Integrity of the Mod(mdg4)-67.2 BTB domain is critical to insulator function in Drosophila melanogaster

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.     

Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis

Homologous chromosomes must pair and establish stable connections during prophase I of meiosis to segregate reliably from each other at anaphase I. In most organisms, the stable connections, called chiasmata, arise from crossovers. In Drosophila males, homologs pair and segregate without crossing over. Chiasmata are replaced by a homolog conjunction complex that includes the Stromalin in Meiosis (SNM) and Modifier of Mdg4 in Meiosis (MNM) proteins. MNM is one of 31 alternative splice products of mod(mdg4), all of which share a common 402-amino-acid N terminus and differ at their C termini. Previous data demonstrated that an MNM-specific exon is required for homolog conjunction, but did not address whether the N-terminal common region, which includes a BTB domain that can mediate coalescence of protein-DNA complexes, is also required. Here we describe a mutation in the common region of mod(mdg4), Z3-3401, that causes qualitatively similar phenotypes as the MNM-specific alleles but disrupts X-Y segregation much more drastically than autosomal segregation. The mutant MNM protein in Z3-3401 is expressed throughout prophase I in spermatocytes but the protein is confined to the cytoplasm, suggesting that the Z3-3401 mutation disrupts a signal required for nuclear localization or retention. Z3-3401 fails to complement a large battery of lethal and semilethal alleles in the common region for meiotic nondisjunction, including an allele containing an amino acid substitution at a conserved residue in the BTB/POZ domain, consistent with a general requirement for the mod(mdg4) common region in homolog segregation.     

Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila

mod(mdg4), also known as E(var)3-93D, is involved in a variety of processes, such as gene silencing in position effect variegation (PEV), the control of gypsy insulator sequences, regulation of homeotic gene expression, and programmed cell death. We have isolated a large number of mod(mdg4) cDNAs, representing 21 different isoforms generated by alternative splicing. The deduced proteins are characterized by a common N terminus of 402 amino acids, including the BTB/POZ-domain. Most of the variable C termini contain a new consensus sequence, including four positioned hydrophobic amino acids and a Cys(2)His(2) motif. Using specific antibodies for two protein isoforms, we demonstrate different distributions of the corresponding proteins on polytene chromosomes. Mutations in the genomic region encoding exons 1-4 show enhancement of PEV and homeotic transformation and affect viability and fertility. Homeotic and PEV phenotypes are enhanced by mutations in other trx-group genes. A transgene containing the common 5' region of mod(mdg4) that is present in all splice variants known so far partially rescues the recessive lethality of mod(mdg4) mutant alleles. Our data provide evidence that the molecular and genetic complexity of mod(mdg4) is caused by a large set of individual protein isoforms with specific functions in regulating the chromatin structure of different sets of genes throughout development.     

Remote homologue identification of Drosophila GAGA factor in mouse

GAGA factor (GAF) is involved in both gene activation and gene repression and plays a role in the modulation of chromatin structure. In Drosophila, Trithroax like (Trl) gene encodes the DNA binding protein called GAGA factor (GAF). Trl-GAF binds to GAGA sites through its C2H2 zinc finger domain and has an N-terminal BTB/POZ domain. Identification of Trl-GAF homologue in mouse helps in deeper understanding of the mechanism and function. Conventional alignment tools such as BLAST and FASTA cannot identify homologues in mouse genome as their sequence identity is below 30%. In the present study, various sequence and structure analyses were followed for the detection of remote homologues of Drosophila GAGA FACTOR in mouse to identify as Zbtb3. Through homology modeling and docking approach, the zinc finger region of mouse Zbtb3 showed conserved residues and favorable DNA binding sites with GAGA sites similar to that of Drosophila GAGA FACTOR.     

The GAGA protein of Drosophila is phosphorylated by CK2

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.     

Detection of new genes participating in the trans-regulation of locus yellow of MDG-4 in Drosophila melanogaster

The aim of the present work was to obtain mutations in the genes involved in regulation of the yellow locus and mdg4. For this purpose, we searched for mutations changing phenotypic expression of the y(2) mutation induced by mdg4 insertion into the regulatory region of the yellow locus. Mutations have been obtained in the earlier described system of prolonged genome instability, sometimes combined with P-M hybrid dysgenesis. The mutation mod(mdg4) in a novel gene, modifier of mdg4, was detected which either enhanced or suppressed a phenotypic expression of several mutations induced by mdg4 insertion. We suggest that mod(mdg4) controls the expression of mdg4. In addition, the mutations in five loci located on the X chromosome have been found which enhanced the mutation phenotype of several y alleles induced by insertions of different mobile elements in the regulatory region of the latter. Possibly, the protein products of these genes designated as enhancers of yellow-1, 2, 3, 4 and 5 are directly or indirectly involved in control of the yellow locus expression.     

The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo.

The Drosophila gypsy retrotransposon disrupts gene activity by blocking the interactions of distal enhancers with target promoters. This enhancer-blocking activity is mediated by a 340 bp insulator DNA within gypsy. The insulator contains a cluster of binding sites for a zinc finger protein, suppressor of Hairy wing [su(Hw)]. Recent studies have shown that a second protein, mod(mdg4), is also important for normal insulator function. Mutations in mod(mdg4) exert paradoxical effects on different gypsy-induced phenotypes. For example, it enhances yellow2 but suppresses cut6. Here, we employ a stripe expression assay in transgenic embryos to investigate the role of mod(mdg4) in gypsy insulator activity. The insulator was inserted between defined enhancers and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. These assays indicate that mod(mdg4) is essential for the enhancer-blocking activity of the insulator DNA. Moreover, reductions in mod(mdg4)+ activity cause the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ. The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. These results provide an explanation for why mod(mdg4) exerts differential effects on different gypsy-induced mutations.     

Effect of mutations in the genes su(Hw) and mod(mdg4) on transvection between alleles of the Yellow locus in Drosophila melanogaster]

A typical example of transvection is a complementation between alleles in the yellow locus: y2 (mdg4 insertion inactivating certain y-enhancers) and y1 (deletion of the y-promoter but not of the enhancer). Transvection was explained by trans-activation of promoter in y2-allele by enhancer of y1-allele. Here we found that the mutation mod(mdg4)1u1 in the modifier of mdg4 locus (a regulatory gene controlling, together with suppressor of Hairy wing) expression of (mdg4) completely suppress the complementation. Removal of an acidic domain from su(Hw) protein product in su(Hw)j mutation partially suppress the complementation. We also have found that mod(mdg4)1u1 mutation trans-inactivates the yellow allele with a wild type phenotype (y+2MC) in heterozygote with the y2 allele, i.e. the negative transvection takes place. In this case, deletion removing an acidic domain even in one copy of su(Hw) suppresses the effect of mod(mdg4)1u1 mutation.     

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.     

EAST affects the activity of Su(Hw) insulators by two different mechanisms in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.