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

Abstract

BackgroundGenetic 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.ResultsWe 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.ConclusionsNF-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.

Inhibition of Protein Kinase C Signaling Maintains Rat Embryonic Stem Cell Pluripotency*

“…The promoter region of human miR-21 is located ϳ(-)3.5 kb upstream of the pre-miR-21 sequence (32). We found that the corresponding region in the rat miR-21 locus is located at the ϳ(-)2.6 kb region and contains a conserved putative NF-B binding motif (GGR(A/G)R(A/G)NNY(C/ T)Y(C/T)C) (33) (Fig. 4C, upper panel).…”

High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

“…Compared to those methods Spec-seq has one significant disadvantage, but several valuable advantages. The disadvantage is that the number of sequences that can be assayed in a single experiment is in the thousands (but see alternative below), whereas PBM and related methods (Berger et al 2006;Warren et al 2006;Nutiu et al 2011), SELEX-seq (Zhao et al 2009;Zykovich et al 2009;Jolma et al 2010Jolma et al , 2013Wong et al 2011), andB1H (Meng et al 2005;Christensen et al 2011;Gupta et al 2014) can assay millions, or more, of sequences simultaneously. But those methods do not measure binding affinity directly, but rather something related to it such as binding site counts in SELEX-seq and B1H or fluorescence intensity in PBM.…”

“…Measurements of affinity changes due to operator sequence variation, by base replacement, by the use of base analogs, or by changing the length of the operator, have been performed almost since the operator sequence was first determined (Goeddel et al 1978;Sadler et al 1983;Betz et al 1986;Sartorius et al 1989;Lehming et al 1990;Sasmor and Betz 1990;Frank et al 1997;Spronk et al 1999;Falcon and Matthews 2001;Kalodimos et al 2002Kalodimos et al , 2004bDaber and Lewis 2009). But those analyses all measured binding affinity to only a few sequences.The lac repressor has not, to our knowledge, been analyzed by current high-throughput methods that can determine specificity over thousands, or even millions, of sequences in parallel (Stormo and Zhao 2010), such as protein-binding microarrays (PBM) (Berger et al 2006;Gordan et al 2013), SELEX-seq [or HT-SELEX (Zhao et al 2009;Zykovich et al 2009;Jolma et al 2010;Wong et al 2011)], bacterial one-hybrid (B1H) (Meng et al 2005;Noyes et al 2008;Christensen et al 2011), and mechanically induced trapping of molecular interactions (MITOMI) (Maerkl and Quake 2007). While those methods offer an expansive overview of binding specificity, the accuracies of the resulting models are highly variable.…”

“…The lac repressor has not, to our knowledge, been analyzed by current high-throughput methods that can determine specificity over thousands, or even millions, of sequences in parallel (Stormo and Zhao 2010), such as protein-binding microarrays (PBM) (Berger et al 2006;Gordan et al 2013), SELEX-seq [or HT-SELEX (Zhao et al 2009;Zykovich et al 2009;Jolma et al 2010;Wong et al 2011)], bacterial one-hybrid (B1H) (Meng et al 2005;Noyes et al 2008;Christensen et al 2011), and mechanically induced trapping of molecular interactions (MITOMI) (Maerkl and Quake 2007). While those methods offer an expansive overview of binding specificity, the accuracies of the resulting models are highly variable.…”

Unraveling determinants of transcription factor binding outside the core binding site

“…Several recent studies that aim to address this gap employed in vitro-based methods (e.g., DIP-seq [Liu et al 2006], PB-seq [Guertin et al 2012], gcPBM Wong et al 2011;Gordan et al 2013], EMSA-seq [Wong et al 2011], SELEXseq [Slattery et al 2011;Jolma et al 2013], MITOMI [Maerkl and Quake 2007;Fordyce et al 2010], HiTS-FLIP [Nutiu et al 2011]) and identified various mechanisms that affect TF binding, including chromatin accessibility (Liu et al 2006;Guertin et al 2012), cofactors that influence binding specificity Slattery et al 2011), TF dimer interactions (Wong et al 2011;Jolma et al 2013), and the effect of sequences flanking the core TF binding site (TFBS) (Maerkl and Quake 2007;Nutiu et al 2011;Gordan et al 2013;Jolma et al 2013;Rajkumar et al 2013) that can be mediated through DNA shape.…”