Molecular and genetic description of a new hypomorphic mutation of Trithorax-like gene and analysis of its effect on Drosophila melanogaster oogenesis

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional protein GAGA involved in many cellular processes. We have isolated and described a new hypomorphic mutation of The Trl gene—Trl _en82 . The mutation is the insertion of a 1.4 kb P-element into the 5′ untranslated region. Trl expression decreased in the ovaries of mutant flies by about 30%; however, it caused abnormalities. The Trl _en82 mutation combined with the null allele of Trl caused female sterility: the females laid a few small eggs with abnormal shape. Many egg chambers demonstrated abnormalities in the Trl _en82 mutants: the oocyte had a regular shape and intruded into the egg chamber region with nurse cells; the rapid transport of nurse cell cytoplasm into the oocyte was disturbed, which resulted in the “dumpless” phenotype of the chambers in mutants; follicular cells often did not completely cover the oocyte and concentrated on its posterior end; and the migration of centripetal cells was affected. We propose that the sterility of the Trl _en82 females is due to the abnormal functioning of follicular cells resulting from low Trlexpression. This proposal is confirmed by normalizing the mutant phenotype of Trl _en82 females after the transfection of Trl cDNA. Note that even an insignificant decrease in Trl expression in such females seriously affected the somatic cell functioning, while a significant decrease in its expression in strong hypomorphic mutants affected both somatic and germline cells in the egg chambers.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.     

Vertebrate Homologue of Drosophila GAGA Factor

Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like (Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c-Krox/Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.     

The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide

SAP18, a polypeptide associated with the Sin3–HDAC co-repressor complex, was identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, dSAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the dSAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl⁶⁷ show the homeotic transformation of segment A6 into A5, indicating that GAGA–dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3–HDAC complex, GAGA might contribute to the regulation of homeotic gene expression.     

Generation and analysis of novel mutations of the Trithorax-like gene in Drosophila melanogaster

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional GAGA factor. The expression of Trl is known to depend on numerous factors, such as the organ, the tissue, the ontogenetic stage, and the ambient temperature. Apparently, this expression is controlled by a complex system of regulatory elements, which so far has been scarcely studied. Our preliminary results indicate that the second intron of the Trl gene bears functionally significant elements. To test this assumption, we generated 23 novel alleles of the gene via P-induced male recombination and analyzed them cytogenetically. Of these mutations, 13 (recessive lethals) are deletions, disrupting the coding gene region. Ten mutations (seven deletions and three duplications) remove parts of the second Trl intron only. Some of these mutant stocks exhibit lower viability at different temperatures. These results suggest that the second intron region harbors functionally significant elements. The deletion mapping results verified the localization of the Trl gene in the 70F1-2 region.     

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3–HDAC co-repressor complex. GAGA–dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.     

GAGA protein is essential for male germ cell development in Drosophila

The Drosophila Trithorax‐like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. In this study, we identify a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy, and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm. genesis 52:738–751, 2014. © 2014 Wiley Periodicals, Inc.     

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brown^Dominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.     

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.