Histone modifications regulate pioneer transcription factor cooperativity.

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

Flexible, symmetry-directed approach to assembling protein cages.

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.