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Transcriptional repression via antilooping in the Drosophila embryo.

ABSTRACT: Transcriptional repressors are thought to inhibit gene expression by interfering with the binding or function of RNA Polymerase II, perhaps by promoting local chromatin condensation. Here, we present evidence for a distinctive mechanism of repression, whereby sequence-specific repressors prevent the looping of distal enhancers to the promoter. Particular efforts focus on the Snail repressor, which plays a conserved role in promoting epithelial-mesenchyme transitions in both invertebrates and vertebrates, including mesoderm invagination in Drosophila, neural crest migration in vertebrates, and tumorigenesis in mammals. Chromosome conformation capture experiments were used to examine enhancer looping at Snail target genes in wild-type and mutant embryos. These studies suggest that the Snail repressor blocks the formation of fruitful enhancer-promoter interactions when bound to a distal enhancer. This higher-order mechanism of transcriptional repression has broad implications for the control of gene activity in metazoan development.

SUBMITTER: Chopra VS

PROVIDER: S-EPMC3386088 | biostudies-other | 2012 Jun

REPOSITORIES: biostudies-other

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Transcriptional repression via antilooping in the Drosophila embryo.

Chopra Vivek S VS Kong Nikki N Levine Michael M

Proceedings of the National Academy of Sciences of the United States of America 20120529 24

Transcriptional repressors are thought to inhibit gene expression by interfering with the binding or function of RNA Polymerase II, perhaps by promoting local chromatin condensation. Here, we present evidence for a distinctive mechanism of repression, whereby sequence-specific repressors prevent the looping of distal enhancers to the promoter. Particular efforts focus on the Snail repressor, which plays a conserved role in promoting epithelial-mesenchyme transitions in both invertebrates and ver ...[more]

PMID: 22645339

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Project description:Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2-6 h) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for Hairy in segmentation, we find groups of targets suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find here that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development.

Project description:BackgroundThe transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.MethodsU937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.ResultsGene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.ConclusionsOur data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.

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Transcriptional repression via antilooping in the Drosophila embryo.

Publications

Transcriptional repression via antilooping in the Drosophila embryo.

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