Stress-induced Duplex Destabilization Research Articles

Whole genome sequence of moderate halophilic marine bacterium Marinobacter litoralis SW-45: Abundance of non-coding RNAs.

A report on the de novo Whole Genome Sequence (WGS) of Marinobacter litoralis SW-45, a moderately salt-tolerant bacterium isolated from the seawater in Malaysia is presented. The strain has a genome size of 3.45 Mb and is capable of producing halophilic lipase, protease and esterase enzymes. Computational prediction of non-coding RNA (ncRNA) genes in M. litoralis SW-45 was performed using standalone software known as the non-coding RNA characterization (nocoRNAc). In addition, a phylogenetic tree showing the evolutionary relationship between the strain and other members of the genus Marinobacter was constructed using 16SrRNA sequence information. A total of 385 ncRNA transcripts, 1124 terminator region, and 2350 Stress Induced Duplex Destabilization sites were predicted. The current WGS shotgun project has provided the relevant genetic information that may be useful for the strain's improvement studies. This manuscript gives the first description of M. litoralis with a complete genome.

Профили вызванной суперспирализацией дестабилизации дуплекса ДНК (SIDD) для промоторов бактериофага T7

Для функционирования регуляторных областей ДНК решающее значение имеет не нуклеотидная последовательность (генетический текст), а их физико-химические и структурные свойства. Именно они обеспечивают кодирование ДНК-белковых взаимодействий, лежащих в основе различных процессов регуляции. Среди таких свойств SIDD (Stress-Induced Duplex Destabilization) — характеристика, описывающая склонность участка дуплекса ДНК к плавлению при заданном уровне суперспирализации. Ранее для данного параметра дуплекса показана роль в функционировании областей регуляции различного типа. В данной работе модель SIDD использована для получения профилей вероятности плавления последовательностей промоторов бактериофага T7. Данный геном характеризуется малым размером (примерно 40 тыс. пар нуклеотидов) и временной организацией экспрессии генов: на первом этапе инфекции ранняя область Т7-ДНК транскрибируется РНК-полимеразой бактерии-хозяина, на более поздних этапах жизненного цикла фагоспецифичная РНК-полимераза последовательно производит транскрипцию областей генов II класса и III класса. При этом механизмы дифференциального узнавания промоторов разных групп ферментом-полимеразой не могут быть основаны исключительно на их нуклеотидной последовательности, в частности в связи с тем, что она очень близка для большинства таких промоторов. В то же время полученные профили SIDD данных промоторов сильно различаются и могут быть разделены на характерные группы, соответствующие функциональным классам промоторов Т7-ДНК. Так, все промоторы ранней области находятся в области влияния одного максимально дестабилизированного участка дуплекса ДНК, соответствующего различным областям конкретных промоторов. Промоторы класса II лишены значительно дестабилизированных областей вблизи точки старта транскрипции. Напротив, промоторы III класса имеют характерные пики профилей вероятности плавления, в каждом случае локализованные в ближней downstream-области. Таким образом, установлены значительные различия профилей для промоторных областей при очень близкой нуклеотидной последовательности (промоторы II и III классов отличаются единичными заменами нуклеотидов), что подтверждает высокую чувствительность рассматриваемого свойства дуплекса к первичной структуре, а также необходимость рассмотрения широкого генетического контекста. Описанные различия профилей вероятности плавления на основе модели SIDD наряду с другими физическими свойствами могут определять дифференциальное узнавание промоторов разных классов РНК-полимеразами.

Comparative Analysis of Mycoplasma gallisepticum vlhA Promoters.

Mycoplasma gallisepticum is an intracellular parasite affecting respiratory tract of poultry that belongs to class Mollicutes. M. gallisepticum features numerous variable lipoprotein hemagglutinin genes (vlhA) that play a role in immune escape. The vlhA promoters have a set of distinct properties in comparison to promoters of the other genes. The vlhA promoters carry a variable GAA repeats region at approximately 40 nts upstream of transcription start site. The promoters have been considered active only in the presence of exactly 12 GAA repeats. The mechanisms of vlhA expression regulation and GAA number variation are not described. Here we tried to understand these mechanisms using different computational methods. We conducted a comparative analysis among several M. gallisepticum strains. Nucleotide sequences analysis showed the presence of highly conserved regions flanking repeated trinucleotides that are not linked to GAA number variation. VlhA genes with 12 GAA repeats and their orthologs in 12 M. gallisepticum strains are more conserved than other vlhA genes and have narrower GAA number distribution. We conducted comparative analysis of physicochemical profiles of M. gallisepticum vlhA and sigma-70 promoters. Stress-induced duplex destabilization (SIDD) profiles showed that sigma-70 group is characterized by the common to prokaryotic promoters sharp maxima while vlhA promoters are hardly destabilized with the region between GAA repeats and transcription start site having zero opening probability. Electrostatic potential profiles of vlhA promoters indicate the presence of the distinct patterns that appear to govern initial stages of specific DNA-protein recognition. Open state dynamics profiles of vlhA demonstrate the pattern that might facilitate transcription bubble formation. Obtained data could be the basis for experimental identification of mechanisms of phase variation in M. gallisepticum.

T7-like bacteriophage promoters: stress-induced duplex destabilization suggests a role in replication

T7-like bacteriophage promoters: stress-induced duplex destabilization suggests a role in replication

Дестабилизация дуплекса ДНК активно реплицирующихся промоторов бактериофагов группы Т7

The relation between the processes of replication and transcription has been actively studied over several decades, but detailed mechanisms for their interaction have not been established reliably. Among the initiating transcription promoters of bacteria and bacteriophages, there are both promoters having an additional function of the secondary origin of replication (OR) and promoters not participating in this process. In this paper, we describe the stability of DNA by Stress-Induced Duplex Destabilization (SIDD) profiles for a complete set of promoters and the primary OR of the bacteriophage T7 genome. It has been shown that, among the native T7 promoters, only those that have an additional function of secondary OR are characterized by high destabilization. These include the phiOL and phiOR promoters adjoining the 5' and 3' terminal repeats of bacteriophage T7, and of six other T7 group phages. In each case, these two promoters are located in the regions of DNA with high destabilization of the duplex. Additionally, the genomes of seven representatives of the T7 group without annotated phiOL and phiOR have been considered. For three of them, high peaks of SIDD profiles have been found near the ends of the genomic DNA that may be due to the presence of similar phiOL and phiOR promoters. Probably, such promoters can be found in the genomes of other bacteriophages. Thus, for the promoters of bacteriophages, we have a confirmation of the relationship of SIDD as a DNA duplex parameter and the DNA replication initiation on promoters, serving as secondary OR.

Significant Role of Segmental Duplications and SIDD Sites in Chromosomal Translocations of Hematological Malignancies: A Multi-parametric Bioinformatic Analysis.

Recurrent non-random chromosomal translocations are hallmark characteristics of leukemogenesis, and however, molecular mechanisms underlying these rearrangements are less explored. The fundamental question is, why and how chromosomes break and reunite so precisely in the genome. Meticulous understanding of mechanism leading to chromosomal rearrangement can be achieved by characterizing breakpoints. To address this hypothesis, a novel multi-parametric computational approach for characterization of major leukemic translocations within and around breakpoint region was performed. To best of our knowledge, this bioinformatic analysis is unique in finding the presence of segmental duplications (SDs) flanking breakpoints of all major leukemic translocation. Breakpoint islands (BpIs) were analyzed for stress-induced duplex destabilization (SIDD) sites along with other complex genomic architecture and physicochemical properties. Our study distinctly emphasizes on the probable correlative role of SDs, SIDD sites and various genomic features in the occurrence of breakpoints. Further, it also highlights potential features which may be playing a crucial role in causing double-strand breaks, leading to translocation.

248. A Bona Fide Mammalian Replicator Enhances All Aspects of Episomal Gene Transfer Into Human Hematopoietic Progenitor Cells

Episomal gene transfer for gene therapy applications is limited at present mainly because of luck of long term persistence of episomes in the nucleus of cells. However, episomal vectors may offer a valid alternative to viral vectors as well as to gene editing strategies. To expand the potential of episomal vectors, we used the Initiation of Replication region, (IR), from β-globin gene locus, which is capable to promote DNA replication ectopically (Wang L et al 2004) and can rescue DNA replication within the context of Scaffold /Matrix Attachment Region (S/MAR) based episomal vector (Giannakoppulos A. et al 2009). We constructed two episomal vectors, derived from the protopype S/MAR based episomal vector pEPI-eGFP (6.7Kb), namely, pEP-IR and its control pEPI-SFFV, by replacing CMV promoter of eGFP by the Spleen Focus forming Virus (SFFV) promoter, while vector pEP-IR carries the IR element just upstream the SFFV promoter. Stress Induced Duplex Destabilization (SIDD) analysis of the novel plasmids indicated that these changes would not disrupt their function as episomal vectors and could be beneficial. All three vectors (pEP-IR and control vectors pEPI-SFFV and pEPI-eGFP) showed excellent episomal properties upon transfer into K562 cells. Additionally, they showed transfection efficiency around 30% upon transfer into CD34+ cells from peripheral blood, from adult, mobilised, healthy donors. CD34+/eGFP+ cells sorted by FACS were placed in semisolid cultures and produced differentiated cell-colonies within 14 days. Non-fluorescent colonies do not contain plasmids, as documented previously too (Papapetrou E. et al 2006). Vector pEP-IR shows an enhancement of all aspects of vector performance: 100% fluorescent colonies against (0%) for pEPI-eGFP as found previously (Papapetrou E. et al 2006) and 56% for pEPI-SFFV, in Fluorescent microscopy; increase of plasmid copy number per fluorescent cell, which was 1.78 for pEP-IR against 1.12 for pEPI-SFFV, as estimated by Real Time PCR. The ratio of the number of fluorescent, stably transfected colonies to the number of transfected, seeded cells presenting the rate of establishment in semisolid cultures of transfected CD34+ cells, was 5.5% for pEPI-SFFV and 15% for pEP-IR. It is thus determined that the relative capacity for establishment for pEP-IR is 3-fold higher than that of control vector pEPI-SFFV. Finally, estimation of eGFP mRNA showed that pEP-IR supports 3 times higher eGFP expression per plasmid copy, compared to pEPI-SFFV. Vector pEPI-IR in a series of over five transfections of CD34+ cells, routinely produced fluorescent cells in the area of 99.8%. Practically, vector pEP-IR is expected to be established in all cells that give rise to the differentiated cell-progeny of CD34+ cells, that is in 10% to 20% of the total sorted CD34+/eGFP+ cells. These estimates for vector pEPI-IR can be of clinical importance.

SIST: stress-induced structural transitions in superhelical DNA.

Supercoiling imposes stress on a DNA molecule that can drive susceptible sequences into alternative non-B form structures. This phenomenon occurs frequently in vivo and has been implicated in biological processes, such as replication, transcription, recombination and translocation. SIST is a software package that analyzes sequence-dependent structural transitions in kilobase length superhelical DNA molecules. The numerical algorithms in SIST are based on a statistical mechanical model that calculates the equilibrium probability of transition for each base pair in the domain. They are extensions of the original stress-induced duplex destabilization (SIDD) method, which analyzes stress-driven DNA strand separation. SIST also includes algorithms to analyze B-Z transitions and cruciform extrusion. The SIST pipeline has an option to use the DZCBtrans algorithm, which analyzes the competition among these three transitions within a superhelical domain. The package and additional documentation are freely available at https://bitbucket.org/benhamlab/sist_codes. dzhabinskaya@ucdavis.edu.

Genomic analysis of the regulatory elements and links with intrinsic DNA structural properties in the shrunken genome of Buchnera

BackgroundBuchnera aphidicola is an obligate symbiotic bacterium, associated with most of the aphididae, whose genome has drastically shrunk during intracellular evolution. Gene regulation in Buchnera has been a matter of controversy in recent years as the combination of genomic information with the experimental results has been contradictory, refuting or arguing in favour of a functional and responsive transcription regulation in Buchnera.The goal of this study was to describe the gene transcription regulation capabilities of Buchnera based on the inventory of cis- and trans-regulators encoded in the genomes of five strains from different aphids (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, Cinara cedri and Cinara tujafilina), as well as on the characterisation of some intrinsic structural properties of the DNA molecule in these bacteria.ResultsInteraction graph analysis shows that gene neighbourhoods are conserved between E. coli and Buchnera in structures called transcriptons, interactons and metabolons, indicating that selective pressures have acted on the evolution of transcriptional, protein-protein interaction and metabolic networks in Buchnera. The transcriptional regulatory network in Buchnera is composed of a few general DNA-topological regulators (Nucleoid Associated Proteins and topoisomerases), with the quasi-absence of any specific ones (except for multifunctional enzymes with a known gene expression regulatory role in Escherichia coli, such as AlaS, PepA and BolA, and the uncharacterized hypothetical regulators YchA and YrbA). The relative positioning of regulatory genes along the chromosome of Buchnera seems to have conserved its ancestral state, despite the genome erosion. Sigma-70 promoters with canonical thermodynamic sequence profiles were detected upstream of about 94% of the CDS of Buchnera in the different aphids. Based on Stress-Induced Duplex Destabilization (SIDD) measurements, unstable σ70 promoters were found specifically associated with the regulator and transporter genes.ConclusionsThis genomic analysis provides supporting evidence of a selection of functional regulatory structures and it has enabled us to propose hypotheses concerning possible links between these regulatory elements and the DNA-topology (i.e., supercoiling, curvature, flexibility and base-pair stability) in the regulation of gene expression in the shrunken genome of Buchnera.

Analysis of stress-induced duplex destabilization (SIDD) properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe

BackgroundReplication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe.ResultsWe have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region.ConclusionSIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

The in silico prediction of promoters in bacterial genomes.

In silico approaches to the identification of bacterial promoters are hampered by poor conservation of their characteristic binding sites. This suggests that the usual position weight matrix models of bacterial promoters are incomplete. A number of methods have been used to overcome this inadequacy, one of which is to incorporate structural properties of DNA. In this paper we describe an extension of the promoter description to include SIDD (stress induced duplex destabilization), DNA curvature and stacking energy. Although we report the best result to date for a realistic promoter prediction task, surprisingly, DNA structural properties did not contribute significantly to this result. We also demonstrate for the first time, that sigma-54 promoters have a stronger association with SIDD than do other promoter types.

Promoter prediction in E. coli based on SIDD profiles and Artificial Neural Networks

BackgroundOne of the major challenges in biology is the correct identification of promoter regions. Computational methods based on motif searching have been the traditional approach taken. Recent studies have shown that DNA structural properties, such as curvature, stacking energy, and stress-induced duplex destabilization (SIDD) are useful in promoter prediction, as well. In this paper, the currently used SIDD energy threshold method is compared to the proposed artificial neural network (ANN) approach for finding promoters based on SIDD profile data.ResultsWhen compared to the SIDD threshold prediction method, artificial neural networks showed noticeable improvements for precision, recall, and F-score over a range of values. The maximal F-score for the ANN classifier was 62.3 and 56.8 for the threshold-based classifier.ConclusionsArtificial neural networks were used to predict promoters based on SIDD profile data. Results using this technique were an improvement over the previous SIDD threshold approach. Over a wide range of precision-recall values, artificial neural networks were more capable of identifying distinctive characteristics of promoter regions than threshold based methods.

Minicircle Performance Depending on S/MAR–Nuclear Matrix Interactions

The ideal vector for cell and tissue modification does not depend on integration but rather behaves as an independent functional unit that replicates as an episome. Based on a scaffold/matrix attachment region (S/MAR), we have introduced, in 2006, an ∼ 4-kb replicating nonviral minicircle able to exploit the cellular replication machinery in a way reminiscent of ARS vectors. Consisting of only one active transcription unit and the S/MAR, it resists silencing as it is free of prokaryotic vector parts and drug selection markers. The rate of final establishment in the nuclear architecture is moderate but comparable to Epstein–Barr virus-based episomes (< 5%). Here, we demonstrate that this parameter can be improved if the host cell chromatin is opened by histone hyperacetylation prior to transfection. It remains unaffected, however, by cell cycle position. Still, this class of episomes revealed intrinsic instability and integration after 5 months of continuous culture. In vivo evolution enabled the effective reduction of S/MAR size from 2 kb to 733 bp (resulting in a minicircle of ∼ 3 kb) with largely improved stability and cloning capacity. Investigation of individual clones served to prove persistent and homogenous expression, which is ascribed to stable association with nuclear attachment sites. Optimum expression levels were shown to depend on the authentic usage of a polyadenylation site 3′ from the S/MAR as anticipated by the stress-induced duplex destabilization algorithm, which finds increasing use to predict the functional parameters of these systems.

Regulation of rat haptoglobin gene expression is coordinated by the nuclear matrix

Using computer stress-induced duplex destabilization (SIDD) analysis and binding experiments, we identified a S/MAR element (-599/-200 bp) (Hp-S/MAR) adjacent to the cis-element (-165/-56 bp) in the rat haptoglobin gene. We examined its functional interactions with the lamins and lamin-associated proteins in the basal state and during acute-phase (AP) response-induced increased transcription. Colocalization, electrophoretic mobility shift assay (EMSA), and re-electrophoresis of nucleoprotein complexes, South-Western and Western blot analysis and coimmunoprecipitation experiments revealed that the lamins, PARP-1, C/EBP beta, and Hp-S/MAR assembled higher order complexes through direct lamin-Hp-S/MAR and probably PARP-1-Hp-S/MAR interactions although C/EBP beta did not bind to the Hp-S/MAR but established direct interaction with PARP-1. The transition from constitutive to increased haptoglobin gene transcription during the AP response was associated with quantitative and qualitative changes in Hp-S/MAR-protein interactions, respectively, observed as increased association of the lamin(s) with the Hp-S/MAR and as the appearance of a 90 kDa Hp-S/MAR-binding protein. Also, during the AP response the contact between C/EBP beta and PARP-1 established in the basal state was lost. DNA chromatography with the haptoglobin cis-element and Western blot analysis suggests that PARP-1 was a coactivator during constitutive and elevated transcription. The results show that the lamin components of the nuclear matrix form a network of functional, dynamic protein-protein and protein-Hp-S/MAR associations with multiple partners, and underline the involvement of PARP-1 in the regulation of haptoglobin gene transcription. We concluded that the interplay of these interactions fine tunes haptoglobin gene expression to meet the changing requirements of liver cells.

Structural and bioinformatic analysis of the Roman snail Cd‐Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress

Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains--apart from the regulatory promoter region--several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions.

PARP-1 Expression in the Mouse is Controlled by an Autoregulatory Loop: PARP-1 Binding to an Upstream S/MAR Element and to a Novel Recognition Motif in its Promoter Suppresses Transcription

This work identifies central components of a feedback mechanism for the expression of mouse poly(ADP-ribose) polymerase-1 (PARP-1). Using the stress-induced duplex destabilization algorithm, multiple base-unpairing regions (BURs) could be localized in the 5′ region of the mouse PARP-1 gene ( muPARP-1). Some of these could be identified as scaffold/matrix-attachment regions (S/MARs), suggesting an S/MAR-mediated transcriptional regulation. PARP-1 binding to the most proximal element, S/MAR 1, and to three consensus motifs, AGGCC, in its own promoter (basepairs –956 to +100), could be traced by electrophoretic mobility-shift assay. The AGGCC-complementary GGCCT motif was detected by cis-diammine-dichloro platinum cross-linking and functionally characterized by the effects of site-directed mutagenesis on its performance in wild type (PARP-1 +/+) and PARP-1 knockout cells (PARP-1 -/-). Mutation of the central AGGCC tract at basepairs –554 to –550 prevented PARP-1/promoter interactions, whereby muPARP-1 expression became up-regulated. Transfection of a series of reporter gene constructs with or without S/MAR 1 (basepairs –1523 to –1007) and the more distant S/MAR 2 (basepairs –8373 to –6880), into PARP-1 +/+ as well as PARP-1 -/- cells, revealed an additional, major level of muPARP-1 promoter down-regulation, triggered by PARP-1 binding to S/MAR 1. We conclude that S/MAR 1 represents an upstream control element that acts in conjunction with the muPARP-1 promoter. These interactions are part of a negative autoregulatory loop.

The Functional Role of S/MARs in Episomal Vectors as Defined by the Stress-Induced Destabilization Profile of the Vector Sequences

The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human β-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells. In Jurkat and other cells, DNA plasmid vectors containing Epstein–Barr virus origin of replication (EBV OriP) and the EBV nuclear antigen-1 gene mediate prolonged episome retention in the host cell nucleus, which, however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative initiation of replication region from the β-globin gene locus, instead of EBV OriP and the EBV nuclear antigen-1 gene, restored replicative capacity and enhanced episome retention mediated by the S/MAR. These effects were associated with a destabilization profile at the initiation of replication region. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin, in this particular setting. We consider that the calculation of stress-induced duplex destabilization may be an informative first step in the design of units that replicate extrachromosomally, particularly as the latter present a safer and, therefore, attractive alternative to integrating viral vectors for gene therapy applications.

The positive aspects of stress: strain initiates domain decondensation (SIDD)

The conventional string-based bioinformatic methods of genomic sequence analysis are often insufficient to identify DNA regulatory elements, since many of these do not have a recognizable motif. Even in case a sequence pattern is known to be associated with an element it may only partially mediate its function. This suggests that properties not correlated with the details of base sequence contribute to regulation. One of these attributes is the DNA strand-separation potential, known as SIDD (stress-induced duplex destabilization) which facilitates the access of tracking proteins and the formation of local secondary structures. Using the type 1 interferon gene cluster as a paradigm, we demonstrate that the imprints in a SIDD profile coincide with chromatin domain borders and with DNAse I hypersensitive sites to which regulatory potential could be assigned. The approach permits the computer-guided identification of yet unknown, mostly remote sites and the design of artificial elements with predictable properties for multiple applications.

Correlations between Scaffold/Matrix Attachment Region (S/MAR) Binding Activity and DNA Duplex Destabilization Energy

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.

Dominant genomic structures: Detection and potential signal functions in the interferon-beta domain

Eukaryotic genomes are divided into chromatin domains, which are thought to represent independent regulatory units. Typically, these domains are flanked by bordering elements that insulate the transcription unit from outside influences. Borders also demarcate the range of action for enhancer-like elements within the domain as they are formed around dominant genomic structures such as DNAse I hypersensitive sites (HS). Here we describe an efficient strategy to localize these elements. Our procedure is based on a computational method and predictions are verified by classical in vivo and in vitro procedures. Exemplified by the interferon-β (IFN-β) domain it proves its potential to provide novel insights into remote control principles of transcription. Sites with secondary-structure forming potential are localized by the analysis of stress-induced duplex destabilization (SIDD) properties and the associating factors are characterized by electrophoretic mobility shift assays (EMSA). These studies reveal far upstream factor binding sites within the IFN-β domains of both humans and mice. A prominent example is YY1, a transcription factor that not only recognizes a core consensus motif, ATGG, but, in addition, the structural context, which is evident from characteristic imprints in the respective SIDD-profiles.