ABC transporters confer multidrug resistance to Drosophila intestinal stem cells
bioRxiv 2018Hannah Dayton, Jonathan DiRusso, Kristopher Kolbert, Olivia Williamson, Aiste Balciunaite, Edridge D’Souza, Kelly Becker, Elizaveta Hosage, Muneera Issa, Victoria Liu, Raghuvir Viswanatha, Shu Kondo, Michele Markstein
Adult stem cells can survive a wide variety of insults from ionizing radiation to toxic chemicals. To date, the multidrug resistant features of stem cells have been characterized only in vertebrates, where there is a critical need to understand how cancer stem cells thwart chemotherapy drugs. These studies reveal that the ability of both normal and cancer stem cells to survive toxins hinges on their high levels of expression of ABC transporters, transmembrane pumps that efflux lipophilic compounds out of cells. This has been observed across a wide spectrum of vertebrate stem cells including breast, blood, intestine, liver, and skin, suggesting that high efflux ability and multidrug resistance may be general features of stem cells that distinguish them from their differentiated daughter cells. Here we show that these previously described vertebrate stem cell features are conserved in Drosophila intestinal stem cells. Using a novel in vivo efflux assay and multiple drug challenges, we show that stem cells in the fly intestine depend on two ABC transporters–one constitutively expressed and the other induced–for efflux and multidrug resistance. These results suggest that stem cell multidrug resistance by ABC transporters is a general stem cell feature conserved over 500 million years of evolution.
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GA-repeats on mammalian X chromosomes support Ohno’s hypothesis of dosage compensation by transcriptional upregulation
bioRxiv 2018Edridge D’Souza, Elizaveta Hosage, Kathryn Weinand, Steve Gisselbrecht, Vicky Marktein, Peter Markstein, Martha Bulyk, Michele Markstein
Over 50 years ago, Susumo Ohno proposed that dosage compensation in mammals would require upregulation of gene expression on the single active X chromosome, a mechanism which to date is best understood in the fruit fly Drosophila melanogaster. Here, we report that the GA-repeat sequences that recruit the conserved MSL dosage compensation complex to the Drosophila X chromosome are also enriched across mammalian X chromosomes, providing genomic support for the Ohno hypothesis. We show that mammalian GA-repeats derive in part from transposable elements, suggesting a mechanism whereby unrelated X chromosomes from dipterans to mammals accumulate binding sites for the MSL dosage compensation complex through convergent evolution, driven by their propensity to accumulate transposable elements. Download Paper: PDF
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. Download Paper: PDF
Recent advances in functional assays of transcriptional enhancers
Genomics 2015Babbitt C., Markstein M., Gray J.
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 gene’s expression in specific cell types at specific times. Until recently, identifying each gene’s 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. Download Paper: PDF
Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila
Development 2014Amcheslavsky A., Nie Y., Li Q., He F., Tsuda L., Markstein M., Ip YT.
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… Download Paper: PDF
Systematic screen of chemotherapeutics in Drosophila stem cell tumors
PNAS 2014Markstein M*, Dettorre S, Cho J, Neumüller RA, Craig-Müller S, Perrimon N*.
*Corresponding AuthorsHere 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… Download Paper: PDF
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Modeling colorectal cancer as a 3-dimensional disease in a dish: the case for drug screening using organoids, zebrafish, and fruit flies
Drug Development Today: Technologies 2013Michele Markstein 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… Download: PDF
Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer
Cancer Cell 2013Roti G, Carlton A, Ross KN, Markstein M, Pajcini K, Su AH, Perrimon N, Pear WS, Kung AL, Blacklow SC, Aster JC, Stegmaier K. Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. Sarco/endoplasmic reticulum calcium ATPase (SERCA) channels emerged at the intersection of… Download Paper: PDF
A Drosophila resource of transgenic RNAi lines for neurogenetics
Genetics 2009Ni JQ, Liu LP, Binari R, Hardy R, Shim HS, Cavallaro A, Booker M, Pfeiffer BD, Markstein M, Wang H, Villalta C, Laverty TR, Perkins LA, Perrimon N. 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… Download Paper: PDF
Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.
Nature Genetics 2008Markstein M*, Pitsouli C, Villalta C, Celniker SE, Perrimon N.
*Corresponding AuthorA 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… Download Paper: PDF
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Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster
Nature Methods 2008Ni JQ*, Markstein M*, Binari R*, Pfeiffer B, Liu LP, Villalta C, Booker M, Perkins L, Perrimon N
*Co-first AuthorsThe conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a… Download Paper: PDF
A regulatory code for neurogenic gene expression in the Drosophila embryo
Development 2004Markstein M*, Zinzen R, Markstein P, Yee KP, Erives A, Stathopoulos A, Levine M*.
*Corresponding AuthorsBioinformatics 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… Download Paper: PDF