Genetic interactions of the suppressor 2 of zeste region genes

A wide variety of gain of function mutations have been induced in the Posterior Sex Comb (Psc)--Aristapedioid (Arp)--Suppressor 2 of zeste (Su(z)2) region of the second chromosome of Drosophila. This region contains at least three apparently related genes, two of which we have been studying. Psc1 has previously been used to identify Psc as a Pc group gene; however, it is a complex mutation with both gain and loss of function character. We report here that the Pc group character of Psc is not due to a gain of function and presumably reflects the function of the wild-type gene. We also provide evidence for a maternal function for Psc, as well as the neighboring Su(z)2 gene. Su(z)2 does not appear to be a Pc group gene as it does not act in a synergistic fashion with other Pc group genes in promoting posteriorly directed transformations. However, we have found that mutations in Su(z)2 do interact in a variety of interesting ways with mutations in Pc group genes.     

Identification of a tetrameric hedgehog signaling complex

Hedgehog (Hh) signal transduction requires a large cytoplasmic multi-protein complex that binds microtubules in an Hh-dependent manner. Here, we show that three members of this complex, Costal2 (Cos2), Fused (Fu), and Cubitus interruptus (Ci), bind each other directly to form a trimeric complex. We demonstrate that this trimeric signaling complex exists in Drosophila lacking Suppressor of Fused (Su(fu)), an extragenic suppressor of fu, indicating that Su(fu) is not required for the formation, or apparently function, of the Hh signaling complex. However, we subsequently show that Su(fu), although not a requisite component of this complex, does form a tetrameric complex with Fu, Cos2, and Ci. This additional Su(fu)-containing Hh signaling complex does not appear to be enriched on microtubules. Additionally, we demonstrate that in response to Hh Ci accumulates in the nucleus without its various cytoplasmic binding partners, including Su(fu). We discuss a model in which Su(fu) and Cos2 each bind to Fu and Ci to exert some redundant effect on Ci such as cytoplasmic retention. This model is consistent with genetic data demonstrating that Su(fu) is not required for Hh signal transduction proper and with the elaborate genetic interactions observed among Su(fu), fu, cos2, and ci.     

'Insulator bodies' are aggregates of proteins but not of insulators

Chromatin insulators are thought to restrict the action of enhancers and silencers. The best-known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator-related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are 'insulator bodies' of many Su(Hw)-insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q-rich domain supports the function of Su(Hw)-dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)-marked nuclear speckles; conversely, the latter accumulate another (C-truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so-called 'insulator bodies'.     

The ability of the Su(Hw) protein to create a platform for ORC binding does not depend on the type of surrounding chromatin

DNA replication begins from multiple sites distributed throughout the genome, named replication origins. Despite the increasing amount of data on the properties of replication origins, it is still unknown what factors are the primary determinants of ORC localization. Su(Hw) is a zinc-finger protein responsible for the activity of the best-studied Drosophila insulators. In the present work, we show that the insulator protein Su(Hw) recruits the histon acetyltransferase complex SAGA and chromatin remodeler dSWI/SNF to Su(Hw)-dependent insulators and creates a platform for ORC binding. We have found Su(Hw) to be necessary for chromatin remodeling and ORC recruitment regardless of the surrounding chromatin type. Thus, the global chromatin state does not affect the molecular mechanism underlying ORC positioning in genome; it is rather the DNA-binding proteins that are the key determinants that create the proper chromatin structure for ORC binding. Su(Hw) is the first example of such protein.     

Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila

Hedgehog (Hh) signaling plays vital roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and several types of cancer. Suppressor of Fused (Su(fu)) is a conserved inhibitory component of the Hh signaling pathway, but how it is regulated remains poorly understood. Here we demonstrate that in Drosophila Hh signaling promotes downregulation of Su(fu) through its target protein HIB (Hh-induced BTB protein). Interestingly, although HIB-mediated downregulation of Su(fu) depends on the E3 ubiquitin ligase Cul3, HIB does not directly regulate Su(fu) protein stability. Through an RNAi-based candidate gene screen, we identify the spliceosome factor Crooked neck (Crn) as a regulator of Su(fu) level. Epistasis analysis indicates that HIB downregulates Su(fu) through Crn. Furthermore, we provide evidence that HIB retains Crn in the nucleus, leading to reduced Su(fu) protein level. Finally, we show that SPOP, the mammalian homologue of HIB, can substitute HIB to downregulate Su(fu) level in Drosophila. Our study suggests that Hh regulates both Ci and Su(fu) levels through its target HIB, thus uncovering a novel feedback mechanism that regulates Hh signal transduction. The dual function of HIB may provide a buffering mechanism to fine-tune Hh pathway activity.     

Rex and a Suppressor of Rex Are Repeated Neomorphic Loci in the Drosophila Melanogaster Ribosomal DNA

We report the molecular characterization of the Posterior sex combs-Suppressor 2 of zeste region of Drosophila melanogaster. The distal breakpoint of the Aristapedioid inversion divides the region into two parts. We have molecularly mapped the lesions associated with several loss of function mutations in the Polycomb group gene Posterior sex combs (Psc) proximal to this breakpoint. In addition, we have found that lesions associated with several loss of function mutations in the Suppressor 2 of zeste [Su(z)2] gene lie distal to this breakpoint. Since the breakpoint does not cause a loss of function in either gene, no essential sequences are shared by these two neighboring genes. There are three dominant gain of function mutations in the region that result in abnormal bristle development. We find that all three juxtapose foreign DNA sequences upstream of the Su(z)2 gene, and that at least two of these mutations (Arp1 and vgD) behave genetically as gain of function mutations in Su(z)2. Northern and in situ hybridization analyses show that the mutations result in increased accumulation of the Su(z)2 mRNA, which we argue is responsible for the bristle loss phenotype.     

The centrosomal protein CP190 is a component of the gypsy chromatin insulator

Chromatin insulators, or boundary elements, affect promoter-enhancer interactions and buffer transgenes from position effects. The gypsy insulator of Drosophila is bound by a protein complex with two characterized components, the zinc finger protein Suppressor of Hairy-wing [Su(Hw)] and Mod(mdg4)2.2, which is one of the multiple spliced variants encoded by the modifier of mdg4 [mod(mdg4)] gene. A genetic screen for dominant enhancers of the mod(mdg4) phenotype identified the Centrosomal Protein 190 (CP190) as an essential constituent of the gypsy insulator. The function of the centrosome is not affected in CP190 mutants whereas gypsy insulator activity is impaired. CP190 associates physically with both Su(Hw) and Mod(mdg4)2.2 and colocalizes with both proteins on polytene chromosomes. CP190 does not interact directly with insulator sequences present in the gypsy retrotransposon but binds to a previously characterized endogenous insulator, and it is necessary for the formation of insulator bodies. The results suggest that endogenous gypsy insulators contain binding sites for CP190, which is essential for insulator function, and may or may not contain binding sites for Su(Hw) and Mod(mdg4)2.2.     

Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates

The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.     

Reduced levels of Su(var)3-9 but not Su(var)2-5 (HP1) counteract the effects on chromatin structure and viability in loss-of-function mutants of the JIL-1 histone H3S10 kinase

It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase is a major regulator of chromatin structure and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of JIL-1 kinase activity, the major heterochromatin markers histone H3K9me2 and HP1 spread in tandem to ectopic locations on the chromosome arms. In this study, we show that the lethality as well as some of the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-9 gene. This effect was observed with three different alleles of Su(var)3-9, strongly suggesting it is specific to Su(var)3-9 and not to second site modifiers. This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene that codes for HP1 in Drosophila where no genetic interactions were detectable between JIL-1 and Su(var)2-5. Taken together, these findings indicate that while Su(var)3-9 histone methyltransferase activity is a major factor in the lethality and chromatin structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1.     

Down-regulation of Notch target gene expression by Suppressor of deltex

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.     

An Arabidopsis cotyledon-specific albino locus: a possible role in 16S rRNA maturation

We report here the isolation and characterization of a cotyledon-specific albino locus of Arabidopsis, WHITE COTYLEDONS (WCO). This recessive mutation in the WCO locus, located on the top of Chromosome 1, results in albino cotyledons but green true leaves. An accumulation profile of chlorophylls and ultrastructure of chloroplasts indicate that WCO is necessary for development of functional chloroplasts in cotyledons but is dispensable in true leaves. This was further supported by the fact that the mutants request feeding of sucrose for their survival at the early seedling stage where true leaves have not emerged, but the mutants which have developed true leaves are able to grow autotrophically without sucrose supplementation. The wco mutants accumulate low levels of chloroplast mRNA encoding photosynthesis-related proteins and have a specific defect in 16S rRNA maturation in a cotyledon-specific manner. Although wco mutants exhibited abnormal chloroplasts and chloroplast gene expression in cotyledons, nuclear genes for photosynthetic components are expressed at similar levels to those found in wild-type siblings. This lack of suppression of the nuclear genes is not due to a defect in the signaling of the so-called "plastid factor" to the nucleus since normal suppression of the nuclear genes was observed in response to the photo-oxidative damage due to norflurazon application.     

Insulation of Enhancer-Promoter Communication by a Gypsy Transposon Insert in the Drosophila cut Gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 Proteins

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein.     

Evolutionarily conserved E(y)2/Sus1 protein is essential for the barrier activity of Su(Hw)-dependent insulators in Drosophila

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for spreading of repressive chromatin. The Su(Hw) protein is responsible for activity of the best-studied Drosophila insulators. Here we demonstrate that an evolutionarily conserved protein, E(y)2/Sus1, is recruited to the Su(Hw) insulators via binding to the zinc-finger domain of Su(Hw). Partial inactivation of E(y)2 in a weak mutation, e(y)2(u1), impairs only the barrier, but not the enhancer-blocking, activity of the Su(Hw) insulators. Whereas neither su(Hw)(-) nor e(y)2(u1) affects fly viability, their combination proves lethal, testifying to functional interaction between Su(Hw) and E(y)2 in vivo. Apparently, different domains of Su(Hw) recruit proteins responsible for enhancer-blocking and for the barrier activity.     

Impairment of cytotype regulation of P-element activity in Drosophila melanogaster by mutations in the Su(var)205 gene

Cytotype regulation of transposable P elements in the germ line of Drosophila melanogaster is associated with maternal transmission of P elements inserted at the left telomere of the X chromosome. This regulation is impaired in long-term stocks heterozygous for mutations in Suppressor of variegation 205 [Su(var)205], a gene implicated in the control of telomere length. Regulation by TP5, a structurally incomplete P element at the X telomere, is more profoundly impaired than regulation by TP6, a different incomplete P element inserted at the same site in a TAS repeat at the X telomere. Genetic analysis with the TP5 element indicates that its regulatory ability is not impaired in flies whose fathers came directly from a stock heterozygous for a Su(var)205 mutation, even when the flies themselves carry this mutation. However, it is impaired in flies whose grandfathers came from such a stock. Furthermore, this impairment occurs even when the Su(var)205 mutation is not present in the flies themselves or in their mothers. The impaired regulatory ability of TP5 persists for at least several generations after TP5 X chromosomes extracted from a long-term mutant Su(var)205 stock are made homozygous in the absence of the Su(var)205 mutation. Impairment of TP5-mediated regulation is therefore not directly dependent on the Su(var)205 mutation. However, it is characteristic of the six mutant Su(var)205 stocks that were tested and may be related to the elongated telomeres that develop in these stocks. Impairment of regulation by TP5 is also seen in a stock derived from Gaiano, a wild-type strain that has elongated telomeres due to a dominant mutation in the Telomere elongation (Tel) gene. Regulation by TP6 is not impaired in the Gaiano genetic background. The regulatory abilities of the TP5 and TP6 elements are therefore not equally susceptible to the effects of elongated telomeres in the mutant Su(var)205 and Gaiano stocks.     

The yeast F(1)F(0)-ATP synthase: analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif

The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.