RNA as a Transcriptional Activator

AbstractTwo recent reports demonstrate that in vivo selection can isolate novel RNAs that activate transcription when tethered to a gene promoter. This highlights the structural plasticity that allows RNA to fulfill many functions normally carried out by proteins

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes

Unanticipated differences between α- and γ-diaminobutyric acid-linked hairpin polyamide-alkylator conjugates

Hairpin polyamide–chlorambucil conjugates containing an {alpha}-diaminobutyric acid ({alpha}-DABA) turn moiety are compared to their constitutional isomers containing the well-characterized {gamma}-DABA turn. Although the DNA-binding properties of unconjugated polyamides are similar, the {alpha}-DABA conjugates display increased alkylation specificity and decreased rate of reaction. Treatment of a human colon carcinoma cell line with {alpha}-DABA versus {gamma}-DABA hairpin conjugates shows only slight differences in toxicities while producing similar effects on cell morphology and G2/M stage cell cycle arrest. However, striking differences in animal toxicity between the two classes are observed. Although mice treated with an {alpha}-DABA hairpin polyamide do not differ significantly from control mice, the analogous {gamma}-DABA hairpin is lethal. This dramatic difference from a subtle structural change would not have been predicted

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-l, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity, The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication

Role of Mismatch Repair Enzymes in GAA•TTC Triplet-repeat Expansion in Friedreich Ataxia Induced Pluripotent Stem Cells

The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA•TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA•TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA•TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA•TTC triplet repeats expanded at a rate of about two GAA•TTC triplet repeats/replication. However, GAA•TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA•TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA•TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA•TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system

Small molecules targeting histone H4 as potential therapeutics for chronic myelogenous leukemia

We recently identified a polyamide-chlorambucil conjugate, 1R-Chl, which alkylates and down-regulates transcription of the human histone H4c gene and inhibits the growth of several cancer cell lines in vitro and in a murine SW620 xenograft model, without apparent animal toxicity. In this study, we analyzed the effects of 1R-Chl in the chronic myelogenous leukemia cell line K562 and identified another polyamide conjugate, 6R-Chl, which targets H4 genes and elicits a similar cellular response. Other polyamide conjugates that do not target the H4 gene do not elicit this response. In a murine model, both 1R-Chl and 6R-Chl were found to be highly effective in blocking K562 xenograft growth with high-dose tolerance. Unlike conventional and distamycin-based alkylators, little or no cytotoxicities and animal toxicities were observed in mg/kg dosage ranges. These results suggest that these polyamide alkylators may be a viable treatment alternative for chronic myelogenous leukemia

Two New Pimelic Diphenylamide HDAC Inhibitors Induce Sustained Frataxin Upregulation in Cells from Friedreich's Ataxia Patients and in a Mouse Model

BACKGROUND: Friedreich's ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems. CONCLUSIONS/SIGNIFICANCE: Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model

NOTICE: this is the author’s version of a work that was accepted for publication in Neurobiology of Disease. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication.Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5 months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia.This work was supported by Repligen Corporation; Muscular Dystrophy Association (MDA) USA; Ataxia UK; Friedreich's Ataxia Research Alliance (FARA); GoFAR; and the Wellcome Trust [089757]

Chromatin structure determines accessibility of a hairpin polyamide–chlorambucil conjugate at histone H4 genes in pancreatic cancer cells

We have shown that a specific pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugate, 1R-Chl, alters the growth characteristics of various cancer cell lines in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without apparent cytotoxicity. This molecule has also shown efficacy in several mouse xenograft models, preventing tumor growth. Previous microarray studies have suggested that members of the histone H4 gene family, H4c and H4j/k, are the primary targets of this molecule, leading to reduced histone mRNA synthesis and growth arrest in cancer cells. In the present study, we examine the effects of 1R-Chl on transcription of other members of the H4 gene family, with the result that mRNA transcription of most genomic copies of H4 are down-regulated by 1R-Chl in a human pancreatic cancer cell line (MIA PaCa-2), but not in a cell line of non-cancerous origin (HEK293 cells). The basis for this differential effect is likely an open chromatin conformation within the H4 genes in cancer cells. Chromatin immunoprecipitation experiments show increased histone acetylation on the histone H4 genes in cancer cells, compared to HEK293 cells, explaining the differential activity of this molecule in cancer versus non-cancer cells