DNA bendability regulates transcription factor binding to nucleosomes [PIONEAR-seq and SELEX-seq]
Cell fates are controlled by 'pioneers', sequence-specific transcription factors (TFs) that bind recognition motifs on nucleosomes ('pioneer binding'). Pioneers occupy a minority of their recognition sequences in the genome, suggesting that the sequence context regulates their binding. Here, we developed PIONEAR-seq, a high-throughput biochemical assay to characterize pioneer binding to nucleosomes. We used PIONEAR-seq to assay 11 human TFs for binding to nucleosomes based on Widom 601 versus genomic sequences. We found that pioneer binding, while mediated primarily by TFs' recognition motifs, senses the broader nucleosome sequence context and that TFs previously found to be dyad or periodic binders on nucleosomes assembled on synthetic sequences exhibit exclusively end binding to nucleosomes based on genomic sequences. We propose a model where the nucleosome exploits the local bendability of the DNA sequence to position pioneer binding, revealing another cis-regulatory layer in eukaryotes. Overall design: SELEX followed by sequencing for the following full-length human Transcription Factors: ASCL1, CEBPB, FOXA1, GATA1, GATA4, KLF4, OCT4, PAX7, PU.1, RUNX2, SOX2 and ZNF143. The DNA ligand libraries for SELEX were based on 4 DNA sequences of 147 bases (ALBN1, CX3CR1, NRCAM, W601) known to position nucleotide either in vivo or in vitro. These sequences were tiled with random 20-mers at several position, to select for preferences in motif binding and motif positioning. The libraries were either left as free DNA for the SELEX-seq assay, or assembled on nucleosome core particles (NCP) for the PIONEAR-seq assay. The TFs were tagged with GST, which was used for the pulldown of the TF and its associated DNA (eventually assembled on NCP in the case of PIONEAR-seq). After the pulldown, the DNA ligands were amplified by PCR and the selection process was repeated 3 further times. After 4 rounds of selection, the DNA ligands were prepared for Illumina sequencing. W601, also known as Widom 601, is a 147-bp DNA sequence of synthetic origin with the highest reported affinity to the histone octamer (PMID: 9514715 ). ALBN1 is a 147-bp DNA sequence based on the genomic sequence of the nucleosome N1 within the ALB enhancer (PMID: 31303471). CX3CR1 is a 147-bp sequence from the CX3 chemokine receptor 1 locus that have been proposed to be a candidate nucleosome target for pioneer factors in hematopoietic differentation (PMID: 31303471). NRCAM is a 147-bp genomic nucleosomal sequence from the Neuron-Glia-CAM- related cell adhesion molecule locus that gets digestable by Mnase upon neuronal reprogramming (PMID: 31303471)