Nucleosomes cover most of the genome and are thought to be displaced by transcription factors (TFs) in regions that direct gene expression. However, the modes of interaction between TFs and nucleosomal DNA remain largely unknown. Here, we have systematically explored interactions between the nucleosome and 220 TFs representing diverse structural families. Consistently with earlier observations, we find that the majority of the studied TFs have less access to nucleosomal DNA than to free DNA. The motifs recovered from TFs bound to nucleosomal and free DNA are generally similar; however, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many TFs preferentially bind close to the end of nucleosomal DNA, or to periodic positions at its solvent-exposed side. TFs often also bind to nucleosomal DNA in a particular orientation. Some TFs specifically interact with DNA located at the dyad position where only one DNA gyre is wound, whereas other TFs prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals striking differences in TF binding to free and nucleosomal DNA, and uncovers a rich interaction landscape between TFs and the nucleosome.

Pioneer transcription factors are required for stem cell pluripotency, cell differentiation, and cell reprogramming 1,2 . Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. Sox2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells 3 . Here we report cryo-electron microscopy structures of the DNA-binding domains of Sox2 and its close homologue Sox11 bound to nucleosomes. These first structures of pioneer factors in complex with nucleosomes show that Sox factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, and this increases DNA accessibility. Sox factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4, including residue lysine-16. This is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. These results indicate that pioneer transcription factors can use binding energy to contribute to initial chromatin opening and facilitate nucleosome remodelling and transcription.Transcription of the human genome is controlled by ~1,600 transcription factors (TFs) 4 . TFs recognize DNA motifs and recruit protein complexes that enable transcription initiation 5 . Binding of most TFs is restricted to regions of the genome that are not packaged into chromatin 6 . Some TFs can however bind to chromatin via contacts to its fundamental unit, the nucleosome 7 . These 'pioneer' TFs can initiate transcription in silent chromatin regions 8 and are required for embryo development, cell differentiation, and cell reprogramming 9,10 . Sox2 and Oct4 are pioneer factors that are widely used for reprogramming of adult cells to induced pluripotent stem cells 2,11,12 . They can interact with nucleosomes in vitro and in vivo 13,14 . Sox2 alone can direct chromatin opening 15 and bind target DNA sites before Oct4 11 in vivo, indicating that Sox2 makes DNA accessible for binding of other factors. Most Sox family factors show pioneer factor function 7 , are essential for developmental processes 16 , and their mutation can lead to severe developmental defects and cancer 17 . How pioneer TFs such as Sox factors bind to the nucleosome and how they make DNA accessible is unknown. Author contributions S.O.D. designed and carried out all experiments and data analysis. F.Z. supported by J.T. identified the original DNA template used in the study. C.D. assisted with cryo-EM data collection. P.C. designed and supervised research. S.O.D. and P.C. interpreted the data and wrote the manuscript, with input from all authors.

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF–TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.

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16Nucleosomes cover most of the genome and are thought to be displaced by