de Martin, X. | Search bioRxiv

bioRxiv2026

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 -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.

molecular biology↗

bioRxiv2023

Recruitment of Homodimeric Proneural Factors by Conserved CAT-CAT E-Boxes Drives Major Epigenetic Reconfiguration in Cortical Neurogenesis

The proneural factors of the basic-helix-loop-helix (bHLH) family of transcription factors coordinate early processes of neurogenesis and neurodifferentiation. Among them, Neurog2 and Neurod2 subsequently act specifying neurons of the glutamatergic lineage. The disruption of proneural factors, their target genes, and the DNA motifs they bind, have been linked to various neuropsychiatric disorders. Proneural factors operate on the DNA forming homodimers or heterodimers with other bHLH factors and binding to specific motifs called E-boxes, which are hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands. These E-box motifs are highly enriched in regulatory elements that become active during corticogenesis. Although neurogenesis and neurodifferentiation appear to rely heavily on the activity of E-boxes, our understanding of the specific dynamics of DNA binding and partner usage throughout neurogenesis and neurodifferentiation remains largely unknown. To shed light on this critical facet of neural development, we conducted a comprehensive analysis leveraging ChIP-seq data of NEUROG2 and NEUROD2, paired with time-matched single-cell RNA-seq and ATAC-seq assays and DNA methylation data, collected from the developing mouse brain. Our analyses revealed that distinct trajectories of chromatin accessibility are selectively linked to specific subsets of NEUROG2 and NEUROD2 binding sites and E-boxes. Notably, while E-boxes composed of CAT-CAG half sites or two CAG half sites are more commonly found within their binding sites, E-boxes consisting of two CAT half sites exhibit a striking enrichment in developmentally dynamic enhancers. These CAT-CAT E-boxes also manifest substantial DNA demethylation effects throughout the process of neurodifferentiation and display the highest levels of evolutionary constraint. Aided by a combination of a detailed DNA-footprinting and structural modeling approach, we propose a compelling model to explain the combinatorial action of bHLH factors across the various stages of neurogenesis. Finally, we hypothesize that NEUROD2 acts as a chromatin remodeler in cortical neurodifferentiation by binding CAT-CAT E-boxes as a homodimer, a mechanism that could be extended to other members of this bHLH class of transcription factors.

neuroscience↗