Search

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy., Competing Interests: P.B.D. and F.Y. own shares in Gene Sciences, Inc., a biotechnology company that is developing therapeutics for prostate cancer.

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α-inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.

Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1alpha, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1alpha siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1alpha siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression.

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus 'tail' has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.

The DNA abnormality found in 98% of Friedreich's ataxia (FRDA) patients is the unstable hyperexpansion of a GAA.TTC triplet repeat in the first intron of the frataxin gene. Expanded GAA.TTC repeats result in decreased transcription and reduced levels of frataxin protein in affected individuals. Beta-alanine-linked pyrrole-imidazole polyamides bind GAA.TTC tracts with high affinity and disrupt the intramolecular DNA.DNA-associated region of the sticky-DNA conformation formed by long GAA.TTC repeats. Fluorescent polyamide-Bodipy conjugates localize in the nucleus of a lymphoid cell line derived from a FRDA patient. The synthetic ligands increase transcription of the frataxin gene in cell culture, resulting in increased levels of frataxin protein. DNA microarray analyses indicate that a limited number of genes are significantly affected in FRDA cells. Polyamides may increase transcription by altering the DNA conformation of genes harboring long GAA.TTC repeats or by chromatin opening.

Mapping and sequencing the genetic blueprint in human, mice, yeast and other model organisms has created challenges and opportunities for chemistry, biology and human medicine. An understanding of the function of each of the approximately 25,000 genes in humans, and the biological circuitry that controls these genes will be driven in part by new technologies from the world of chemistry. Many cellular events that lead to cancer and the progression of human disease represent aberrant gene expression. Small molecules that can be programmed to mimic transcription factors and bind a large repertoire of DNA sequences in the human genome would be useful tools in biology and potentially in human medicine. Polyamides are synthetic oligomers programmed to read the DNA double helix. They are cell permeable, bind chromatin and have been shown to downregulate endogenous genes in cell culture.

Human T-cell leukemia virus type-1 (HTLV-1) depends on the virally encoded transcription factor Tax for efficient viral replication and gene expression. In a complex with CREB, Tax contacts the minor groove of the promoter DNA at guanine and cytosine rich sequences that flank three of the off-consensus cyclic-AMP response elements (CREs). In this study, we used six Tax-directed pyrrole-imidazole polyamides specifically designed to block Tax binding to DNA at each GC sequence of the three viral CREs. We found that four of these polyamides disrupt binding of the Tax/CREB complex in vitro, and that these same molecules also inhibit Tax-mediated transcription in vitro on chromatin-assembled templates. However, of these four Tax/CREB-specific polyamides, only one polyamide appears to be uniquely Tax specific. We show that polyamides can enter the nuclei of HTLV-1 infected T-cells, and two of the four polyamides down-regulated virion production in these cells. Together, these data illustrate the importance of studying polyamide inhibition of gene expression in vitro and in vivo, as the function of the polyamides in living cells is not fully understood. Finally, our data indicates that targeted disruption of the Tax/CREB complex, or other complexes which assemble on the HTLV-1 promoter, may provide a novel approach for inhibiting viral replication in vivo.

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.

Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that bind in the minor groove of DNA. Previous studies have established that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible for molecular recognition by Py-Im polyamides, and that nucleosomes remain fully folded upon ligand binding. Two polyamides that bind within the sea urchin 5S gene nucleosome positioning sequence inhibit both heat-induced nucleosome sliding and transcription by bacteriophage T7 RNA polymerase from the nucleosomal template, but not from histone-free DNA. These polyamides prevent repositioning of the histone octamer by RNA polymerase, and thereby inhibit passage of the elongating polymerase through nucleosomal DNA. These results establish unambiguously the requirement for octamer mobility for transcription of nucleosomal templates by T7 RNA polymerase.

When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.

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-1, 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.

A site-specific triple-helical DNA complex has previously been shown to inhibit DNA binding by eukaryotic transcription factor Sp1. To examine the functional consequences of such inhibition, homopurine target sequences for oligonucleotide-directed triple-helix formation were inserted in various configurations relative to Sp1 transcription activator binding sites, upstream of the TATA element of recombinant eukaryotic promoters. The resulting promoters were tested for activity in the presence or absence of recombinant human Sp1 in a Drosophila in vitro transcription system lacking endogenous Sp1. When triple-helical complexes were assembled on the promoters by incubation with specific oligodeoxyribonucleotides, promoter-specific repression of basal transcription was observed in the absence of Sp1. Transcriptional repression required the preassembly of triple-helical complexes before addition of nuclear extract. The degree of basal repression was a function of the number and proximity of triple-helical complexes relative to the basal promoter complex. Repression did not result from triple-helix-induced template degradation. Addition of recombinant Sp1 did not cause derepression. These results suggest that triple-helical complexes can repress transcription primarily by blocking promoter DNA assembly into initiation complexes rather than by occluding Sp1 binding. One of several plausible mechanisms for triple-helix-induced repression involves changes in DNA flexibility. Evidence in favor of this model is provided by a permutation-dependent gel mobility assay in which formation of site-specific triple-helical complexes is shown to stiffen double-helical DNA.