Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

<p>Abstract</p> <p>Background</p> <p>The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes.</p> <p>Results</p> <p>Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors.</p> <p>Conclusions</p> <p>This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.</p

Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation

10.1371/journal.pgen.1000106PLoS Genetics46

Hypervariable intronic region in NCX1 is enriched in short insertion-deletion polymorphisms and showed association with cardiovascular traits

<p>Abstract</p> <p>Background</p> <p>Conserved non-coding regions (CNR) have been shown to harbor gene expression regulatory elements. Genetic variations in these regions may potentially contribute to complex disease susceptibility.</p> <p>Methods</p> <p>We targeted CNRs of cardiovascular disease (CVD) candidate gene, <it>Na(+)-Ca(2+) exchanger (NCX1) </it>with polymorphism screening among CVD patients (n = 46) using DHPLC technology. The flanking region (348 bp) of the 14 bp indel in intron 2 was further genotyped by DGGE assay in two Eastern-European CVD samples: essential hypertension (HYPEST; 470 cases, 652 controls) and coronary artery disease, CAD (CADCZ; 257 cases, controls 413). Genotype-phenotype associations were tested by regression analysis implemented in PLINK. Alignments of primate sequences were performed by ClustalW2.</p> <p>Results</p> <p>Nine of the identified <it>NCX1 </it>variants were either singletons or targeted by commercial platforms. The 14 bp intronic indel (rs11274804) was represented with substantial frequency in HYPEST (6.82%) and CADCZ (14.58%). Genotyping in Eastern-Europeans (n = 1792) revealed hypervariable nature of this locus, represented by seven alternative alleles. The alignments of human-chimpanzee-macaque sequences showed that the major human variant (allele frequency 90.45%) was actually a human-specific deletion compared to other primates. In humans, this deletion was surrounded by other short (5-43 bp) deletion variants and a duplication (40 bp) polymorphism possessing overlapping breakpoints. This indicates a potential indel hotspot, triggered by the initial deletion in human lineage. An association was detected between the carrier status of 14 bp indel ancestral allele and CAD (<it>P </it>= 0.0016, OR = 2.02; Bonferroni significance level alpha = 0.0045), but not with hypertension. The risk for the CAD development was even higher among the patients additionally diagnosed with metabolic syndrome (<it>P </it>= 0.0014, OR = 2.34). Consistent with the effect on metabolic processes, suggestive evidence for the association with heart rate, serum triglyceride and LDL levels was detected (<it>P </it>= 0.04).</p> <p>Conclusions</p> <p>Compared to SNPs targeted by large number of locus-specific and genome-wide assays, considerably less attention has been paid to short indel variants in the human genome. The data of genome dynamics, mutation rate and population genetics of short indels, as well as their impact on gene expressional profile and human disease susceptibility is limited. The characterization of <it>NCX1 </it>intronic hypervariable non-coding region enriched in human-specific indel variants contributes to this gap of knowledge.</p

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks

A Combination of Genomic Approaches Reveals the Role of FOXO1a in Regulating an Oxidative Stress Response Pathway

Background: While many of the phenotypic differences between human and chimpanzee may result from changes in gene regulation, only a handful of functionally important regulatory differences are currently known. As a first step towards identifying transcriptional pathways that have been remodeled in the human lineage, we focused on a transcription factor, FOXO1a, which we had previously found to be up-regulated in the human liver compared to that of three other primate species. We concentrated on this gene because of its known role in the regulation of metabolism and in longevity. Methodology: Using a combination of expression profiling following siRNA knockdown and chromatin immunoprecipitation in a human liver cell line, we identified eight novel direct transcriptional targets of FOXO1a. This set includes the gene for thioredoxin-interacting protein (TXNIP), the expression of which is directly repressed by FOXO1a. The thioredoxininteracting protein is known to inhibit the reducing activity of thioredoxin (TRX), thereby hindering the cellular response to oxidative stress and affecting life span. Conclusions: Our results provide an explanation for the repeated observations that differences in the regulation of FOXO transcription factors affect longevity. Moreover, we found that TXNIP is down-regulated in human compared to chimpanzee, consistent with the up-regulation of its direct repressor FOXO1a in humans, and with differences in longevity between th

Thermodynamics-Based Models of Transcriptional Regulation by Enhancers: The Roles of Synergistic Activation, Cooperative Binding and Short-Range Repression

Quantitative models of cis-regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled, or heuristic approximations of the underlying regulatory mechanisms. We have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence, as a function of transcription factor concentrations and their DNA-binding specificities. It uses statistical thermodynamics theory to model not only protein-DNA interaction, but also the effect of DNA-bound activators and repressors on gene expression. In addition, the model incorporates mechanistic features such as synergistic effect of multiple activators, short range repression, and cooperativity in transcription factor-DNA binding, allowing us to systematically evaluate the significance of these features in the context of available expression data. Using this model on segmentation-related enhancers in Drosophila, we find that transcriptional synergy due to simultaneous action of multiple activators helps explain the data beyond what can be explained by cooperative DNA-binding alone. We find clear support for the phenomenon of short-range repression, where repressors do not directly interact with the basal transcriptional machinery. We also find that the binding sites contributing to an enhancer's function may not be conserved during evolution, and a noticeable fraction of these undergo lineage-specific changes. Our implementation of the model, called GEMSTAT, is the first publicly available program for simultaneously modeling the regulatory activities of a given set of sequences

Widespread Site-Dependent Buffering of Human Regulatory Polymorphism

The average individual is expected to harbor thousands of variants within non-coding genomic regions involved in gene regulation. However, it is currently not possible to interpret reliably the functional consequences of genetic variation within any given transcription factor recognition sequence. To address this, we comprehensively analyzed heritable genome-wide binding patterns of a major sequence-specific regulator (CTCF) in relation to genetic variability in binding site sequences across a multi-generational pedigree. We localized and quantified CTCF occupancy by ChIP-seq in 12 related and unrelated individuals spanning three generations, followed by comprehensive targeted resequencing of the entire CTCF–binding landscape across all individuals. We identified hundreds of variants with reproducible quantitative effects on CTCF occupancy (both positive and negative). While these effects paralleled protein–DNA recognition energetics when averaged, they were extensively buffered by striking local context dependencies. In the significant majority of cases buffering was complete, resulting in silent variants spanning every position within the DNA recognition interface irrespective of level of binding energy or evolutionary constraint. The prevalence of complex partial or complete buffering effects severely constrained the ability to predict reliably the impact of variation within any given binding site instance. Surprisingly, 40% of variants that increased CTCF occupancy occurred at positions of human–chimp divergence, challenging the expectation that the vast majority of functional regulatory variants should be deleterious. Our results suggest that, even in the presence of “perfect” genetic information afforded by resequencing and parallel studies in multiple related individuals, genomic site-specific prediction of the consequences of individual variation in regulatory DNA will require systematic coupling with empirical functional genomic measurements

Deciphering Normal Blood Gene Expression Variation—The NOWAC Postgenome Study

There is growing evidence that gene expression profiling of peripheral blood cells is a valuable tool for assessing gene signatures related to exposure, drug-response, or disease. However, the true promise of this approach can not be estimated until the scientific community has robust baseline data describing variation in gene expression patterns in normal individuals. Using a large representative sample set of postmenopausal women (N = 286) in the Norwegian Women and Cancer (NOWAC) postgenome study, we investigated variability of whole blood gene expression in the general population. In particular, we examined changes in blood gene expression caused by technical variability, normal inter-individual differences, and exposure variables at proportions and levels relevant to real-life situations. We observe that the overall changes in gene expression are subtle, implying the need for careful analytic approaches of the data. In particular, technical variability may not be ignored and subsequent adjustments must be considered in any analysis. Many new candidate genes were identified that are differentially expressed according to inter-individual (i.e. fasting, BMI) and exposure (i.e. smoking) factors, thus establishing that these effects are mirrored in blood. By focusing on the biological implications instead of directly comparing gene lists from several related studies in the literature, our analytic approach was able to identify significant similarities and effects consistent across these reports. This establishes the feasibility of blood gene expression profiling, if they are predicated upon careful experimental design and analysis in order to minimize confounding signals, artifacts of sample preparation and processing, and inter-individual differences

The Genetic Signatures of Noncoding RNAs

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses