Distinct DNA-binding syntax and chromatin remodeling capacities of bHLH transcription factors in cell differentiation, reprogramming and cancer

Basic helix–loop–helix (bHLH) transcription factors orchestrate cell differentiation, reprogramming, and oncogenic transformation, yet the molecular determinants that govern their DNA-binding specificity and capacity to remodel chromatin remain incompletely understood. Here, we assemble and unify all available bHLH induction ChIP-seq datasets (74 experiments covering 17 factors) and integrate them with matched chromatin accessibility, nucleosome positioning, CpG methylation, transcriptomic profiling, methyl-HT-SELEX, and structural modeling. Using an exact hexanucleotide–based approach, we define the sequence grammar that shapes bHLH–DNA interactions and identify distinct motif architectures associated with binding to accessible versus inaccessible chromatin. CAT- and CAG-preferring bHLH factors—including proneural and myogenic regulators—display robust pioneer-like behavior, characterized by preferential engagement of closed chromatin through specific E-box variants, cooperative motif clustering, and characteristic spacing patterns. TWIST factors exhibit a unique 5-bp E-box periodicity linked to dedifferentiation programs, whereas CAC-preferring oncogenic bHLHs remain largely restricted to open chromatin. By integrating methylation and SELEX data, we reveal that CpG methylation drives a systematic shift from the canonical CAC–CAC motif toward CAT–CAC E-boxes, providing a mechanistic explanation for MYC enhancer invasion in cancer and uncovering parallel behavior in the HEY family. Nucleosome chemical mapping and structural predictions further show that nearly all bHLH factors bind preferentially at nucleosome flanks, independent of α-helix length, challenging prior models of nucleosomal engagement. Together, these results establish a unified framework linking sequence syntax, chromatin state, and transcriptional outcome across the bHLH family, providing mechanistic principles for understanding lineage specification, reprogramming, and oncogenic enhancer remodeling.