CRE-subnucleosome2

The binding of transcription factors (TFs) to cis-regulatory elements (CREs) is a major step in the control of gene expression. The mammalian genome is packaged into nucleosomes, which inhibit the binding of TFs to DNA. Chromatin remodelers are recruited at CREs to open the chromatin and facilitate the binding of TFs, but how this occurs is not well understood. Partners 1, 2 and 5 recently identified a new class of subnucleosomal particles associated with 50-80 bp of DNA, located at discrete positions at promoters, enhancers, and CTCF binding sites. We demonstrated that BRG1, the catalytic subunit of the SWI/SNF chromatin remodelers, is required to generate these particles at enhancers in mouse embryonic stem (ES) cells. We further showed that these 50-80 bp subnucleosomes interact in vivo with OCT4, a master TF controlling the ES cell phenotype. This interaction occurs independently of the presence of OCT4 binding motifs within the particle, suggesting that OCT4 binds a new histone domain only accessible on the subnucleosome. The 50-80 bp subnucleosomes are thus genuine components of open chromatin. They allow the spreading of OCT4 within a genomic interval up to ten times larger than the region bound by OCT4 onto histone-free DNA, at the level of its consensus binding motif. These results identify a new mechanism used by OCT4, and potentially other TFs, to reinforce their interaction with CREs. In the CRE-subnucleosome2 project, we will analyse the nature and function of these subnucleosomal particles and define the structural basis of the OCT4-subnucleosome interaction. We will purify the subnucleosomal particles present at each category of CRE and characterise them using cryo-electron microscopy, proteomics, in vitro binding studies and computational modelling. We will identify all TFs interacting with these particles and the chromatin remodelers that generate the subnucleosomes at each category of CREs.