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.

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.DOI: http://dx.doi.org/10.7554/eLife.26691.001

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.