ChromSMF: integrated profiling of histone modifications, protein-DNA interactions and DNA methylation on multi-kilobase DNA molecules

Chromatin accessibility and histone post-translational modifications are widely used to identify and characterize cis -regulatory elements. Yet, these are typically measured separately, precluding direct linkage between them. Here, we present Chromatin-informed Single Molecule Footprinting (ChromSMF), a method that simultaneously quantifies histone modifications, transcription factor binding, and nucleosome occupancy, while measuring DNA methylation and sequence variations on the same multi-kilobase DNA molecules. ChromSMF combines antibody-guided tethering of the adenine methyltransferase Hia5 to modified histones, protein-DNA footprinting using the cytosine methyltransferase M.CviPI, and direct methylation detection by Nanopore sequencing. We benchmark ChromSMF across five histone modifications, uncovering relationships between epigenetic states and chromatin opening across entire cis -regulatory landscapes. We further present a computational framework for integrated analysis of multiple layers of epigenetic regulation on individual haplotypes. Together, ChromSMF provides an integrated genome-wide genomic method to investigate the combinatorial function of multiple genetic and epigenetic factors on gene regulation across diverse cellular contexts.