The ability of IAPs to bind to and ubiquitinate mitochondrial proteins such as dOmi may be a key conserved function to counterbalance the lethal effects of these proteins if accidentally released into the cytosol.

Drosophila SAP18, a member of the Sin3/Rpd3 histone deacetylase complex, interacts with Bicoid and inhibits its activity.

Bicoid directs anterior development in Drosophila embryos by activating different genes along the anterior-posterior axis. However, its activity is down-regulated at the anterior tip of the embryo, in a process known as retraction. Retraction is under the control of the terminal polarity system, and results in localized repression of Bicoid target genes. Here, we describe a Drosophila homolog of human SAP18, a member of the Sin3A/Rpd3 histone deacetylase complex. dSAP18 interacts with Bicoid in yeast and in vitro, and is expressed early in development coincident with Bicoid. In tissue culture cells, dSAP18 inhibits the ability of Bicoid to activate reporter genes. These results suggest a model in which dSAP18 interacts with Bicoid to silence expression of Bicoid target genes in the anterior tip of the embryo.

Groucho augments the repression of multiple Even skipped target genes in establishing parasegment boundaries.

Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped has previously been shown to contain a Groucho-independent repression activity. In the Even skipped protein, outside the Groucho-independent repression domain, we have identified a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein. Even skipped interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Even skipped with a mutated Groucho interaction motif, which abolished binding to Groucho, showed a significantly reduced ability to rescue the even skipped null phenotype when driven by the complete even skipped regulatory region. Replacing this motif with a heterologous Groucho interaction motif restored the rescuing function of Even skipped in segmentation. Further functional assays demonstrated that the Even skipped C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain was active on two target genes that are normally repressed by Even skipped at different concentrations, paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Even skipped to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions.

even skipped is required to produce a trans-acting signal for larval neuroblast proliferation that can be mimicked by ecdysone.

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.

Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila.

During the development of the nervous system embryonic neurons are incorporated into neural networks that underlie behaviour. For example, during embryogenesis in Drosophila, motor neurons in every body segment are wired into the circuitry that drives the simple peristaltic locomotion of the larva. Very little is known about the way in which the necessary central synapses are formed in such a network or how their properties are controlled. One possibility is that presynaptic and postsynaptic elements form relatively independently of each other. Alternatively, there might be an interaction between presynaptic and postsynaptic neurons that allows for adjustment and plasticity in the embryonic network. Here we have addressed this issue by analysing the role of synaptic transmission in the formation of synaptic inputs onto identified motorneurons as the locomotor circuitry is assembled in the Drosophila embryo. We targeted the expression of tetanus toxin light chain (TeTxLC) to single identified neurons using the GAL4 system. TeTxLC prevents the evoked release of neurotransmitter by enzymatically cleaving the synaptic-vesicle-associated protein neuronal-Synaptobrevin (n-Syb) [1]. Unexpectedly, we found that the cells that expressed TeTxLC, which were themselves incapable of evoked release, showed a dramatic reduction in synaptic input. We detected this reduction both electrophysiologically and ultrastructurally.

Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.

Drosophila homeobox gene eve enhances trol, an activator of neuroblast proliferation in the larval CNS.

The regulation of stem cell division by developmental cues is critical for the assembly and function of multicellular organisms. Stem cell division in the Drosophila brain is controlled by trol, which is required for activation of proliferation by quiescent neuroblasts at the appropriate stage of larval development. We show that the transcriptional regulator eve is part of the trol activation pathway by identifying eve as a dominant enhancer of a weak trol allele, trolb22. Known eve mutations are capable of enhancing the lethality of trolb22 and uncovering a defective neuroblast proliferation phenotype. Additionally, genetic and molecular analysis reveals that an independent mutation which acts as a dominant enhancer of trol is also an allele of eve. The enhancement of trolb22 lethality can be suppressed by the presence of an eve transgene. Interestingly, extra copies of eve supplied by the eve transgene also enhance trolb22 lethality, suggesting that the level of Eve protein may be critical for neuroblast activation. Finally, activation of neuroblast proliferation is normal in eve4 heterozygotes, suggesting that the proliferation defect observed in trolb22;eve/+ animals is due to a synergistic interaction.

Two distinct types of repression domain in engrailed: one interacts with the groucho corepressor and is preferentially active on integrated target genes.

Active transcriptional repression has been characterized as a function of many regulatory factors. It facilitates combinatorial regulation of gene expression by allowing repressors to be dominant over activators under certain conditions. Here, we show that the Engrailed protein uses two distinct mechanisms to repress transcription. One activity is predominant under normal transient transfection assay conditions in cultured cells. A second activity is predominant in an in vivo active repression assay. The domain mediating the in vivo activity (eh1) is highly conserved throughout several classes of homeoproteins and interacts specifically with the Groucho corepressor. While eh1 shows only weak activity in transient transfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relative activities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of the assay to the Groucho interaction domain, albeit to a lesser extent. This suggests that it utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on integrated target genes.

A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo.

The engrailed homeoprotein is a dominantly acting or 'active' transcriptional repressor both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), it actively represses it, resulting in a ftz mutant phenocopy. We have mapped functional regions of engrailed using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays (K. Han and J. L. Manley (1993) EMBO J. 12, 2723-2733), we find that two evolutionarily conserved regions contribute to activity. The one of these that does not flank the HD is particularly crucial to repression activity in vivo. We find that this domain is present not only in all engrailed-class homeoproteins but also in all known members of several other classes, including goosecoid, Nk1, Nk2 and msh. Thus engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. We further show using rescue transgenes that the widely conserved in vivo repression domain is required for the normal function of engrailed in the embryo.

Early even-skipped stripes act as morphogenetic gradients at the single cell level to establish engrailed expression.

even-skipped (eve) has been proposed to set up parasegment borders at the anterior edge of each of its seven stripes by providing a sharp expression boundary, where engrailed is activated on one side and wingless on the other. By expressing bell-shaped early eve stripes without the sharp boundary provided by narrow, late stripes, we find that the early gradient is sufficient for generating stable parasegment borders. Based on several lines of evidence, we propose that the anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not. While the early eve gradient is sufficient to establish parasegmental borders without refined, late expression, late eve expression has a role in augmenting this boundary to provide for strong, continuous stripes or engrailed expression. In addition, we show that the early eve gradient is sufficient, at its posterior edge, for subdividing the ftz domain into engrailed expressing and non-expressing cells.

Inserting the Ftz homeodomain into engrailed creates a dominant transcriptional repressor that specifically turns off Ftz target genes in vivo.

The Engrailed homeodomain protein is an 'active' or dominant transcriptional repressor in cultured cells. In contrast, the Fushi Tarazu homeodomain protein is an activator, both in cultured cells and in Drosophila embryos, where it activates several known target genes, including its own gene. This auto-activation has been shown to depend on targeting to a fushi tarazu enhancer by the Fushi Tarazu homeodomain. We combined Fushi Tarazu targeting and Engrailed active repression in a chimeric regulator, EFE. When EFE is ubiquitously expressed, it overrides endogenous Fushi Tarazu and causes a fushi tarazu mutant phenotype. Normal Fushi Tarazu target genes are affected as they are in fushi tarazu mutants. One such target gene is repressed by EFE even where Fushi Tarazu is not expressed, suggesting that the repression is active. This is confirmed by showing that the in vivo activity of EFE depends on a domain that is required for active repression in culture. A derivative that lacks this domain, while it cannot repress the endogenous fushi tarazu gene, can still reduce the activity of the fushi tarazu autoregulatory enhancer, suggesting that it competes with endogenous Fushi Tarazu for binding sites in vivo. However, this passive repression is much less effective than active repression.

Active repression of transcription by the engrailed homeodomain protein.

The Drosophila engrailed gene product (En) is a homeodomain-containing protein that contributes to segmental patterning. In transfection assays it acts as a transcriptional repressor. We show that En is an active repressor, blocking activation by mammalian and yeast activators that bind to sites some distance away from those bound by En. Active repression is distinct from the effects of passive homeodomain-containing proteins, which repress when competing with activators for binding sites and activate when competing with En. Active repression activity maps outside the En homeodomain, and this activity can be transferred to a heterologous DNA binding domain.

Expression of a transfected mouse muscle-creatine kinase gene is induced upon growth factor deprivation of myogenic but not of nonmyogenic cells.

To determine whether mitogen-regulated expression of skeletal muscle genes is independent of cell type, muscle and nonmuscle cells were transfected with cloned 5'-flanking sequences of muscle creatine kinase (MCK) fused to a heterologous reporter gene and tested for expression in high and low mitogen culture conditions. Consistent with the behavior of endogenous MCK, a -3300MCK-CAT gene is expressed at high levels in differentiated muscle cells but at low to undetectable levels in proliferating myoblasts and in either mitogen-deprived or stimulated nonmuscle cells of mesodermal, ectodermal, or endodermal origin. A -776MCK-CAT gene behaves similarly with respect to its cell type specificity but it supports only an intermediate expression level in response to mitogen deprivation in skeletal muscle cells. These data suggest that the -3300 to +7 nucleotide region of mouse MCK contains one or more elements which are activable by mitogen deprivation only in myogenic cells.

Activation and repression of transcription by homoeodomain-containing proteins that bind a common site.

The product of the fushi tarazu (ftz) gene is shown to be a site-dependent activator of transcription. In vitro-defined binding sites act as ftz-dependent enhancers in cultured cells. Another homoeodomain-containing protein, the engrailed gene product, competes for homoeodomain-binding sites and counteracts ftz activation.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer.

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.

The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.

Regulation of creatine kinase induction in differentiating mouse myoblasts.

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.

The mouse muscle creatine kinase cDNA and deduced amino acid sequences: comparison to evolutionarily related enzymes.

The nucleotide sequence of cloned DNA corresponding to full-length mouse muscle creatine kinase mRNA has been determined. This 1415 base pair DNA sequence and the deduced 381 amino acid sequence of the protein have been compared to creatine kinase sequences from other vertebrate species and to invertebrate guanidino kinase sequences. These comparisons show that the vertebrate muscle creatine kinases constitute a remarkably conserved protein family with a unit evolutionary period of 30. The creatine kinases also retain marked sequence similarity with the more distantly related invertebrate guanidino kinases. A portion of the sequence, presumably part of the ATP binding site, shows similarity to other nucleotide binding proteins with diverse functions. Comparisons of the untranslated regions of the creatine kinase cDNA sequences show that the 5' untranslated regions are more highly conserved than are the 3' untranslated regions; this may point to some regulatory function in the 5' region.