Genome-wide in vitro reconstitution to study nucleosome positioning and chromatin architecture

Access to genetic information within the cell nucleus is regulated by the distribution of nucleosomes, which are the basic unit of chromatin. Local access to specific genomic regions is facilitated by repositioning nucleosomes to enable transcription and other nuclear processes. Nucleosome positioning is primarily regulated by ATP-dependent chromatin remodeling enzymes (CRs) that belong to the Snf2-type helicase family. These enzymes disrupt histone-DNA contacts by consuming ATP. The functions of CRs can be redundant or essential, complicating their study in vivo. To address this, we employ a unique bottom-up approach, in which we reconstitute chromatin in vitro using purified proteins and a yeast genomic plasmid library. To elucidate the diverse remodeling functions of CRs, we add purified CRs in combination with various transcription factors to the in vitro reconstituted chromatin. The resulting changes in nucleosome positioning are monitored using MNase-seq. Depending on the type of CR used, we observe distinct nucleosome positioning patterns. Furthermore, we have expanded our in vitro reconstitution approach to explore the 3D genome organization of reconstituted chromatin, discovering a role for CRs in the 3D genome organization of S. cerevisiae.