Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia.

Friedreich ataxia (FRDA) is caused by hyperexpansion of GAA*TTC repeats located in the first intron of the FXN gene, which inhibits transcription leading to the deficiency of frataxin. The FXN gene is an excellent target for therapeutic intervention since (i) 98% of patients carry the same type of mutation, (ii) the mutation is intronic, thus leaving the FXN coding sequence unaffected and (iii) heterozygous GAA*TTC expansion carriers with approximately 50% decrease of the frataxin are asymptomatic. The discovery of therapeutic strategies for FRDA is hampered by a lack of appropriate molecular models of the disease. Herein, we present the development of a new cell line as a molecular model of FRDA by inserting 560 GAA*TTC repeats into an intron of a GFP reporter minigene. The GFP_(GAA*TTC)(560) minigene recapitulates the molecular hallmarks of the mutated FXN gene, i.e. inhibition of transcription of the reporter gene, decreased levels of the reporter protein and hypoacetylation and hypermethylation of histones in the vicinity of the repeats. Additionally, selected histone deacetylase inhibitors, known to stimulate the FXN gene expression, increase the expression of the GFP_(GAA*TTC)(560) reporter. This FRDA model can be adapted to high-throughput analyses in a search for new therapeutics for the disease.

Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments.

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.

Energetics and affinity of the histone octamer for defined DNA sequences.

Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and histone octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.

Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides.

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.

Chemical approaches to control gene expression.

A current goal in molecular medicine is the development of new strategies to interfere with gene expression in living cells in the hope that novel therapies for human disease will result from these efforts. This review focuses on small-molecule or chemical approaches to manipulate gene expression by modulating either transcription of messenger RNA-coding genes or protein translation. The molecules under study include natural products, designed ligands, and compounds identified through functional screens of combinatorial libraries. The cellular targets for these molecules include DNA, messenger RNA, and the protein components of the transcription, RNA processing, and translational machinery. Studies with model systems have shown promise in the inhibition of both cellular and viral gene transcription and mRNA utilization. Moreover, strategies for both repression and activation of gene transcription have been described. These studies offer promise for treatment of diseases of pathogenic (viral, bacterial, etc.) and cellular origin (cancer, genetic diseases, etc.).

Asymmetric DNA binding by a homodimeric bHLH protein.

Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.

Rapid identification of key amino-acid-DNA contacts through combinatorial peptide synthesis.

BACKGROUND: Basic helix-loop-helix (bHLH) transcription factors are characterized by a conserved four-helix bundle that recognizes a specific hexanucleotide DNA sequence in the major groove. Previous studies have shown that amino acids in the basic region make base-specific contacts, whereas the HLH region is responsible for dimerization. Structural data suggest that portions of the loop region may be proximal to the DNA; however, the role of the loop in DNA-binding affinity and specificity has not been investigated. RESULTS: Protein-DNA recognition by the Drosophila bHLH transcription factor Deadpan was probed using combinatorial solid-phase peptide synthesis methods. A series of bHLH peptide libraries that modulate amino acid content and length in the loop region was screened with DNA and peptide affinity columns, and analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). A functional bHLH peptide with reduced loop length was found, and Lys80 was unambiguously identified as the sole loop residue critical for DNA binding. Unnatural amino acids were substituted at this position to assess contributions of the terminal amino group and the alkyl chain length to DNA-binding affinity and specificity. CONCLUSIONS: Using combinatorial solid-phase peptide synthesis methods and MALDI-MS, we were able to rapidly identify a key amino acid involved in DNA binding by a bHLH protein. Our approach provides a powerful alternative to current recombinant DNA methods to identify and probe the energetics of protein-DNA interactions.

Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides.

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.

Identification of a minimal domain of 5 S ribosomal RNA sufficient for high affinity interactions with the RNA-specific zinc fingers of transcription factor IIIA.

Transcription factor IIIA of Xenopuslaevis serves a dual function during oogenesis and early development: this zinc finger protein binds to the internal promoter element of the 5 S ribosomal RNA genes and acts as a positive transcription factor; additionally, the protein functions in 5 S RNA storage. The central four zinc fingers (zf4-7) of the nine-finger protein have been shown to bind 5 S rRNA with comparable or higher affinity than the full-length protein. The role of finger seven in binding affinity has been examined by deletion analysis. A zf4-6 protein binds 5 S RNA with about a sevenfold reduction in binding affinity, compared to zf4-7. The effect of non-specific competitor DNA on binding affinities of the zinc finger peptides was examined and found to have a significant effect on the measured affinities of these peptides for full-length and truncated versions of 5 S RNA. The interaction of zf4-6 with full-length 5 S RNA was far more sensitive to non-specific competitor concentration than was the zf4-7:5 S RNA interaction, suggesting that finger seven contributes to both affinity and specificity in this protein:RNA interaction. In order to map zinc finger binding sites on the 5 S RNA molecule, we generated truncated versions of the RNA and tested these molecules for their binding affinities with zf4-7 and zf4-6. Previous studies showed that a 75 nucleotide long RNA, comprising loop A, helix II, helix V, region E and helix IV, bound zf4-7 with high affinity. Selection and amplification binding assays (selex) have now been used to generate smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA, comprising loop A, helix V, region E and helix IV, but lacking helix II, retains high affinity for zf4-6. These data are consistent with the proposal that fingers 4-6 bind this central core of 5 S RNA and that finger seven binds the helix II region.

Inhibition of Ets-1 DNA binding and ternary complex formation between Ets-1, NF-kappaB, and DNA by a designed DNA-binding ligand.

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-kappaB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1. NF-kappaB.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-kappaB also inhibits NF-kappaB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Characterization of the DNA binding properties of the bHLH domain of Deadpan to single and tandem sites.

The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCl) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (Kd = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCl and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.

Minor groove DNA-protein contacts upstream of a tRNA gene detected with a synthetic DNA binding ligand.

Transcription factor IIIB (TFIIIB) is composed of the TATA box binding protein (TBP) and class III gene-specific TBP-associated factors (TAFs). TFIIIB is brought to a site centered approximately 35 bp upstream from the transcription start site of tRNA genes via protein-protein interactions with the intragenic promoter-recognition factor TFIIIC. Since TBP interacts with TATA elements through the minor groove of DNA, we asked whether TFIIIB interacts with DNA in the minor groove. Polyamides containing pyrrole (Py) and imidazole (Im) amino acids are synthetic DNA ligands that bind to predetermined sequences in the minor groove of double helical DNA. These small molecules have been shown to interfere with protein-DNA interactions in the minor groove. A series of DNA constructs was generated in which the binding site for a Py-Im polyamide was placed at various distances upstream from a tRNA gene transcription start site. We find that a match polyamide will effectively inhibit tRNA gene transcription when its binding site is located within 33 bp of the transcription start site of the Xenopus TyrD tRNA gene. Moreover, in the presence of polyamide, RNA polymerase III is redirected to a new transcription initiation site located approximately one DNA helical turn downstream from the native start site. Our results suggest that a subunit of TFIIIB, possibly TBP, makes an essential minor groove DNA contact centered approximately 30 bp upstream from the tRNA gene.

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-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.

Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis.

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.

Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: determinants of affinity and sequence specificity.

The high resolution solution structure of a protein containing the three amino-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound to its cognate DNA duplex was determined by nuclear magnetic resonance spectroscopy. The protein, which is designated zf1-3, binds with all three fingers in the DNA major groove, with a number of amino acids making base-specific contacts. The DNA structure is close to B-form. Although the mode of interaction of zf1-3 with DNA is similar to that of zif268 and other structurally characterized zinc finger complexes, the TFIIIA complex exhibits several novel features. Each zinc finger contacts four to five base-pairs and the repertoire of known base contact residues is extended to include a tryptophan at position +2 of the helix (finger 1) and arginine at position +10 (finger 3). Sequence-specific base contacts are made over virtually the entire length of the finger 3 helix. Lysine and histidine side-chains involved in base recognition are dynamically disordered in the solution structure; in the case of lysine, in particular, this could significantly decrease the entropic cost of DNA binding. The TGEKP(N) linker sequences, which are highly flexible in the unbound protein, adopt ordered conformations on DNA binding. The linkers appear to play an active structural role in stabilization of the protein-DNA complex. Substantial protein-protein contact surfaces are formed between adjacent fingers. As a consequence of these protein-protein interactions, the orientation of finger 1 in the major groove differs from that of the other fingers. Contributions to high affinity binding by zf1-3 come from both direct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA complex. The structures provide a molecular level explanation for the large body of footprinting and mutagenesis data available for the TFIIIA-DNA complex.

Domain packing and dynamics in the DNA complex of the N-terminal zinc fingers of TFIIIA.

The three N-terminal zinc fingers of transcription factor IIIA bind in the DNA major groove. Substantial packing interfaces are formed between adjacent fingers, the linkers lose their intrinsic flexibility upon DNA binding, and several lysine side chains implicated in DNA recognition are dynamically disordered.

Mitotic repression of the transcriptional machinery.

Nuclear RNA transcription is silenced when eukaryotic cells enter mitosis. Until recently, this repression was thought to derive solely from the condensation of interphase chromatin into mitotic chromosomes. Recent studies, however, have shown that changes in chromatin structure and occupancy of promoter elements by both general and gene-specific transcription factors also play a role in transcriptional silencing. In addition, studies with simplified systems reveal that reversible phosphorylation of the basal transcriptional machinery represses transcription at mitosis.

Regulation of gene expression by small molecules.

Small molecules that target specific DNA sequences have the potential to control gene expression. Ligands designed for therapeutic application must bind any predetermined DNA sequence with high affinity and permeate living cells. Synthetic polyamides containing N-methylimidazole and N-methylpyrrole amino acids have an affinity and specificity for DNA comparable to naturally occurring DNA-binding proteins. We report here that an eight-ring polyamide targeted to a specific region of the transcription factor TFIIIA binding site interferes with 5S RNA gene expression in Xenopus kidney cells. Our results indicate that pyrrole-imidazole polyamides are cell-permeable and can inhibit the transcription of specific genes.

Differential kinetics of transcription complex assembly distinguish oocyte and somatic 5S RNA genes of Xenopus.

Differential transcription of the Xenopus gene families encoding the oocyte and somatic 5S ribosomal RNAs can be reproduced in vitro with cell-free extracts prepared from Xenopus oocytes and unfertilized eggs. The transcriptional activities of these genes as assayed in these in vitro systems are a consequence of large differences in the rates of assembly of active transcription complexes. The somatic 5S genes sequester limiting transcription factors much more rapidly than the corresponding oocyte 5S genes and, as a consequence, are far more active. However, once transcription complexes are formed, these complexes are stable on both of these genes. Previous studies have established that transcription factors IIIA and IIIC are sufficient to form a stable protein-DNA complex on the somatic 5S gene. The rate of formation of the stable TFIIIA+C complex for the oocyte gene is far slower than that for the somatic 5S gene. Insertion of the DNA binding site for TFIIIC2 (the B-block promoter element from tRNA genes) into the 3' flanking region of a synthetic oocyte 5S gene increases the transcription efficiency and rate of transcription complex assembly of this gene relative to the parent gene lacking the B-block element. Our results support a model in which competition for limiting transcription factors plays a pivotal role in establishing differential transcription of the two classes of 5S genes during early embryogenesis.

Importance of minor groove binding zinc fingers within the transcription factor IIIA-DNA complex.

The gene-specific transcription factor IIIA (TFIIIA) binds to the internal promoter element of the 5 S rRNA gene through nine zinc fingers which make specific DNA contacts. Seven of the nine TFIIIA zinc fingers participate in major groove DNA contacts while two fingers, 4 and 6, have been proposed to bind in or across the minor groove. Pyrrole-imidazole polyamides are minor groove binding ligands that recognize predetermined DNA sequences with affinity and specificity comparable to natural DNA-binding proteins. We have examined the DNA binding activity of nine finger TFIIIA and shorter recombinant analogs in the presence of polyamides that bind six base-pair sequences (Kd = 0.03 to 1.7 nM) in the minor groove of the binding site for zinc finger 4. DNase I footprint titrations demonstrate that the polyamides and a recombinant protein containing the three amino-terminal zinc fingers of TFIIIA (zf1-3) co-occupy the TFIIIA binding site, in agreement with the known location of zf1-3 in the major groove. In contrast, the polyamides block the specific interaction of TFIIIA or zf1-4 with the 5 S RNA gene, supporting a model for minor groove occupancy by zinc finger 4. Minor groove binding polyamides targeted to specific DNA sequences may provide a novel chemical approach to probing multidomain protein-DNA interactions.