Michele Markstein

Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo

Metazoan genomes contain vast tracts of cis-regulatory DNA that have been identified typically through tedious functional assays. As a result, it has not been possible to uncover a cis-regulatory code that links primary DNA sequences to gene expression patterns. In an initial effort to determine whether coordinately regulated genes share a common “grammar,” we have examined the distribution of Dorsal recognition sequences in the Drosophila genome. Dorsal is one of the best-characterized sequence-specific transcription factors in Drosophila. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters are shown to be associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network.

Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.

A Drosophila Resource of Transgenic RNAi Lines for Neurogenetics

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the “VALIUM” series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

A regulatory code for neurogenic gene expression in the Drosophila embryo

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.

Systematic screen of chemotherapeutics in Drosophila stem cell tumors

Significance In this article we report a large-scale chemical screen in adult Drosophila to find inhibitors of stem-cell–derived tumors. To our surprise, we found that some Food and Drug Administration-approved chemotherapy drugs have the dual property of reducing growth of stem-cell–derived tumors while also stimulating hyperproliferation of their wild-type counterparts. Since hyperproliferation is one of the hallmarks of cancer cells, this side effect could contribute to refueling the growth of the very tumors that these chemotherapeutics are intended to inhibit. We show that this side effect is driven by the evolutionarily conserved Janus kinase-signal transducers and activators of transcription (JAK-STAT) inflammatory pathway, raising the possibility that the JAK-STAT pathway may also be activated in humans who are treated with some chemotherapeutics. Here we report the development of an in vivo system to study the interaction of stem cells with drugs using a tumor model in the adult Drosophila intestine. Strikingly, we find that some Food and Drug Administration-approved chemotherapeutics that can inhibit the growth of Drosophila tumor stem cells can paradoxically promote the hyperproliferation of their wild-type counterparts. These results reveal an unanticipated side effect on stem cells that may contribute to tumor recurrence. We propose that the same side effect may occur in humans based on our finding that it is driven in Drosophila by the evolutionarily conserved Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. An immediate implication of our findings is that supplementing traditional chemotherapeutics with anti-inflammatories may reduce tumor recurrence.

Modeling colorectal cancer as a 3-dimensional disease in a dish: the case for drug screening using organoids, zebrafish, and fruit flies

This review discusses recent shifts in the understanding of colorectal cancer as a stem cell based disease, based on findings that tie patient prognosis to the presence of cancer stem cells in colorectal tumors. Currently no drugs specifically target CSCs in colorectal tumors. However, recent advances in the culturing of colorectal stem cells using mammalian organoids, zebrafish, and Drosophila offer promising avenues for anti-CSC drug discovery.

Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses. DOI: http://dx.doi.org/10.7554/eLife.26398.001

Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila.

Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of EdU incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression of Charlatan blocked differentiation reversibly, but loss of Charlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation.

Recent advances in functional assays of transcriptional enhancers

In this special edition of Genomics, we present reviews of the current state of the field in identifying and functionally understanding transcriptional enhancers in cells and developing tissues. Typically several enhancers coordinate the expression of an individual target gene, each controlling that genes expression in specific cell types at specific times. Until recently, identifying each genes enhancers had been challenging because enhancers do not occupy prescribed locations relative to their target genes. Recently there have been powerful advances in DNA sequencing and other technologies that make it possible to identify the majority of enhancers in virtually any cell type of interest. The reviews in this edition of Genomics highlight some of these new and powerful approaches.

Abstract 4189: Cancer drugs on the fly: whole-animal chemical screening in Drosophila identifies unexpected role for EGFR in RAF intestinal stem cell tumors

A major challenge in cancer therapeutics is to identify drugs that target cancer stem cells. This presents a problem for traditional cell-based screening approaches because the complex cellular interactions underlying stem cell biology are difficult to recapitulate in vitro. Conversely, mice, which are arguably among the best animal models for cancer—are not amenable to high-throughput screening. However, many aspects of stem cell initiated tumorigenesis can be modeled in the fruit fly Drosophila melanogaster, which due to its small size is well suited for large-scale screens. The Drosophila intestine, in particular, presents a compelling model to study stem-cell derived intestinal tumors because Drosophila intestinal stem cells share many features with their mammalian counterparts including a propensity to form tumors in response to mutations in WNT, NOTCH, or EGFR signaling. We therefore developed an in vivo screening pipeline using adult Drosophila, to identify compounds that can suppress the growth of stem cell initiated tumors. In previous work we showed that ectopically expressing a constitutively active human c-RAF transgene, RAF(gof) in Drosophila intestinal stem cells results in fast growing tumors that are sensitive to several FDA approved chemotherapeutics. These results establish the clinical relevance of using Drosophila to model stem cell cancers. Here we present results from a screen of known bioactives, that reveal an unexpected role of the EGF receptor, EGFR, in RAF(gof) intestinal stem cell tumors. Based on the linear order of genes in the canonical RAF pathway—EGFR→RAS→RAF→MEK→ERK—we expected that drugs that inhibit genes acting downstream of RAF would score as hits in the screen, whereas genes that act upstream of RAF would not. Indeed, that same logic is applied when assessing whether to give human patients certain pathway specific drugs: patients with activating mutations in RAS or RAF are typically not given drugs that target the upstream EGFR. Consistent with our expectation, we found that drugs that inhibit genes downstream of RAF, effectively stopped Raf(gof) tumor growth. However, to our surprise, we also found that inhibitors of EGFR blocked the growth of RAF(gof) tumors. Using RNAi against EGFR, we genetically validated the chemical results. Our preliminary evidence suggests that EGFR is required in a parallel pathway, upstream of AKT. These findings are significant because they suggest that in some cases, human patients with RAF activated tumors may benefit from drug therapies that target EGFR. Importantly, our results demonstrate that taking an unbiased approach in assessing which drug therapies to apply to a given cancer, may yield unexpectedly good results. Citation Format: Michele Markstein, Hannah Dayton, Samantha Dettorre. Cancer drugs on the fly: whole-animal chemical screening in Drosophila identifies unexpected role for EGFR in RAF intestinal stem cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4189.