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

Establishment of Rab-11 Induced Inflammatory Regulation as Therapeutic Targets in Colon Cancer Progression

Colon cancer is the third-deadliest cancer in the United States. Better understanding the cancer microenvironment/niches is crucial to the development of successful therapeutic targets. An RNAi screening using enterocyte specific driver was performed in Drosophila melanogaster intestine to search for niches regulating the intestine stem cell homeostasis. A small GTPase, Rab11 caused strong intestine stem cell (ISC) proliferation and tissue hyperplasia upon knockdown, due to increased production of inflammatory cytokines and growth factors. Increased inflammatory cytokines and proliferation were also observed in mouse Rab11a knockout (KO) intestine, indicating Rab11 regulatory role in the inflammation-induced hyperplasia is evolutionarily conserved and may also apply to human. We hypothesized that Rab11 is required to maintain cytokines in an appropriate state and its expression is down regulated in cancers. We investigated dextran sulfate sodium and chemical induced mouse colon cancer. Rab11 was largely reduced/absent in cancer tissues whereas well present in the normal tissue. We also investigated the correlation of Rab11 level and human cancer progression by immunofluorescence staining, and found that close to 50% and 40% of the cases studied had reduced Rab11 level by 20% and 30%, respectively. The greater the reduction is, the higher chance it is associated with more progressed cancer. Rab11, therefore, functions to suppress cancer progression and can be potentially developed towards a better diagnosis and treatment target for colon cancer. We will screen FDA approved drugs for ISC proliferation regulation, using a fly intestine tumor model established by expressing a human activated RAFGOFgene and a luciferase gene in the fly gut precursor cells. Selected drugs will be applied to test the Rab11 induced hyperplasia in fly, and further validated by mouse and human organoids derived from Rab11 KO mouse or human colon cancer tissues

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

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

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

Decoding cis-regulatory DNAs in the Drosophila genome

Cis-regulatory DNAs control the timing and sites of gene expression during metazoan development. Changes in gene expression are responsible for the morphological diversification of metazoan body plans. However, traditional methods for the identification and characterization of cis-regulatory DNAs are tedious. During the past year, computational methods have been used to identify novel cis-DNAs within the entire Drosophila genome. These methods change the way that cis-DNAs will be analyzed in future studies and offer the promise of unraveling complex gene networks

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 gene\u27s expression in specific cell types at specific times. Until recently, identifying each gene\u27s 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

Whole-genome analysis of dorsal-ventral patterning in the Drosophila embryo

The maternal Dorsal regulatory gradient initiates the differentiation of several tissues in the early Drosophila embryo. Whole-genome microarray assays identified as many as 40 new Dorsal target genes, which encode a broad spectrum of cell signaling proteins and transcription factors. Evidence is presented that a tissue-specific form of the NF-Y transcription complex is essential for the activation of gene expression in the mesoderm. Tissue-specific enhancers were identified for new Dorsal target genes, and bioinformatics methods identified conserved cis-regulatory elements for coordinately regulated genes that respond to similar thresholds of the Dorsal gradient. The new Dorsal target genes and enhancers represent one of the most extensive gene networks known for any developmental process

Systematic screen of chemotherapeutics in Drosophila stem cell tumors

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

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