Specific DNA-binding Proteins Research Articles

β-Catenin induces T-cell transformation by promoting genomic instability (HEM4P.237)

Abstract Deregulated activation of β-catenin has been correlated with genomic instability in cancer. During thymocyte development β-catenin activates transcription in partnership with Tcf-1, an essential T-cell specific DNA-binding protein. We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination-activating-gene (RAG) and c-Myc activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG-recombination events and resembled oncogenic translocations in ponies. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. Interestingly, ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including sites near the translocation. Pre-transformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG induced DNA double strand breaks (DSBs). Importantly these cells were able to survive despite DNA damage as activated β-catenin promoted an anti-apoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas and this is associated with compromised DSB repair and increased survival of thymocytes with damaged DNA.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Using hidden Markov models to predict DNA-binding proteins with sequence and structure information

In the post-genome period, the protein domain structures are published rapidly, but they have not been studied comprehensively. To figure out the cell function, the protein---DNA interactions decrypt the protein domain structures in recent research. Several machine-learning based methods are applied to the issue; however, they are not efficient to translate the tertiary structure characteristics of proteins into appropriate features for predicting the DNA-binding proteins. In this work, a novel machine-learning approach based on hidden Markov models identifies the characteristics of DNA-binding proteins with their amino acid sequences and tertiary structures. After we distill the features from DNA-binding proteins, a support vector machine based classifier predicts general DNA-binding proteins with the accuracy of 88.45 % through fivefolds cross-validation. Furthermore, we construct a response element specific classifier for predicting response element specific DNA-binding proteins, and the performance achieves the precision of 96.57 % with recall rate as 88.83 % in average. To verify the prediction of DNA-binding proteins, we used the DNA-binding proteins from MCF-7 that are likely to bind with estrogen response elements (ERE), and the results show that our methods can apply to practice.

Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

Positive and Negative Design for Nonconsensus Protein-DNA Binding Affinity in the Vicinity of Functional Binding Sites

Recent experiments provide an unprecedented view of protein-DNA binding in yeast and human genomes at single-nucleotide resolution. These measurements, performed over large cell populations, show quite generally that sequence-specific transcription regulators with well-defined protein-DNA consensus motifs bind only a fraction among all consensus motifs present in the genome. Alternatively, proteins in vivo often bind DNA regions lacking known consensus sequences. The rules determining whether a consensus motif is functional remain incompletely understood. Here we predict that genomic background surrounding specific protein-DNA binding motifs statistically modulates the binding of sequence-specific transcription regulators to these motifs. In particular, we show that nonconsensus protein-DNA binding in yeast is statistically enhanced, on average, around functional Reb1 motifs that are bound as compared to nonfunctional Reb1 motifs that are unbound. The landscape of nonconsensus protein-DNA binding around functional CTCF motifs in human demonstrates a more complex behavior. In particular, human genomic regions characterized by the highest CTCF occupancy, show statistically reduced level of nonconsensus protein-DNA binding. Our findings suggest that nonconsensus protein-DNA binding is fine-tuned around functional binding sites using a variety of design strategies.

Two enzymes, TilS and HprT, can form a complex to function as a transcriptional activator for the cell division protease gene ftsH in Bacillus subtilis

The FtsH protein is an ATP-dependent cytoplasmic membrane protease involved in the control of membrane protein quality, cell division and heat shock response in Bacillus subtilis and many other bacteria. TilS, the tRNA(Ile2) lysidine synthetase, is a tRNA-binding protein that can modify pre-tRNA(Ile2). HprT, the hypoxanthine-guanine phosphoribosyltransferase, is implicated in purine salvage. Both tilS and hprT are essential for cell viability of B. subtilis. In this report, by co-purification experiments and gel filtration analyses, we show that there is complex formation between co-expressed TilS and HprT. Electrophoretic mobility shift assays and in vitro transcription analyses demonstrated that the TilS/HprT complex functions as a specific DNA-binding protein that can stimulate ftsH transcription in vitro. Two regions located upstream of the ftsH promoter have been identified as the TilS/HprT-binding sites and shown to be required for TilS/HprT-dependent ftsH transcription in vitro and in vivo. Results from gel supershift assays support the notion that the TilS/HprT complex likely employs its distinct segments for interaction with these two distinct TilS/HprT-binding sites, respectively. In conclusion, we present the first evidence that bi-functional TilS and HprT can form a complex to function as a transcriptional activator to stimulate ftsH transcription.

Abstract 2236: A selection strategy for the design of small proteins that can distinguish DNA repair intermediates.

Abstract Recognition of DNA damage by repair proteins is crucial for genomic stability. Several repair enzymes recognize aberrant DNA structural features, including nicks, gaps, and abasic sites, in a sequence-independent DNA manner. Reported here is a strategy to model these protein-DNA interactions using small proteins. A phage-displayed library of approximately one hundred million distinct zinc fingers was engineered at the DNA level based on the DNA binding domain of the repair protein poly(ADP-ribose) polymerase. Eight codons, representing amino acid residues in ‘loop’ regions of the zinc finger structure, were randomized using an NNK codon scheme. The DNA cassette was cloned into a commercially obtained T7 phagemid vector. The resultant phage library was exposed to nicked DNA molecules, which were attached to magnetic beads via a biotin-streptavidin linkage. In four rounds of selection that featured negative selection against non-nicked DNA and streptavidin coated beads, viral retention on the affinity resin increased from a negligible quantity to 62%. DNA sequencing following the final round revealed near convergence to a single, 39-mer peptide. This selected peptide will be generated by bacterial expression and assayed for binding activity using multiple strategies. While phage display of sequence specific DNA binding proteins is well-precedented, selection of peptides that bind specific DNA structures has not been reported. Molecules discovered by this strategy will help illuminate the structure-function relationships of proteins that recognize distinct DNA structures and may lead to new anticancer approaches. Citation Format: Rachel M. Guerra, Kristina M. Langenborg, Kayla M. Gross, Emily V. Sher, John W. Gilboy, Dylan Plaskon, Kevin P. Rice. A selection strategy for the design of small proteins that can distinguish DNA repair intermediates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2236. doi:10.1158/1538-7445.AM2013-2236

Bayesian hierarchical model of protein-binding microarrayk-mer data reduces noise and identifies transcription factor subclasses and preferredk-mers

Sequence-specific transcription factors (TFs) regulate the expression of their target genes through interactions with specific DNA-binding sites in the genome. Data on TF-DNA binding specificities are essential for understanding how regulatory specificity is achieved. Numerous studies have used universal protein-binding microarray (PBM) technology to determine the in vitro binding specificities of hundreds of TFs for all possible 8 bp sequences (8mers). We have developed a Bayesian analysis of variance (ANOVA) model that decomposes these 8mer data into background noise, TF familywise effects and effects due to the particular TF. Adjusting for background noise improves PBM data quality and concordance with in vivo TF binding data. Moreover, our model provides simultaneous identification of TF subclasses and their shared sequence preferences, and also of 8mers bound preferentially by individual members of TF subclasses. Such results may aid in deciphering cis-regulatory codes and determinants of protein-DNA binding specificity. Source code, compiled code and R and Python scripts are available from http://thebrain.bwh.harvard.edu/hierarchicalANOVA. Supplementary data are available at Bioinformatics online.

Chromatin structure outside and inside the nucleus

The structure of the 30-nm chromatin fiber has provided, over the years, an important reference in chromatin studies. Originally derived from electron microscopic studies of soluble chromatin fibers released by restriction digestion, the gross structural features of such fragments have been supported by biophysical methods such as low angle X-ray and neutron scattering, sedimentation, light scattering, and electric dichroism. Electron microscopy and sedimentation velocity measurements demonstrated that reconstituted chromatin fibers, prepared from repeating arrays of high affinity nucleosome positioning sequences, retain the same overall features as observed for native chromatin fibers. It had been suggested that the 30 nm fiber might be the form assumed in vivo by transcriptionally silent chromatin, but individual gene or genome-wide studies of chromatin released from nuclei do not reveal any such simple correlation. Furthermore, even though the 30 nm fiber has been thought to represent an intermediate in the hierarchical folding of DNA into chromosomes, most analyses of chromatin folding within the nucleus do not detect any regular extended compact structures. However, there are important exceptions in chicken erythroid cell nuclei as well as in transcribed regions that form extended loops. Localized domains within the nucleus, either at the surface of chromosome domains or constrained as a specialized kind of constitutive heterochromatin by specific DNA binding proteins, may adopt 30 nm fiber-like structures.

How DNA-Binding Proteins find their Target Sites in Human Cells

Precise and timely expression of specific genes is fundamental for the viability of any organism. Specific DNA-binding proteins (transcription factors, TFs) transiently associate to promoter sequences on the genome to activate or repress transcription of specific genes. The way TFs explore the nuclear space, find, and bind to their target sequences is the key to decipher transcriptional control but is still subject of debate. We exploit a live-cell model system to identify what factors primarily orchestrate TFs target search. Our assay is based on human cells containing, at a single locus, specific DNA binding sites (‘target') for an exogenous DNA-binding protein (TetR, ‘searcher'). First, single-particle-tracking experiments revealed that individual TetR proteins move in a very composite way. They transiently interact with nonspecific DNA sequences, via their DNA-binding-domain, with residence times ranging from hundreds of millisecond up to few seconds. Furthermore, a subset of proteins shows confinement within micron-sized regions even when DNA-binding is impaired, which suggests that the organization of mammalian nuclei plays a role in search dynamics. Next, we directly quantified the association rate constant of proteins to their targets in living cells. Based on our binding assay, the measured rate constant resulted much slower than expected and local decondensation of chromatin, at the target site locus, did not affect the association rate. In other words, the effective size of a DNA binding site is much smaller than the size of the molecular interaction. The picture emerging from our experiments shows that, in human cells, specific DNA-binding proteins move in a complex environment where they may remain locally confined. They interact transiently also with nonspecific DNA sequences and their target search mechanism is consistent with a non-compact exploration of the nuclear space with very inefficient binding.

A study of DNA design dependency of segmented DNA-induced gold nanoparticle aggregation towards versatile bioassay development

DNA-decorated gold nanoparticles (AuNPs) are important sensing probes for designing versatile assays for detecting DNA hybridization, DNA binders and DNA associated biological processes. In this study, we report DNA design rules for a pair of segmented DNA-conjugated AuNPs that can undergo “transient” aggregation due to cooperative base-pairing force of sticky ends on DNA and salt screening. A wildtype estrogen receptor response element (ERE) was used as a model DNA which is segmented into two half-sites, each carrying a complementary sticky end and a half-site DNA, were conjugated onto AuNPs to form two sets of complementary DNA–AuNPs conjugates. A number of DNA design parameters are studied for their effects on the aggregation kinetics, namely DNA conformation (i.e., mixed structure of ssDNA and dsDNA), number of sticky-ends bases, spacer length, as well as the symmetrical and asymmetrical interplay between the complementary set of segmented DNA–AuNPs conjugates. Firstly, we found that dsDNA serves as a more effective spacer than ssDNA in preventing base coordination of the nucleotides to the AuNPs surface due to its rigidity that in turn helps to improve the accessibility of sticky-ends for faster aggregation. Secondly, base-pairing force in facilitating the salt-induced AuNPs aggregation is tunable by the number of sticky-ends, which is closely related to the length and structural composition of the DNA spacer. Thirdly, symmetrically spaced sticky-ends enable quicker non-crosslinking aggregation than the asymmetrical combination due to their closer initial interparticle distance. Based on the optimized DNA design, we appended the efficient aggregation that leads to transient formation of a full ERE sequence for detecting estrogen receptor β (ER β) by exploiting protein binding retarded particle aggregation. With a competition assay, binding affinity of ERβ to different DNA sequences can be easily screened using a single set of complementary segmented DNA–AuNPs probes. The DNA design dependency of the DNA–AuNPs aggregation studied herein has enabled potential applications for rapid and highly specific DNA-binding protein detection, which could be extended to detect a wide range of DNA binders and its related biological processes.

Transcription activator-like effector hybrids for conditional control and rewiring of chromosomal transgene expression

The ability to conditionally rewire pathways in human cells holds great therapeutic potential. Transcription activator-like effectors (TALEs) are a class of naturally occurring specific DNA binding proteins that can be used to introduce targeted genome modifications or control gene expression. Here we present TALE hybrids engineered to respond to endogenous signals and capable of controlling transgenes by applying a predetermined and tunable action at the single-cell level. Specifically, we first demonstrate that combinations of TALEs can be used to modulate the expression of stably integrated genes in kidney cells. We then introduce a general purpose two-hybrid approach that can be customized to regulate the function of any TALE either using effector molecules or a heterodimerization reaction. Finally, we demonstrate the successful interface of TALEs to specific endogenous signals, namely hypoxia signaling and microRNAs, essentially closing the loop between cellular information and chromosomal transgene expression.

MuMoD: a Bayesian approach to detect multiple modes of protein–DNA binding from genome-wide ChIP data

High-throughput chromatin immunoprecipitation has become the method of choice for identifying genomic regions bound by a protein. Such regions are then investigated for overrepresented sequence motifs, the assumption being that they must correspond to the binding specificity of the profiled protein. However this approach often fails: many bound regions do not contain the ‘expected’ motif. This is because binding DNA directly at its recognition site is not the only way the protein can cause the region to immunoprecipitate. Its binding specificity can change through association with different co-factors, it can bind DNA indirectly, through intermediaries, or even enforce its function through long-range chromosomal interactions. Conventional motif discovery methods, though largely capable of identifying overrepresented motifs from bound regions, lack the ability to characterize such diverse modes of protein–DNA binding and binding specificities. We present a novel Bayesian method that identifies distinct protein–DNA binding mechanisms without relying on any motif database. The method successfully identifies co-factors of proteins that do not bind DNA directly, such as mediator and p300. It also predicts literature-supported enhancer–promoter interactions. Even for well-studied direct-binding proteins, this method provides compelling evidence for previously uncharacterized dependencies within positions of binding sites, long-range chromosomal interactions and dimerization.

Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

BackgroundReplication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as “replication domains”, and recent findings indicate that replication timing is under developmental and cell type-specific regulation.Methodology/Principal FindingsWe examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1.ConclusionsOur results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome.

Atomistic modeling of protein–DNA interaction specificity: progress and applications

An accurate, predictive understanding of protein-DNA binding specificity is crucial for the successful design and engineering of novel protein-DNA binding complexes. In this review, we summarize recent studies that use atomistic representations of interfaces to predict protein-DNA binding specificity computationally. Although methods with limited structural flexibility have proven successful at recapitulating consensus binding sequences from wild-type complex structures, conformational flexibility is likely important for design and template-based modeling, where non-native conformations need to be sampled and accurately scored. A successful application of such computational modeling techniques in the construction of the TAL-DNA complex structure is discussed. With continued improvements in energy functions, solvation models, and conformational sampling, we are optimistic that reliable and large-scale protein-DNA binding prediction and engineering is a goal within reach.

AdeR, a PucR-Type Transcription Factor, Activates Expression of l-Alanine Dehydrogenase and Is Required for Sporulation of Bacillus subtilis

The Bacillus subtilis ald gene encodes L-alanine dehydrogenase, which catalyzes the NAD(+)-dependent deamination of L-alanine to pyruvate for the generation of energy and is required for normal sporulation. The transcription of ald is induced by alanine, but the mechanism underlying alanine induction remains unknown. Here we report that a gene (formerly yukF and now designated adeR) located upstream of ald is essential for the basal and alanine-inducible expression of ald. The disruption of the adeR gene caused a sporulation defect, whereas the complementation of an adeR mutation with an intact adeR gene restored the sporulation ability. adeR expression was not subject to autoregulation and alanine induction. Deletion and mutation analyses revealed that an inverted repeat, centered at position -74.5 relative to the transcriptional initiation site of ald, was required for ald expression and also likely served as a ρ-independent transcription terminator. Electrophoretic mobility shift assays showed that purified His-tagged AdeR was a specific DNA-binding protein and that this inverted repeat was required for AdeR binding. AdeR shows no significant amino acid sequence similarity to the known transcriptional activators of ald genes from other bacteria. AdeR appears to be a member of the PucR family of transcriptional regulators. Its orthologs of unknown function are present in some other Bacillus species. Collectively, these findings support the notion that AdeR is a transcriptional activator which mediates ald expression in response to alanine availability and is important for normal sporulation in B. subtilis.

Profile of Keith Joung

Profile of Keith Joung

Development of RNA Stiffness Parameters and Analysis on Protein-RNA Binding Specificity: Comparison with DNA

It has been well established that the elastic character of DNA plays an important role in protein-DNA binding specificity. In this work, we have analyzed the role of elasticity to understand the binding specificity of protein-RNA complexes. We have developed a sequence dependent stiffness scale for the trinucleotides in RNA and revealed the similarities and differences compared with DNA. We found that the stiffness of 15 trinucleotides has inverse effects and nine nucleotides are significantly different between RNA and DNA. The analysis on the relationship between RNA stiffness and RNA-binding specificity shows that the influence of elasticity is minimal in protein-RNA recognition, whereas it plays an important role in protein-DNA binding specificity. We observed a moderate correlation between stiffness and dissociation constant in U1A RBD1 protein and PP7 coat protein, whereas the correlation is poor for many other complexes. These results show that along with RNA stiffness, other interactions, such as shape complementarity, electrostatic interactions, hydrogen bonds and direct contacts between RNA and protein atoms are important for protein- RNA recognition. Keywords: Binding specificity, elasticity, protein-RNA recognition, stiffness, trinucleotides, protein-DNA, parameters, GLD-1, nucleotides, native gel electrophoresis

NuST: analysis of the interplay between nucleoid organization and gene expression

Different experimental results suggest the presence of an interplay between global transcriptional regulation and chromosome spatial organization in bacteria. The identification and clear visualization of spatial clusters of contiguous genes targeted by specific DNA-binding proteins or sensitive to nucleoid perturbations can elucidate links between nucleoid structure and gene expression patterns. Similarly, statistical analysis to assess correlations between results from independent experiments can provide the integrated analysis needed in this line of research. NuST (Nucleoid Survey tools), based on the Escherichia coli genome, gives the non-expert the possibility to analyze the aggregation of genes or loci sets along the genome coordinate, at different scales of observation. It is useful to discover correlations between different sources of data (e.g. expression, binding or genomic data) and genome organization. A user can use it on datasets in the form of gene lists coming from his/her own experiments or bioinformatic analyses, but also make use of the internal database, which collects data from many published studies. NuST is a web server (available at http://www.lgm.upmc.fr/nust/). The website is implemented in PHP, SQLite and Ajax, with all major browsers supported, while the core algorithms are optimized and implemented in C. NuST has an extensive help page and provides a direct visualization of results as well as different downloadable file formats. A template Perl code for automated access to the web server can be downloaded at http://www.lgm.upmc.fr/nust/downloads/, in order to allow the users to use NuST in systematic bioinformatic analyses.

Using systems and structure biology tools to dissect cellular phenotypes

The Center for the Multiscale Analysis of Genetic Networks (MAGNet, http://magnet.c2b2.columbia.edu) was established in 2005, with the mission of providing the biomedical research community with Structural and Systems Biology algorithms and software tools for the dissection of molecular interactions and for the interaction-based elucidation of cellular phenotypes. Over the last 7 years, MAGNet investigators have developed many novel analysis methodologies, which have led to important biological discoveries, including understanding the role of the DNA shape in protein-DNA binding specificity and the discovery of genes causally related to the presentation of malignant phenotypes, including lymphoma, glioma, and melanoma. Software tools implementing these methodologies have been broadly adopted by the research community and are made freely available through geWorkbench, the Center's integrated analysis platform. Additionally, MAGNet has been instrumental in organizing and developing key conferences and meetings focused on the emerging field of systems biology and regulatory genomics, with special focus on cancer-related research.