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Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
+44 (0)1865 483269
Regulation of Gene Expression
We are made up of cells that differ dramatically in their size, shape, function, longevity and ability to keep dividing even though, with few exceptions, every cell contains the same genome. The great diversity of cells observed in an adult human is a consequence of the different cell types expressing different profiles of genes. Failure of correct gene regulation during foetal development usually results in death or severe congenital defects, and in adult life leads to disease including cancer.
My research group uses a combination of Drosophila genetics, molecular biology, biochemistry and immunohistochemistry to study the mechanisms that transcription factors use to regulate gene expression during animal development.
Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active'' chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing.
The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner.
Factors affecting transcriptional elongation have been characterized extensively in in vitro, single cell (yeast) and cell culture systems; however, data from the context of multicellular organisms has been relatively scarce. While studies in homogeneous cell populations have been highly informative about the underlying molecular mechanisms and prevalence of polymerase pausing, they do not reveal the biological impact of perturbing this regulation in an animal. The core components regulating pausing are expressed in all animal cells and are recruited to the majority of genes, however, disrupting their function often results in discrete phenotypic effects. Mutations in genes encoding key regulators of transcriptional pausing have been recovered from several genetic screens for specific phenotypes or interactions with specific factors in mice, zebrafish and flies. Analysis of these mutations has revealed that control of transcriptional pausing is critical for a diverse range of biological pathways essential for animal development and survival.
The fruit fly Drosophila melanogaster is a versatile model organism that has been used in biomedical research for over a century to study a broad range of phenomena. There are many technical advantages of using Drosophila over vertebrate models; they are easy and inexpensive to culture in laboratory conditions, have a much shorter life cycle, they produce large numbers of externally laid embryos and they can be genetically modified in numerous ways. Research using Drosophila has made key advances in our understanding of regenerative biology and will no doubt contribute to the future of regenerative medicine in many different ways.
Bio-electrosprays (BESs) provide a means of precisely manipulating cells and thus have the potential for many clinical uses such as the generation of artificial tissues/organs. Previously we tested the biological safety of this technology with a variety of living cells and also embryos from the vertebrate model organisms Danio rerio (zebrafish) and Xenopus tropicalis (frog). However, the viability and fertility of the treated embryos could not be fully assessed due to animal licensing laws. Here we assay the viability and fertility of Drosophila melanogaster (fruit fly) embryos in conjunction with the bio-electrospray procedure. Bio-electrosprayed Drosophila embryos developed into fully fertile adult flies that were indistinguishable from wild-type. Thus, we demonstrate that the bio-electrospray procedure does not induce genetic or physical damage that significantly affects the development or fertility of a multicellular organism. This study along with our previous investigations demonstrates the potential of this approach to be developed for the precise manipulation of sensitive biological materials.
The Groucho (Gro)/transducin-like enhancer of split family of transcriptional corepressors are implicated in many signalling pathways that are important in development and disease, including those mediated by Notch, Wnt and Hedgehog. Here, we describe a genetic screen in Drosophila that yielded 50 new gro alleles, including the first protein-null allele, and has two mutations in the conserved Q oligomerization domain that have been proposed to have an essential role in corepressor activity. One of these latter mutations, encoding an amino-terminal protein truncation that lacks part of the Q domain, abolishes oligomerization in vitro and renders the protein unstable in vivo. Nevertheless, the mutation is not a null: maternal mutant embryos have intermediate segmentation phenotypes and relatively normal terminal patterning suggesting that the mutant protein retains partial corepressor activity. Our results show that homo-oligomerization of Gro is not obligatory for its action in vivo, and that Gro represses transcription through more than one molecular mechanism.
The Drosophila Groucho (Gro) protein was the founding member of the family of transcriptional co-repressor proteins that now includes the transducin-like enhancer of split (TLE) and Gro-related gene (Grg) proteins in vertebrates. Gro family proteins do not bind DNA directly, but are recruited by a diverse profile of transcription factors, including members of the Hes, Runx, Nkx, LEF1/Tcf, Pax, Six and c-Myc families. The primary structure of Gro proteins includes five identifiable regions, of which the most highly conserved are the amino-terminal glutamine-rich Q domain and the carboxy-terminal WD-repeat domain. The Q domain contains two coiled-coil motifs that facilitate oligomerization into tetramers and binding to some transcription factors. The WD domain folds to form a beta-propeller, which mediates protein-protein interactions. Many transcription factors interact with the WD domain via a short peptide motif that falls into either of two classes: WRPW and related tetrapeptides; and the 'eh1' motif (FxIxxIL). Gro family proteins are broadly expressed during development and in the adult. They have essential functions in many developmental pathways (including Notch and Wnt signaling) and are implicated in the pathogenesis of some cancers. The molecular mechanisms through which Gro proteins act to repress transcription are not yet well understood. It is becoming clear that Gro proteins have different modes of action in vivo dependent on biological context and these include direct and indirect modification of chromatin structure at target genes.
The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). Here, we identify several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) beta propeller domain in vitro and in vivo. We define these interactions at the molecular level with crystal structures of the WD domain of human TLE1 bound to either WRPW or eh1 peptides. The two distinct peptide motifs adopt markedly different bound conformations but occupy overlapping sites across the central pore of the beta propeller. Our structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo.
Segmental patterning in Drosophila relies on a cascade of transcription factors that subdivide the embryo into successively more precise domains [1, 2]. We have identified a missense mutation (W049) in the gene encoding the transcriptional elongation factor Spt5 (reviewed in [3-5]) which, when homozygous in the maternal germ line, leads to defects in segmental patterning of the embryo. W049 alters the C-terminal domain of Spt5 and affects its activity in vitro, impairing its abilities to confer sensitivity to the transcriptional inhibitor DR13 and to stimulate transcription at limiting nucleotide concentrations. In vivo, W049 shows locus-specific effects on transcription: expression of gap genes remains wild-type, but striped patterning of the primary pair-rule genes even-skipped and runt is disrupted. even-skipped stripes are broadened in the mutant embryos indicating that Spt5 is likely to be a direct, negative regulator of this target gene. Our results suggest control of transcriptional elongation by repressors contributes to striped gene expression in the embryo. By contrast, expression of heat shock-induced proteins is reduced in the mutant embryos. These results provide genetic evidence for Spt5 function during heat shock induction and demonstrate that Spt5 acts both positively and negatively on transcription in vivo depending on context.
Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors which are components of the Notch signalling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGC ACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. We demonstrate that the 3 bp flanking the E-box core are intrinsic to DNA recognition by these proteins and that the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo.