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.     

Epigenetic gene regulation by noncoding RNAs

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.     

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.     

Retrogenes Moved Out of the Z Chromosome in the Silkworm

Previous studies on organisms with well-differentiated X and Y chromosomes, such as Drosophila and mammals, consistently detected an excess of genes moving out of the X chromosome and gaining testis-biased expression. Several selective evolutionary mechanisms were shown to be associated with this nonrandom gene traffic, which contributed to the evolution of the X chromosome and autosomes. If selection drives gene traffic, such traffic should also exist in species with Z and W chromosomes, where the females are the heterogametic sex. However, no previous studies on gene traffic in species with female heterogamety have found any nonrandom chromosomal gene movement. Here, we report an excess of retrogenes moving out of the Z chromosome in an organism with the ZW sex determination system, Bombyx mori. In addition, we showed that those "out of Z" retrogenes tended to have ovary-biased expression, which is consistent with the pattern of non-retrogene traffic recently reported in birds and symmetrical to the retrogene movement in mammals and fruit flies out of the X chromosome evolving testis functions. These properties of gene traffic in the ZW system suggest a general role for the heterogamety of sex chromosomes in determining the chromosomal locations and the evolution of sex-biased genes.     

The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.     

Drosophila retinal pigment cell death is regulated in a position-dependent manner by a cell memory gene

The stereotyped organization of the Drosophila compound eye depends on the elimination by apoptosis of about 25% of the inter-ommatidial pigment cell precursors (IOCs) during metamorphosis. This program of cell death is under antagonistic effects of the Notch and the EGFR pathways. In addition, uncharacterized positional cues may underlie death versus survival choices among IOCs. Our results provide new genetic evidences that cell death is regulated in a position- dependent manner in the eye. We show that mutations in Trithorax-like (Trl) and lola-like/batman specifically block IOC death during eye morphogenesis. These genes share characteristics of both Polycomb-Group and trithorax-Group genes, in that they are required for chromatin-mediated repression and activation of Hox genes. However, Trl function in triggering IOC death is independent from a function in repressing Hox gene expression during eye development. Analysis of mosaic ommatidiae containing Trl mutant cells revealed that Trl function for IOC death is required in cone cells. Strikingly, cell death suppression in Trl mutants depends on the position of IOCs. Our results further support a model whereby death of IOCs on the oblique sides of ommatidiae requires Trl-dependent reduction of a survival signal, or an increase of a death signal, emanating from cone cells. Trl does not have the same effect on horizontal IOCs whose survival seems to involve additional topological constraints.     

Inferring the history of interchromosomal gene transposition in Drosophila using n-dimensional parsimony

Gene transposition puts a new gene copy in a novel genomic environment. Moreover, genes moving between the autosomes and the X chromosome experience change in several evolutionary parameters. Previous studies of gene transposition have not utilized the phylogenetic framework that becomes possible with the availability of whole genomes from multiple species. Here we used parsimonious reconstruction on the genomic distribution of gene families to analyze interchromosomal gene transposition in Drosophila. We identified 782 genes that have moved chromosomes within the phylogeny of 10 Drosophila species, including 87 gene families with multiple independent movements on different branches of the phylogeny. Using this large catalog of transposed genes, we detected accelerated sequence evolution in duplicated genes that transposed when compared to the parental copy at the original locus. We also observed a more refined picture of the biased movement of genes from the X chromosome to the autosomes. The bias of X-to-autosome movement was significantly stronger for RNA-based movements than for DNA-based movements, and among DNA-based movements there was an excess of genes moving onto the X chromosome as well. Genes involved in female-specific functions moved onto the X chromosome while genes with male-specific functions moved off the X. There was a significant overrepresentation of proteins involving chromosomal function among transposed genes, suggesting that genetic conflict between sexes and among chromosomes may be a driving force behind gene transposition in Drosophila.     

Guilt by association: non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila.

Dosage compensation is a striking example of the interplay between gene-specific regulation and chromosomal architecture. This process has evolved to make X-linked gene expression equivalent in males with one X chromosome and females with two. Examining species at the molecular level has shown that dosage compensation is mediated by sex-specific factors that decorate the X chromosomes to regulate chromatin structure and gene expression. In Drosophila, dosage compensation is achieved, at least in part, through site-specific histone H4 acetylation, which is modulated by a male- and X-specific protein complex. The discovery of non-coding RNAs that 'paint' dosage-compensated X chromosomes in mammals and in Drosophila suggests that RNAs play an intriguing, unexpected role in the regulation of chromatin structure and gene expression.     

The Drosophila GAGA factor is required for dosage compensation in males and for the formation of the male-specific-lethal complex chromatin entry site at 12DE

Drosophila melanogaster males have one X chromosome, while females have two. To compensate for the resulting disparity in X-linked gene expression between the two sexes, most genes from the male X chromosome are hyperactivated by a special dosage compensation system. Dosage compensation is achieved by a complex of at least six proteins and two noncoding RNAs that specifically associate with the male X. A central question is how the X chromosome is recognized. According to a current model, complexes initially assemble at approximately 35 chromatin entry sites on the X and then spread bidirectionally along the chromosome where they occupy hundreds of sites. Here, we report that mutations in Trithorax-like (Trl) lead to the loss of a single chromatin entry site on the X, male lethality, and mislocalization of dosage compensation complexes.     

Low occurrence of gene transposition events during the evolution of the genus Drosophila

Abstract.— The role played by gene transpositions during the evolution of eukaryotic genomes is still poorly understood and indeed has been analyzed in detail only in nematodes. In Drosophila , a limited number of transpositions have been detected by comparing the chromosomal location of genes between different species. The relative importance of gene transposition versus other types of chromosomal rearrangements, for example, inversions, has not yet been evaluated. Here, we use physical mapping to perform an extensive search for long-distance gene transpositions and assess their impact during the evolution of the Drosophila genome. We compare the relative order of 297 molecular markers that cover 60% of the euchromatic fraction of the genome between two related Drosophila species and conclude that the frequency of gene transpositions is very low, namely one order of magnitude lower than that of nematodes. In addition, gene transpositions seem to be events almost exclusively associated with genes of repetitive nature such as the Histone gene complex ( HIS-C ).     

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.     

The MCP silencer of the Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.