Tonia Von Ohlen

Phosphorylation of Groucho Mediates RTK Feedback Inhibition and Prolonged Pathway Target Gene Expression

BACKGROUND Signaling by receptor tyrosine kinase (RTK) pathways plays fundamental roles in processes of cell-fate determination, often through the induction of specific transcriptional responses. Yet it is not fully understood how continuous target gene expression, required for irreversible cell-fate specification, is preserved after RTK signaling has ended. Here we address this question using the Drosophila embryo, a model system that has been instrumental in elucidating the developmental functions of RTK signal transduction. RESULTS The Groucho corepressor is phosphorylated and downregulated in response to RTK signaling. Here we show that RTK pathways use Groucho phosphorylation as a general mechanism for inducing expression of pathway target genes encoding cell-fate determinants as well as feedback antagonists, indicating that relief of Groucho-dependent repression is an integral element of RTK signaling networks. We further demonstrate that after mitogen-activated protein kinase (MAPK) has been deactivated, sustained phosphorylation of Groucho is essential for persistent RTK-induced target gene expression and cell-fate determination in several developmental contexts. CONCLUSIONS Phosphorylation of Groucho by MAPK plays a dual role in the regulation of RTK responses: (1) it mediates rapid feedback inhibition, and (2) it provides a stable memory mechanism of past MAPK activity. We propose that, in this manner, phosphorylation of Groucho enables transiently active RTK pathways to fix the spatiotemporal expression profiles of downstream targets over time.

Use of Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections.

ABSTRACT Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

Identification of Critical Host Mitochondrion-Associated Genes during Ehrlichia chaffeensis Infections

ABSTRACT Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis (HME). To determine what host components are important for bacterial replication, we performed microarray analysis on Drosophila melanogaster S2 cells by comparing host gene transcript levels between permissive and nonpermissive conditions for E. chaffeensis growth. Five-hundred twenty-seven genes had increased transcript levels unique to permissive growth conditions 24 h postinfection. We screened adult flies that were mutants for several of the “permissive” genes for the ability to support Ehrlichia replication. Three additional D. melanogaster fly lines with putative mutations in pyrimidine metabolism were also tested. Ten fly lines carrying mutations in the genes CG6479, separation anxiety, chitinase 11, CG6364 (Uck2), CG6543 (Echs1), withered (whd), CG15881 (Ccdc58), CG14806 (Apop1), CG11875 (Nup37), and dumpy (dp) had increased resistance to infection with Ehrlichia. Analysis of RNA by quantitative real-time reverse transcription-PCR (qRT-PCR) confirmed that the bacterial load was decreased in these mutant flies compared to wild-type infected control flies. Seven of these genes (san, Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) encoded proteins that had mitochondrial functions or could be associated with proteins with mitochondrial functions. Treatment of THP-1 cells with double-stranded RNA to silence the human UCK2 gene indicates that the disruption of the uridine-cytidine kinase affects E. chaffeensis replication in human macrophages. Experiments with cyclopentenyl cytosine (CPEC), a CTP synthetase inhibitor and cytosine, suggest that the nucleotide salvage pathway is essential for E. chaffeensis replication and that it may be important for the provision of CTP, uridine, and cytidine nucleotides.

Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS

Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, we used multiple approaches to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. Here, we show that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, we have identified a novel domain, the Pst domain, that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, we show that Ind can and does repress vnd expression, but does so in a stage specific manner. We conclude from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border.

Ehrlichia chaffeensis Infections in Drosophila melanogaster

ABSTRACT Ehrlichia chaffeensis is an obligate, intracellular bacterium, transmitted by the tick Amblyomma americanum, and is the causative agent of human monocytic ehrlichiosis infections. We previously demonstrated that E. chaffeensis is capable of growing in Drosophila S2 cells. Therefore, we tested the hypothesis that E. chaffeensis can infect adult Drosophila melanogaster. Adult Drosophila organisms were experimentally challenged with intra-abdominal injections of bacteria. Ehrlichia-infected flies showed decreased survival compared to wild-type flies, and bacteria isolated from flies could reinfect mammalian macrophages. Ehrlichia infections activated both the cellular and humoral immune responses in the fly. Hemocytes phagocytosed bacteria after injection, and antimicrobial peptide pathways were induced following infection. Increased pathogenicity in flies carrying mutations in genes in both the Toll and Imd pathways suggests that both immune defense pathways participate in host defense. Induction of Drosophila cellular and humoral responses and the in vivo replication of E. chaffeensis suggests that D. melanogaster is a suitable host for E. chaffeensis. In the future, it will be a useful tool to unlock some of the in vivo mysteries of this arthropod-borne bacterium.

Identification of an upstream regulatory element reveals a novel requirement for Ind activity in maintaining ind expression

A maternally established gradient of nuclear Dorsal protein is the first step in subdivision of the Drosophila neurectoderm into stripes of homeodomain gene expression. Dorsal in combination with the EGF and TGFbeta signaling pathways are key regulators of the expression of the genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) in the developing neurectoderm. These three genes encode homeodomain transcription factors that can repress each other, which ensures adjacent, non-overlapping expression domains. Expression of vnd, ind, and msh is maintained after decline in EGF and TGFbeta signaling, but the relevant positive transcriptional regulators have not yet been defined. Here, we show that Ind can bind DNA with the same sequence specificity as its murine ortholog Gsh1. We have identified a novel upstream regulatory element at the ind locus containing predicted Ind binding sites, and we show that Ind activity is both necessary and sufficient for reporter gene expression from this element. We conclude that Ind can act as a transcriptional activator, and that positive autoregulation of Ind is a mechanism for persistent ind expression within the developing embryonic nervous system.

The Drosophila homeodomain transcription factor, Vnd, associates with a variety of co‐factors, is extensively phosphorylated and forms multiple complexes in embryos

Vnd is a dual transcriptional regulator that is essential for Drosophila dorsal–ventral patterning. Yet, our understanding of the biochemical basis for its regulatory activity is limited. Consistent with Vnd’s ability to repress target expression in embryos, endogenously expressed Vnd physically associates with the co‐repressor, Groucho, in Drosophila Kc167 cells. Vnd exists as a single complex in Kc167 cells, in contrast with embryonic Vnd, which forms multiple high‐molecular‐weight complexes. Unlike its vertebrate homolog, Nkx2.2, full‐length Vnd can bind its target in electrophoretic mobility shift assay, suggesting that co‐factor availability may influence Vnd’s weak regulatory activity in transient transfections. We identify the high mobility group 1‐type protein, D1, and the novel helix–loop–helix protein, Olig, as novel Vnd‐interacting proteins using co‐immunoprecipitation assays. Furthermore, we demonstrate that both D1 and Olig are co‐expressed with Vnd during Drosophila embryogenesis, consistent with a biological basis for this interaction. We also suggest that the phosphorylation state of Vnd influences its ability to interact with co‐factors, because Vnd is extensively phosphorylated in embryos and can be phosphorylated by activated mitogen‐activated protein kinase in vitro. These results highlight the complexities of Vnd‐mediated regulation.

Ind represses msh expression in the intermediate column of the Drosophila neuroectoderm, through direct interaction with upstream regulatory DNA.

The Drosophila neurectoderm is initially subdivided across the dorsoventral (DV) axis into three domains that are defined by the expression of three homeodomain containing proteins. These are from ventral to dorsal: Ventral nervous system defective (vnd), Intermediate neuroblasts defective (ind) and Muscle segment homeobox (msh). This is remarkably similar to the distribution of the orthologous homeodomain proteins in the developing neural tube of mice and Zebrafish. This pattern is partially governed by a ‘ventral dominance’ mechanism, in which Vnd represses ind and Ind represses msh. A major unanswered question in this process is: How does Ind direct positioning of the ventral border of msh expression. Toward this goal, we have identified regulatory DNA essential for expression of msh in the early neurectoderm. In addition, we demonstrated that Ind acts directly in this element by a combination of genetic and molecular experiments. Specifically, expression is expanded ventrally in ind mutant embryos and Ind protein directly and specifically bound to the msh regulatory DNA, and this interaction was required to limit the ventral boundary of msh expression. Developmental Dynamics 238:2735–2744, 2009.

Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme Containing either Sigma 32 or Sigma 70

Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for human monocytic ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens.

Phosphorylation of Ind by MAP kinase enhances Ind-dependent transcriptional repression

The Drosophila neuroectoderm is initially subdivided into three longitudinal domains that give rise to columns of neuroblasts. This subdivision is coordinately accomplished by the action of the signaling pathways, Dorsal and Epidermal Growth Factor Receptor (EGFR), in conjunction with the homeodomain proteins, Ventral nervous system defective, Intermediate neuroblasts defective (Ind) and Muscle Segment Homeobox. We previously demonstrated that Ind expression is activated in response to the EGFR pathway. Here we show that EGF signaling subsequently mediates the direct phosphorylation of Ind by MAP kinase, which enhances the capacity of Ind to repress target genes, such as achaete. Specifically, we show that reduced EGF signaling results in diminished repression of achaete in the intermediate column, despite the presence of high levels of Ind protein. We also demonstrate that ectopic activation of MAP kinase results in the lateral expansion of the Ind expression domain with a corresponding reduction in achaete expression. This regulation is also dependent on the co-repressor, Dichaete. Our data indicate that EGF signaling, acting through MAP kinase, impinges on multiple aspects of Ind regulatory activity. While it has been often demonstrated that MAP kinase phosphorylation of transcriptional repressors attenuates their repressor activity, here we provide an example of phosphorylation enhancing repressor activity.