1Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
Correspondence: bfp2@psu.edu
PMID: 29563167
GEO ID: GSE93662
General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. Sequence recognition via direct base readout is assumed to suffice for specificity, and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-bp resolution using several ChIP-exo based assays. We find that computationally-predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the non-unique portions of GRF motifs, and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions, but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions, and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels.
ChIP-seq; Chromatin; DNA shape; GRFs; Protein-DNA interactions