2026-10-13T13:12:08Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33350391 2026-09-22T09:23:23Z category_24169 portal_886 item_type_3 month_year_09_2026

Supporting data for "Structural basis of KLF4-mediated nucleosome engagement and cell-fate reprogramming" Hoi Man Kevin Ng (13022160) Structural biology (incl. macromolecular modelling) KLF 4 WT nucleosome core fragment Chromatin / metabolism structural biology tools <p dir="ltr">Pioneer transcription factors (TFs) bind nucleosomal DNA, locally invade chromatin structure, and reprogram the epigenome to change cell identity. Krüppel-like factor 4 (KLF4), a member of the KLF/SP family of C2H2 zinc-finger TFs, is essential for the establishment and maintenance of pluripotency. KLF4 can recognize methylated DNA and bind nucleosomes. However, the structural mechanisms by which multi-finger C2H2 arrays engage nucleosomal binding sites, alter chromatin conformation, and contribute to cell-fate transitions remain poorly understood. Here, using a combination of rational mutagenesis, biochemical analyses, cryogenic electron microscopy, and iPSC reprogramming experiments, we decode and design KLF4's engagement with chromatin to pioneer cell fate decisions.</p><p dir="ltr">First, we dissect contributions of KLF4’s three DNA-binding zinc fingers to naked and nucleosomal DNA recognition. Structure-guided mutagenesis reveals a division of labour within the zinc-finger array. Zinc fingers 2 and 3 are required for efficient binding to both naked and nucleosomal DNA, whereas zinc finger 1 contributes predominantly to naked-DNA recognition. Disruption of the DNA-contacting interface of zinc fingers 2 and 3 impairs nucleosome engagement and abolishes pluripotency reprogramming, while mutations in zinc finger 1 preserve both activities. We further characterize a KLF4 double mutation in zinc finger 2 with altered base- and methylation-reader functions that promotes pluripotency induction in the absence of Sox2 and Oct4. These findings indicate that altered recognition of DNA bases and methylation states represents an important principle for engineering KLF4 activity and reprogramming potential.</p><p dir="ltr">Second, we determined a cryo-EM structure of KLF4 bound to a nucleosome. The structure reveals how KLF4 engages nucleosomal DNA near the entry/exit site and induces localized DNA unwrapping, exposing its complete recognition sequence. Zinc fingers 2 and 3 form a dual-wedge arrangement, establishing direct contacts with histones and displacing DNA from the H2A and H3 surfaces. By contrast, zinc finger 1 forms limited DNA contacts and does not measurably engage the histone surface. Six amino acids mediate the principal histone contacts, and structure-guided mutation of these residues disrupts both nucleosome binding and fibroblast-to-pluripotency reprogramming activity. We further show that H3K56 acetylation enhances KLF4–nucleosome binding and promotes DNA unwrapping, providing a mechanistic link between epigenetic histone modification and pioneer-factor activity.</p><p dir="ltr">Lastly, in addition to the entry-exit engagement state, we identify a second KLF4 binding mode in which the DNA-binding domain contacts the nucleosomal acidic patch. This interaction is observed with both a Widom 601 nucleosome and a native LIN28b nucleosome, suggesting that this binding mode may be independent of DNA sequence. An α-helical density positioned above the acidic patch is consistent with the involvement of one KLF4 zinc finger mediating this interaction. This binding configuration suggests an additional function for C2H2 DNA-binding domains beyond sequence recognition and raises the possibility that KLF4 may influence nucleosome–nucleosome interactions by engaging the acidic patch.</p><p dir="ltr">Together, these findings establish the structural and functional principles governing KLF4-nucleosome recognition. They demonstrate how coordinated zinc-finger interactions, histone contacts, and epigenetic modifications regulate pioneer-factor activity and cell-fate transitions, providing a framework for understanding and re-engineering epigenetic reprogramming and cell-fate control.</p> 2026-09-22T09:23:23Z Dataset Dataset 10.25442/hku.33350391.v1 https://figshare.com/articles/dataset/Supporting_data_for_Structural_basis_of_KLF4-mediated_nucleosome_engagement_and_cell-fate_reprogramming_/33350391 CC BY-NC 4.0 Open Access after 2027-08-27