Influence DNA Binding Research Articles

Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2: Implications for DNA binding, stability and dynamics.

FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.

Predictions of DNA mechanical properties at a genomic scale reveal potentially new functional roles of DNA flexibility

Abstract Mechanical properties of DNA have been implied to influence many of its biological functions. Recently, a new high-throughput method, called loop-seq, which allows measuring the intrinsic bendability of DNA fragments, has been developed. Using loop-seq data, we created a deep learning model to explore the biological significance of local DNA flexibility in a range of different species from different kingdoms. Consistently, we observed a characteristic and largely dinucleotide-composition-driven change of local flexibility near transcription start sites. In the presence of a TATA-box, a pronounced peak of high flexibility can be observed. Furthermore, depending on the transcription factor investigated, flanking-sequence-dependent DNA flexibility was identified as a potential factor influencing DNA binding. Compared to randomized genomic sequences, depending on species and taxa, actual genomic sequences were observed both with increased and lowered flexibility. Furthermore, in Arabidopsis thaliana, mutation rates, both de novo and fixed, were found to be associated with relatively rigid sequence regions. Our study presents a range of significant correlations between characteristic DNA mechanical properties and genomic features, the significance of which with regard to detailed molecular relevance awaits further theoretical and experimental exploration.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents

ObjectivesWe recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. MethodsMinimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. ResultsADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. ConclusionOur findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD’s influence on NFκB–DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB–DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

Structure-Based Approaches to Classify the Functional Impact of ZBTB18 Missense Variants in Health and Disease.

The Cys2His2 type zinc finger is a motif found in many eukaryotic transcription factor proteins that facilitates binding to genomic DNA so as to influence cellular gene expression. One such transcription factor is ZBTB18, characterized as a repressor that orchestrates the development of mammalian tissues including skeletal muscle and brain during embryogenesis. In humans, it has been recognized that disease-associated ZBTB18 missense variants mapping to the coding sequence of the zinc finger domain influence sequence-specific DNA binding, disrupt transcriptional regulation, and impair neural circuit formation in the brain. Furthermore, general population ZBTB18 missense variants that influence DNA binding and transcriptional regulation have also been documented within this domain; however, the molecular traits that explain why some variants cause disease while others do not are poorly understood. Here, we have applied five structure-based approaches to evaluate their ability to discriminate between disease-associated and general population ZBTB18 missense variants. We found that thermodynamic integration and Residue Scanning in the Schrodinger Biologics Suite were the best approaches for distinguishing disease-associated variants from general population variants. Our results demonstrate the effectiveness of structure-based approaches for the functional characterization of missense alleles to DNA binding, zinc finger transcription factor protein-coding genes that underlie human health and disease.

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self-renewal.

The self‐renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site‐specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF‐independent self‐renewal. In contrast, phosphomimetic substitution of S131 enhances LIF‐independent self‐renewal, without influencing DNA binding. Modelling the DNA–homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self‐renewal.

General population ZBTB18 missense variants influence DNA binding and transcriptional regulation.

Genetic variation of the multi-zinc finger BTB domain transcription factor ZBTB18 can cause a spectrum of human neurodevelopmental disorders, but the underlying mechanisms are not well understood. Recently, we reported that pathogenic, de novo ZBTB18 missense mutations alter its DNA-binding specificity and gene regulatory functions, leading to human neurodevelopmental disease. However, the functional impact of the general population ZBTB18 missense variants is unknown. Here, we investigated such variants documented in the Genome Aggregation Database (gnomAD) to discover that ZBTB gene family members are intolerant to loss-of-function and missense mutations, but not synonymous mutations. We studied ZBTB18 as a protein-DNA complex to find that general population missense variants are rare, and disproportionately map to non-DNA-contact residues, in contrast to the majority of disease-associated variants that map to DNA-contact residues, essential to motif binding. We studied a selection of variants (n = 12), which spans the multi-zinc finger region to find 58.3% (7/12) of variants displayed altered DNA binding, 41.6% (5/12) exhibited altered transcriptional activity in a luciferase reporter assay, 33.3% (4/12) exhibited altered DNA binding and transcriptional activity, whereas 33.3% (4/12) displayed a negligible functional impact. Our results demonstrate that general population variants, while rare, can influence ZBTB18 function, with potential consequences for neurodevelopment, homeostasis, and disease.

Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise

The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of tens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR’s core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed specific DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

Timing of adrenal regression controlled by synergistic interaction between Sf1 SUMOylation and Dax1.

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function. A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient-Sf12KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone).

The role of monogamous bivalency and Fc interactions in the binding of anti-DNA antibodies to DNA antigen

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. These antibodies can bind DNA avidly by monogamous bivalency, a mechanism which requires the interaction of both Fab combining regions with antigenic determinants on the same polynucleotide. To explore further this mechanism, we tested Fab and F(abʹ)2 fragments prepared from IgG from patient plasmas in an ELISA with native DNA antigen, detecting antibody with a peroxidase conjugated anti-Fab reagent. These studies showed that Fab fragments, which can only bind monovalently, had negligible activity. Although bivalent F(abʹ)2 fragments would be predicted to bind DNA, these fragments also showed poor anti-DNA activity. Control studies showed that the fragments retained antibody activity to tetanus toxoid and an EBV antigen preparation. Together, these findings suggest that anti-DNA avidity depends on monogamous bivalency, with the antibody Fc portion also influencing DNA binding, in a mechanism which can be termed Fc-dependent monogamous bivalency.

Allosteric Pathways in the Multi-Domain Thyroid Hormone Receptor

Allostery is an important regulatory mechanism in biology. Such mechanisms fine-tune the response of the nuclear hormone receptors (NRs) to extracellular stimuli. The structural topology of NRs consists of a mostly unstructured N-terminal (NTD)/activation function-1 domain (AF-1), a DNA binding domain (DBD) that recognizes and binds specific DNA sequences and a C-terminal ligand binding domain (LBD). Besides DNA, these domains also interact with specific factors that are subunits of the basal transcriptional machinery. Through multiple sources of data, we have observed that NRs are not simply docking sites for cellular molecules. Instead, we have characterized multiple allosteric pathways that link ligand, DNA and coactivator-binding sites. Here, we will present data from biophysical and cell-based studies on the thyroid-hormone receptor (TR). TR is a type II NR and functions as a heterodimer with the retinoid X receptor (RXR). Thyroid hormone (T3) is essential for development, differentiation, and metabolic balance in higher organisms and humans. Improper functioning of TR has been associated with pathophysiological conditions such as prostate cancer, colon cancer and hypothyroidism. Yet, the mechanism of transcriptional regulation by TR is not completely understood. Here, we present data that describes specific allosteric communication between the multiple TR domains. Specifically, we show how the AF1 influences DNA binding by the DBD and that the DNA-DBD interactions influence AF1 structural conformations. Furthermore, we show that there are direct interactions between the DBD and LBD and these are dependent on the DNA-bound state of the DBD. Our study on TR allostery also links the DNA and coactivator. Finally, propose a novel ‘frustrated-fit’ model to explain the negative cooperativity between TR and RXR ligands in the TR:RXR heterodimeric complex. For our studies, we utilize a combination of crystallography, experimental and theoretical biophysics and cell-based reporter gene assays.

Microphthalmia-associated transcription factor mutations are associated with white-spotted coat color in swamp buffalo.

SummaryA candidate gene analysis of the microphthalmia‐associated transcription factor (MITF) gene was used in an attempt to identify the genetic basis for a white‐spotted coat color phenotype in the Asian swamp buffalo (Bubalus bubalis carabanensis). Ninety‐three buffaloes—32 solid, 38 spotted and 23 white individuals—were Sanger‐sequenced for all MITF exons as well as highly conserved intronic and flanking regions. MITF cDNA representing skin and iris tissue from six spotted, nine solid and one white buffaloes was also Sanger‐sequenced to confirm detected mutations. Two independent loss‐of‐function mutations, a premature stop codon (c.328C>T, p.Arg110*) and a donor splice‐site mutation (c.840+2T>A, p.Glu281_Leu282Ins8), both of which cause white‐spotted coat color in swamp buffaloes, were identified. The nonsense mutation leads to a premature stop codon in exon 3, and likely removal of the resulting mRNA via nonsense‐mediated decay pathway, whereas the donor splice‐site mutation leads to aberrant splicing of exon 8 that encodes part of a highly conserved region of MITF. The resulting insertion of eight amino acid residues is expected to perturb the leucine zipper part in the basic helix‐loop‐helix leucine zipper (bHLH‐Zip) domain and will most likely influence dimerization and DNA binding capacity. Electrophoretic mobility shift assay was performed using mutant and wild‐type MITF proteins and showed that the mutant MITF protein resulting from the splice‐site mutation decreased in vitro DNA binding capacity compared to wild‐type MITF. White‐spotted buffalo bulls are sacrificed in funeral ceremonies in Tana Toraja, Indonesia, because they are considered holy, and our results show that genetic variation causes a tie to the cultural use of these buffaloes.

A structural approach reveals how neighbouring C2H2 zinc fingers influence DNA binding specificity

Development of an accurate protein–DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C2H2 zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C2H2-ZF–DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C2H2-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering.

A Tale of Two Proteins

Genetic regulatory proteins sense input signals to modulate transcription. In these proteins, intrinsic disorder (ID) is emerging as a common theme. Prokaryotic regulators have short ID segments, whereas eukaryotic regulators have extended regions that lack predicted 2° structure. Two examples illustrate the impact of ID on gene regulation: Lac repressor (LacI) in E. coli and the Hox protein Ultrabithorax (Ubx) in D. melanogaster. ID in the LacI hinge was first indicated by proteolytic susceptibility and later confirmed by physical studies. This short hinge region in free LacI is unfolded, but forms a folded dimeric structure that inserts into the minor groove and bends DNA to facilitate helix‐turn‐helix (HTH) domain contacts critical for high affinity binding. Restriction of hinge motions by a disulfide bond limits response to inducer, indicating the key role of the hinge in DNA binding and induction. Similarly, DNA binding by the structured Ubx HTH is augmented by an adjacent, disordered sequence, and Ubx‐DNA binding is also regulated by extensively disordered regions found well outside the HTH. These ID regions enable Ubx to interact with itself and a wide range of partners to further influence DNA binding. ID‐HTH interactions also mediate the non‐natural assembly of Ubx into strong, flexible fibers with intriguing materials properties. The importance of intrinsic disorder in genetic regulatory proteins appears widespread and may derive from the need to be flexible both in response and, particularly in eukaryotes, in partner selection.

Teaching Upper-Division Biochemistry Students the Role of Noncovalent Interactions and Ligand Structure in DNA Binding

The experiment described here uses the binding of cyanine dyes to DNA as a model system for demonstrating the role that both ligand structure and noncovalent interactions play in DNA binding. Upper-division biochemistry laboratory students observe changes in absorbance and fluorescence spectra when small molecules bind either in the minor groove of DNA or by intercalation. Students specifically note that ligand charge, substituents, and flexibility greatly influence binding mode. Based on these observations, students then determine the binding mode of an unknown small molecule. This experiment also fosters independent thought and problem-solving abilities. Students must self-direct binding-mode identification of their unknown without any guidance from the instructor. After completing this experiment, students demonstrate a clear understanding of the factors that influence DNA binding.

Genomic Regions Flanking E-Box Binding Sites Influence DNA Binding Specificity of bHLH Transcription Factors through DNA Shape

DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein-binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case studies, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the three-dimensional structure of DNA binding sites. Thus, the local shape of target sites might play a widespread role in achieving regulatory specificity within TF families.

71 Genomic regions flanking E-box binding sites influence DNA binding specificity of bHLH transcription factors through DNA shape

Transcriptional regulation of gene expression is enacted mainly through binding of transcription factors (TFs) to specific, short DNA sites in cis-regulatory regions of genes. Most TFs are members of protein families that share a common DNA-binding domain and thus recognize similar DNA-binding sequences. It is not well understood why paralogous TFs often bind different genomic target sites in vivo to effect different regulatory programs, despite apparently recognizing the same sequence motifs. Here, we designed custom protein-binding microarrays (PBMs) to analyze the DNA-binding specificities of two Saccharomyces cerevisiae basic helix-loop-helix (bHLH) proteins, Tye7 and Cbf1, as a model system. Our data reveal that E-box DNA-binding sequences (CAnnTG), when tested in the context of their native genomic flanking sequences, are bound differently by Cbf1 and Tye7. Computational models of the PBM data indicate that DNA sequence features located in the genomic sequences outside the E-box contribute to DNA-binding specificity in vitro. Our analyses suggest that these flanking regions affect DNA-binding specificity indirectly by influencing the three-dimensional structure of the E-box binding sites. Finally, we show that these subtle differences in intrinsic sequence preferences of Cbf1 and Tye7 in vitro help to explain their differential DNA-binding preferences in vivo. Our results provide further evidence that the local shape of DNA-binding sites may be an important feature in distinguishing the DNA-binding preferences among paralogous TFs and thus may play a widespread role in determining how transcriptional regulatory specificity within TF families is achieved.

Focus on Molecules: Neural retina leucine zipper (NRL)

The protein NRL (neural retina leucine zipper) (Uniprot Accession #P54845) is a basic motif-leucine zipper (bZIP) transcription factor of theMaf sub-family, involved in cellular differentiation and gene regulation, that has an essential intrinsic role in the development of rod photoreceptors (Mears et al., 2001 (cited within Nichols et al., 2010)). The gene is located on human chromosome 14q between bands 11.1 to 11.2 and encodes for a 237 amino acid proteinwith a molecular weight of 26 kDa that is located within the cellular nucleus. The leucine zipper domain is located between positions 187e208 (Fig. 1), with characteristic intertwined alpha helices forming a coil leading to a bifurcation at the DNA binding sites. The NRL protein is able to dimerise, forming both homoand heterodimers. The dimerisation composition influences DNA binding, with each dimer subunit independently able to recognise one half of the recognition sequence, leading to increased binding specificity and control of transcription. The additional DNA binding site in the NRL protein is provided by a basic domain located between positions 159e185 (Fig. 1A) on the amino terminal side of the domain.

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Polyglutamine Expansion Reduces the Association of TATA-binding Protein with DNA and Induces DNA Binding-independent Neurotoxicity

TATA-binding protein (TBP) is essential for eukaryotic gene transcription. Human TBP contains a polymorphic polyglutamine (polyQ) domain in its N terminus and a DNA-binding domain in its highly conserved C terminus. Expansion of the polyQ domain to >42 glutamines typically results in spinocerebellar ataxia type 17 (SCA17), a neurodegenerative disorder that resembles Huntington disease. Our recent studies have demonstrated that polyQ expansion causes abnormal interaction of TBP with the general transcription factor TFIIB and induces neurodegeneration in transgenic SCA17 mice (Friedman, M. J., Shah, A. G., Fang, Z. H., Ward, E. G., Warren, S. T., Li, S., and Li, X. J. (2007) Nat. Neurosci. 10, 1519-1528). However, it remains unknown how polyQ expansion influences DNA binding by TBP. Here we report that polyQ expansion reduces in vitro binding of TBP to DNA and that mutant TBP fragments lacking an intact C-terminal DNA-binding domain are present in transgenic SCA17 mouse brains. polyQ-expanded TBP with a deletion spanning part of the DNA-binding domain does not bind DNA in vitro but forms nuclear aggregates and inhibits TATA-dependent transcription activity in cultured cells. When this TBP double mutant is expressed in transgenic mice, it forms nuclear inclusions in neurons and causes early death. These findings suggest that the polyQ tract affects the binding of TBP to promoter DNA and that polyQ-expanded TBP can induce neuronal toxicity independent of its interaction with DNA.