Computational Inference of Genome-Wide Protein-DNA Interactions Using High-Throughput Genomic Data

Transcriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape.

We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations.

We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites.

Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets.

This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

Fluorescent microplate-based analysis of protein-DNA interactions I:immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.

Fluorescent microplate-based analysis of protein-DNA interactions II:immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Mapping protein-DNA interactions using ChIP-sequencing

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.

DNA interaction and phosphotransfer of the C-4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein.

Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild-type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N-terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF-RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML-RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

Palladium(II) complexes as biologically potent metallo-drugs: Synthesis, spectral characterization, DNA interaction studies and antibacterial activity

Four novel mononuclear Pd(II) complexes have been synthesized with the biologically active Schiff base ligands (L-1-L-4) derived from 3-amino-2-methyl-4(3H)-quinazolinone. The structure of the complexes has been proposed by elemental analysis, molar conductance, IR, H-1 NMR, mass, UV-Vis spectrometric and thermal studies. The investigation of interaction of the complexes with calf thymus DNA (CT-DNA) has been performed with absorption and fluorescence spectroscopic studies. The nuclease activity was done using pUC19 supercoiled DNA by gel-electrophoresis. All the ligands and their Pd(II) complexes have also been screened for their antibacterial activity by discolor diffusion technique. (C) 2013 Elsevier B.V. All rights reserved.

Dinuclear zinc bis(thiosemicarbazone) complexes: Synthesis, in vitro anticancer activity, cellular uptake and DNA interaction study

Four dinucleating bis(thiosemicarbazone) ligands and their zinc complexes have been synthesized and characterized by multinuclear NMR (H-1 and C-13), IR, UV-Vis, ESI-MS and fluorescence spectroscopic techniques. Their purity was assessed by elemental analysis. Cytotoxicity was tested against five human cancer cell lines using the sulphorhodamine B (SRB) assay, where one of the complexes, 1,3-bis{biacetyl-2'-(4 `'-N-pyrrolidinylthiosemicarbazone)-3'-(4 `'-N-pyrrolidinylthiosemicarbazone) zinc(II)} propane (6), was found to be quite cytotoxic against MCF-7 (breast cancer) and HepG2 (hepatoma cancer) cell lines, with a potency similar to that of the well known anticancer drug adriamycin. It is evident from the cellular uptake studies that the uptake is same for the active complex 6 and the inactive complex 8 (1,6-bis{biacetyl- 2'-(4 `'-N-pyrrolidinylthiosemicarbazone)-3'-(4 `'-N-pyrrolidinylthiosemicarbazone) zinc(II)} hexane) in MCF-7 and HepG2 cell lines. In vitro DNA binding and cleavage studies revealed that all complexes bind with DNA through electrostatic interaction, and cause no significant cleavage of DNA. (C) 2'13 Elsevier B. V. All rights reserved.

mu-Oxamido binuclear copper (II) complexes: Synthesis, crystal structure, DNA interaction and antibacterial studies

Four binuclear copper (II) complexes Cu(oxpn)Cu(B)](2+) (2-5) bridged by N, N'-bis3-(methylamino) propyl] oxamide (oxpn), where, B is N, N-donor heterocyclic bases (viz. 2,2'-bipyridine (bpy, 2), 1,10-phenathroline (phen, 3), dipyrido3,2-d:2',3'-f]quinoxaline (dpq, 4) and dipyrido3,2-a:2',3'-c]phenazine (dppz, 5) are synthesized, characterized by different spectroscopic and single crystal X-ray data technique. The phen (3) and dpq (4) complexes were structurally characterized by X-ray data analysis. Their DNA binding, oxidative cleavage and antibactirial activities were studied. The dpq (4) and dppz (5) complexes are avid binders to the Calf thymus DNA (CT-DNA). The phen (3), dpq (4) and dppz (5) complexes show efficient oxidative cleavage of supercoiled DNA (SC DNA) through hydroxyl radical ((OH)-O-center dot) pathway in the presence of Mercaptopropionic acid (MPA). (C) 2013 Elsevier Ltd. All rights reserved.