Genome-wide and cell-specific epigenetic analysis challenges the role of polycomb in Drosophila spermatogenesis.

The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation.

Co-transcriptional architecture in a Y loop in Drosophila melanogaster.

The Y loops of Drosophila spermatocytes are formed by the expression of huge individual transcription units on the Y chromosome and their large size provides a unique system for the investigation of the organisation of transcription in intact nuclei. By labelling ribonucleo-protein (RNP) components, the loop chromatin and nascent transcripts in Y loop C, we reveal a highly structured organisation of RNP domains associated with nascent transcripts. We distinguish two types of RNP domain, a proximal domain that runs alongside the chromatin of loop C and a distal RNP domain that wraps around the proximal domain and the loop chromatin. The proximal domain is marked by the Pasilla protein, and separate distal subdomains are marked by the S5 antigen and Boule. We discuss the implications of this highly structured co-transcriptional architecture for the organisation of the process of transcription.

A comprehensive map of insulator elements for the Drosophila genome.

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins-dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF-to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.

Alternative splicing modulates Ubx protein function in Drosophila melanogaster.

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments-encoded by individual exons-that modify the distance between the homeodomain and a cofactor-interaction module termed the "YPWM" motif. To investigate the functional implications of Ubx alternative splicing, here we analyze the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, we obtain specific anomalies affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd). Altogether, our results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, we suggest that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development.

Insulators: linking genome architecture to gene regulation.

Insulator elements have long been associated with a proposed domain boundary function, ensuring appropriate associations between regulatory elements and transcription units through the physical organisation of the genome into looped domains. Recent experiments in Drosophila have, however, highlighted a more direct involvement of insulators in transcriptional regulation.

The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning.

Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF-32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. Here we find that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlapped with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal-B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, we demonstrate the importance of dCTCF in fly development and in the regulation of abdominal segmentation.

Genomic analysis of heat-shock factor targets in Drosophila.

We have used a chromatin immunoprecipitation-microarray (ChIP-array) approach to investigate the in vivo targets of heat-shock factor (Hsf) in Drosophila embryos. We show that this method identifies Hsf target sites with high fidelity and resolution. Using cDNA arrays in a genomic search for Hsf targets, we identified 141 genes with highly significant ChIP enrichment. This study firmly establishes the potential of ChIP-array for whole-genome transcription factor target mapping in vivo using intact whole organisms.

Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis.

We have investigated the role of TGIF, a TALE-class homeodomain transcription factor, in Drosophila development. In vertebrates, TGIF has been implicated, by in vitro analysis, in several pathways, most notably as a repressor modulating the response to TGFbeta signalling. Human TGIF has been associated with the developmental disorder holoprosencephaly. Drosophila TGIF is represented by the products of two tandemly repeated highly similar genes, achintya and vismay. We have generated mutations that delete both genes. Homozygous mutant flies are viable and appear morphologically normal, but the males are completely sterile. The defect lies at the primary spermatocyte stage and differentiation is blocked prior to the onset of the meiotic divisions. We show that mutants lacking TGIF function fail to activate transcription of many genes required for sperm manufacture and of some genes required for entry into the meiotic divisions. This groups TGIF together with two other genes producing similar phenotypes, always early and cookie monster, as components of the machinery required for the activation of the spermatogenic programme of transcription. TGIF is the first sequence-specific transcription factor identified in this pathway. By immunolabelling in mouse testes we show that TGIF is expressed in the early stages of spermatogenesis consistent with a conserved role in the activation of the spermatogenesis transcription programme.

Talin is essential for integrin function in Drosophila.

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.