Multiple Transcription Factor Binding Sites Research Articles

Nucleosomal DNA Dynamics Mediate Oct4 Pioneer Factor Binding

Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus, the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence-specific manner. Here, we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF that is essential for understanding how chromatin is unraveled during cell fate conversions.

Information Integration and Energy Expenditure in Gene Regulation

The quantitative concepts used to reason about gene regulation largely derive from bacterial studies. We show that this bacterial paradigm cannot explain the sharp expression of a canonical developmental gene in response to a regulating transcription factor (TF). In the absence of energy expenditure, with regulatory DNA at thermodynamic equilibrium, information integration across multiple TF binding sites can generate the required sharpness, but with strong constraints on the resultant "higher-order cooperativities." Even with such integration, there is a "Hopfield barrier" to sharpness; for n TF binding sites, this barrier is represented by the Hill function with the Hill coefficient n. If, however, energy is expended to maintain regulatory DNA away from thermodynamic equilibrium, as in kinetic proofreading, this barrier can be breached and greater sharpness achieved. Our approach is grounded in fundamental physics, leads to testable experimental predictions, and suggests how a quantitative paradigm for eukaryotic gene regulation can be formulated.

An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example

BackgroundPost-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification.ResultsTraditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline.ConclusionA methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.

Functional anatomy of distant-acting mammalian enhancers

Transcriptional enhancers are a major class of functional element embedded in the vast non-coding portion of the human genome. Acting over large genomic distances, enhancers play critical roles in the tissue and cell type-specific regulation of genes, and there is mounting evidence that they contribute to the aetiology of many human diseases. Methods for genome-wide mapping of enhancer regions are now available, but the functional architecture contained within human enhancer elements remains unclear. Here, we review recent approaches aimed at understanding the functional anatomy of individual enhancer elements, using systematic qualitative and quantitative assessments of mammalian enhancer variants in cultured cells and in vivo. These studies provide direct insight into common architectural characteristics of enhancers including the presence of multiple transcription factor-binding sites and the mixture of both transcriptionally activating and repressing domains within the same enhancer. Despite such progress in understanding the functional composition of enhancers, the inherent complexities of enhancer anatomy continue to limit our ability to predict the impact of sequence changes on in vivo enhancer function. While providing an initial glimpse into the mutability of mammalian enhancers, these observations highlight the continued need for experimental enhancer assessment as genome sequencing becomes routine in the clinic.

Multiple Transcription Factor Binding Sites Predict AID Targeting in Non-Ig Genes

Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ≈ 25% of expressed genes in germinal center B cells. Observations in Ung(-/-) Msh2(-/-) mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung(-/-) Msh2(-/-) mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-β binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Noise Can Induce Steady-State Bimodality in Positive Feedback Loops Without Cooperativity

Positive feedback is a common network motif in gene regulatory networks that is widely recognized to lead to bistability and, as a consequence, to hysteresis and switch-like responses. A non-linear, cooperative promoter response provides the necessary ingredient to generate bistability in deterministic descriptions of positive feedback. Using a synthetic system, we show experimentally that positive feedback is capable of inducing a bimodal, switch-like response with non-cooperative feedback, even when the underlying deterministic dynamics do not admit bistability. In accordance with theoretical models, the bimodal response requires the promoter within the feedback loop to be noisy, with infrequent, large bursts of expression. In addition, the transcription factor (TF) involved in the feedback loop has to be short-lived. Using a stochastic model and experimentally measured in vivo parameters of the promoter response in the absence of feedback, we can quantitatively describe the feedback response. We also find that multiple TF binding sites in a promoter can be important for the bimodal response not because of molecular cooperativity in TF binding, but because of increased noise in the promoter. Because many promoters possess multiple binding sites and many TFs are unstable, positive feedback loops in many gene regulatory networks may exhibit bimodal responses, but not necessarily because of deterministic bistability as is commonly thought.

A comparative analysis of relative occurrence of transcription factor binding sites in vertebrate genomes and gene promoter areas

The detection of transcription factor binding sites (TFBS) in genomic sequences is a basic task for elucidating the transcriptional aspects of gene regulation. Evaluation procedures applicable to the TFBS prediction outputs need improvement. Predicted TFBS located outside of the transcription associated areas are often neglected from the functional and the evolutionary points of view, therefore deserving a systematic overview. We calculated theoretical occurrences of 184 TFBS according to their position weight matrices and the dinucleotide statistics of the completed vertebrate genomes, then performed a TFBS prediction in the corresponding complete genomic sequences and their repeat-free, repetitive and regulatory fractions. Repeat-free fractions of the closely related mammalian genomes were characterized by strong similarities in TFBS occurrences. A significant over-representation of multiple TFBS was found in both repetitive and non-repetitive genome fractions. F-values and real TFBS occurrences calculated for human, chimp, mouse, rat, zebrafish and fugu genomes are available for free download at http://www.gmu.edu/departments/mmb/baranova/pages/bioinformatics

Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication

The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses.

Regulation of Forestomach-specific Expression of the Murine Adenosine Deaminase Gene

The maturation of stratified squamous epithelium of the upper gastrointestinal tract is a highly ordered process of development and differentiation. Information on the molecular basis of this process is, however, limited. Here we report the identification of the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult mouse, Ada is highly expressed in the terminally differentiated epithelial layer of upper gastrointestinal tract tissues. The data reported here represent the identification and detailed analysis of a 1. 1-kilobase (kb) sequence located 3.4-kb upstream of the transcription initiation site of the murine Ada gene, which is sufficient to target cat reporter gene expression to the forestomach in transgenic mice. This 1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level comparable to the endogenous Ada gene. This expression is localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites.

Stimulation of 92-kDa Gelatinase B Promoter Activity by ras Is Mitogen-activated Protein Kinase Kinase 1-independent and Requires Multiple Transcription Factor Binding Sites Including Closely Spaced PEA3/ets and AP-1 Sequences

The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha-ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5' flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5' deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning -634 to -531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (-540) motif, AP-1 sites (-533, -79), a NF-kappa B (-600) consensus sequence, and a GT box (-52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activate protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/est and AP-1 sites and is MEK1-independent.