Extensive characterization of NF-κB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits

Background Genetic studies have provided ample evidence of the influence of non-coding DNA polymorphisms on trait variance, particularly those occurring within transcription factor binding sites. Protein binding microarrays and other platforms that can map these sites with great precision have enhanced our understanding of how a single nucleotide polymorphism can alter binding potential within an in vitro setting, allowing for greater predictive capability of its effect on a transcription factor binding site. Results We have used protein binding microarrays and electrophoretic mobility shift assay-sequencing (EMSA-Seq), a deep sequencing based method we developed to analyze nine distinct human NF-κB dimers. This family of transcription factors is one of the most extensively studied, but our understanding of its DNA binding preferences has been limited to the originally described consensus motif, GGRRNNYYCC. We highlight differences between NF-κB family members and also put under the spotlight non-canonical motifs that have so far received little attention. We utilize our data to interpret the binding of transcription factors between individuals across 1,405 genomic regions laden with single nucleotide polymorphisms. We also associated binding correlations made using our data with risk alleles of disease and demonstrate its utility as a tool for functional studies of single nucleotide polymorphisms in regulatory regions. Conclusions NF-κB dimers bind specifically to non-canonical motifs and these can be found within genomic regions in which a canonical motif is not evident. Binding affinity data generated with these different motifs can be used in conjunction with data from chromatin immunoprecipitation-sequencing (ChIP-Seq) to enable allele-specific analyses of expression and transcription factor-DNA interactions on a genome-wide scale.Wellcome Trust (London, England) (grant 075491/Z/04)European Commission (Seventh Framework Programme FP7/2007-2013: Model-In (222008))European Commission (Seventh Framework Programme FP7 ITN Network INTEGER (214902))Medical Research Council (Canada) (MRC project grant G0700818

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance

Identification of p-Smad2/3 inducible miRNAs.

<p>(<b>A</b>) Western blot analysis of p-Smad2 and PCNA (loading control) levels in TAG1 ES cells untreated (0), treated with SB-431542 (SB) for 16 hrs (16) or with SB for 16 hrs followed by doxycycline (Dox) for 12 hrs (16+12). (<b>B</b>) Relative fold induction of mature miRNAs in TAG1 cells treated as in (<b>A</b>) detected by small RNA deep-sequencing. (<b>C</b>) qPCR validation of the deep-sequencing data, normalized to the non-Smad2/3 responsive miR-26a. Error bars represent ±SEM of triplicate reactions.</p

Expression of p-Smad2/3 inducible miRNAs in the early embryo.

<p>(<b>A</b>) qPCR was performed on RNA from pooled mouse E6.5 embryos to detect the relative expression levels of the p-Smad2/3 inducible miRNAs. Error bars show ±SEM of triplicate assays. ‘^†’ denotes the direct p-Smad2/3 target miRNAs. (<b>B–F</b>) <i>In situ</i> hybridizations were performed on E6.5 embryos using Locked Nucleic Acid modified oligonucleotides against miRs-382-5p (<b>B, E</b>), -181d-5p (<b>C, F</b>) and -499-5p (negative control) (<b>D</b>). Images are representative of 32 embryos for -382-5p, 29 for -181d-5p, and 22 for -499-5p as indicated in the images (n). Dotted lines in (<b>B, C</b>) indicate positions of sections presented in (<b>E, F</b>), respectively. Scale bars for whole mounts indicate 100 µm (<b>B, C, D</b>), and for sections 50 µm (<b>E, F</b>). Extra-embryonic (Exe), epiblast (Ep) and endoderm (En).</p

Pri-miRNA genes encoding the p-Smad2/3-inducible miRNAs.

†<p>Directly regulated p-Smad2/3-responsive miRNAs.</p

Predicted gene structure of pri-miRs-181c/d and -341∼3072 and validation of FoxH1 binding sites.

<p>(<b>A, B</b>) Schematic representations of the predicted gene structures of pri-miR-181c/d (<b>A</b>) and pri-miR-341∼3072 (<b>B</b>). The putative FoxH1 binding sites (Asymmetric Enhancer Elements, ASE) are indicated in purple. Sequence and species conservation of the ASE, in purple boxes, are shown below the respective gene maps. Positions of miRNAs are indicated with blue lines and in yellow for miR-382 (<b>B</b>). ‘AAAA’ in red denotes the predicted poly(A) tails. A red box highlights sequence fragments used for each pri-miRNA expression luciferase construct. TSS – transcriptional start site. (<b>C</b>) Candidate putative miR-ASE sites were tested by luciferase assay in ES cells treated with SB-431542 (SB) for 12 hrs followed by Activin for 5 hrs or with a further SB treatment of 5 hrs (SB 17 hrs). The nucleotides mutated to generate the miR-ASE(B)MUT construct are presented in the panel to the right of the graph. Student’s t-test was used for the statistical analysis (**p<0.01). Assays were performed in 3 biological replicates measured in quadruplicate (n = 3). Error bars show ±SEM.</p