We have thus far shown how OCT4 and SOX2, two pioneer TFs essential for stem cell pluripotency and capable of reprogramming differentiated cells, access joint motifs in the context of a nucleosome. We developed an assay to determine the accessibility profile for OCT4/SOX2 at base-pair resolution throughout an entire nucleosome. This revealed preferred binding at the entry/exit sites of nucleosomal DNA enabling structural analysis in order to dissect the molecular mechanism. The resulting cryo-EM structures of OCT4-SOX2 bound nucleosome of two representative motif locations are, to our knowledge, the first structures of a TF engaging a nucleosome, revealing novel principles that govern TF access throughout chromatin. The structural and functional dissection reveals that OCT4 and SOX2 together remodel the nucleosome and distort the DNA trajectory, without changing the histone octamer core.
We have extended this analysis now to a second family of transcription factors, the basic helix-loop-helix (bHLH) TF family whose members are able to bind a generic CANNTG DNA motif (known as E-boxes) occurring ~15 mio times in the human genome. Only a fraction (~1%) of E-boxes are actually occupied at given time, and the principles governing binding were unknown. Our work focused on two phylogenetically and structurally diverse members: the bHLH leucine-zipper (bHLH-LZ) TF, MYC-MAX, a key regulator of cell proliferation and oncogene, and the bHLH PAS-domain containing (bHLH-PAS) TF, CLOCK-BMAL1, an important transcriptional circuit driver of the cellular circadian clock. While CLOCK-BMAL1 has been linked to opening and occupying chromatinised sites as a pioneer factor, MYC-MAX is assumed to be dependent on other proteins to access chromatin. We find that MYC-MAX and CLOCK-BMAL1 preferentially bind E-boxes near the ends of the nucleosomal DNA, yet show different accessibility profiles depending on the dimerization domains present. Structural studies including endogenous nucleosome positioning sequences such as Lin28 demonstrate that MYC-MAX, MAX-MAX and CLOCK-BMAL1 release DNA from the histones to gain access to nucleosomes, clarifying a previously controversial readout mechanism. We observe unexpected and extensive histone interactions between the bHLH-dimerization domains, particularly for CLOCK-BMAL1. This work introduces TF/histone interactions as important and ubiquitous specifier of bHLH binding. These findings challenge the notion of unique TFs/nucleosome contact interfaces, and instead establish that different motif registers fine-tune bHLH affinity for nucleosomes through different interfaces, as well as determine the extent of competition with other proteins for nucleosome access.
