Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia

Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 05/2010; 5(5):e10656. DOI: 10.1371/journal.pone.0010656
Source: PubMed


The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes") that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.
To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.
Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.
We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

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    • "HpFur, therefore, adds to this list of regulators able to readout specific determinants also in the minor groove. This has interesting implications, as minor groove binding drugs have been suggested as alternative treatment approaches for critical pathologies and successfully used for targeting the minor groove-binding transcription factors involved in disease (39,52). "
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    ABSTRACT: Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.
    Nucleic Acids Research 12/2013; 42(5). DOI:10.1093/nar/gkt1258 · 9.11 Impact Factor
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    • "We have chosen the oligopeptide antibiotic, distamycin A, which inhibits the pathogenesis of vaccinia virus in culture [28]. It displaces essential transcription factors like SRF and MEF2 [29], and inhibits the binding of high mobility group proteins HMGA1 to P-Selectin promoter [30]. It also inhibits the binding of DNA to nuclear scaffold and linker histones [31]. "
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    ABSTRACT: The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.
    PLoS ONE 02/2013; 8(2):e57693. DOI:10.1371/journal.pone.0057693 · 3.23 Impact Factor
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    • "A549 cells (ATCC) were maintained in Dulbelcco’s Modified Eagle Medium with 10% fetal bovine serum (Invitrogen Life Sciences), 1% L-glutamine (GIBCO/Invitrogen Life Sciences), and 2% penicillin/streptomycin (10,000 units penicillin/ml and 10 mg streptomycin/ml, Sigma). Transfection experiments were performed using standard protocols [38]. The transfection reagent was Fugene 6 (Roche Applied Science, Indianapolis, IN), used following the manufacturer’s instructions, with a 3:1 ratio of Fugene to total plasmid DNA. "
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    Respiratory research 06/2012; 13(1):42. DOI:10.1186/1465-9921-13-42 · 3.09 Impact Factor
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