WDR5 remodels NANOG condensates to drive transcriptional programs and sustain stem cell identity

Domain: biology
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Stem cell pluripotency relies on a finely tuned interplay between transcription factors and epigenetic regulators. Here, we identify a direct interaction between NANOG, a master pluripotency transcription factor, and WDR5, a core chromatin regulator essential for maintaining stem cell identity. Mechanistically, WDR5 remodels irregular NANOG aggregates into dynamic, liquid-liquid phase-separated condensates at pluripotency-associated promoters to activate target genes. Structural analyses show that the NANOG homeodomain engages WDR5 through an extended interface distinct from previously characterized short linear motifs. The NANOG R153A mutation disrupts this interaction, leading to impaired condensate formation, reduced chromatin co-occupancy, and diminished levels of active histone marks, ultimately compromising embryonic stem cell pluripotency. Pharmacological inhibition of the WDR5-NANOG interaction suppresses leukemia stem cell expansion in vivo, highlighting its therapeutic potential. Collectively, this study reveals that WDR5-dependent regulation of NANOG condensate dynamics links phase-separated assemblies to transcriptional control of stem cell identity in physiological and pathological contexts.

Introduction
Stem cells possess the potential to maintain their pluripotent state, self-renew, and differentiate into specialized cell types1. These capabilities underpin their essential roles in embryonic development2, tissue regeneration3,4, and tumor progression5. Core transcription factors, such as NANOG, OCT4, and SOX2, play critical roles in maintaining pluripotency by orchestrating complex transcriptional programs6–8. Among them, NANOG stands out as a master regulator of pluripotency9–11. For instance, NANOG cooperates with BACH1 to recruit MLL/SET1 complexes, promoting H3K4me3 deposition and regulating enhancer-promoter activity critical for maintaining pluripotency12. Additionally, NANOG directly recruits BRG1, a catalytic subunit of the SWI/SNF chromatin remodeling complex, thereby facilitating transcription factor accessibility at critical pluripotency loci13.

NANOG comprises three domains: an intrinsically disordered N-terminal domain (NTD), a central structured DNA-binding homeodomain (HD), and a C-terminal domain (CTD) containing tryptophan repeats. While NANOG is well recognized to function as a transcription factor through its HD, emerging evidence suggests that both the NTD and CTD also play critical regulatory roles via their distinct structural features. The CTD was first reported to mediate NANOG homodimerization14 and subsequently shown to form higher-order oligomers and fibrillar assemblies that promote DNA bridging and long-range chromatin interactions15. NANOG also forms phase-separated condensates, mediated by its NTD, with coactivators such as MED1 and BRD4 at super-enhancers, a process that facilitates the robust transcriptional activation for maintaining pluripotency in embryonic stem cells (ESCs)16,17. Additionally, NANOG-TAZ condensates specifically reinforce pluripotency-associated transcriptional programs in cancer stem cells (CSCs), thereby enhancing their stem-like properties and therapy resistance18. This liquid-liquid phase-separated (LLPS)-driven regulatory mechanism underscores NANOG’s functional versatility, providing a unifying principle linking transcription factor dynamics and gene expression regulation across both physiological and pathological contexts.

Beyond transcription factors, epigenetic regulation constitutes another essential regulatory layer for maintaining pluripotency. The WD40 repeat protein WDR5 serves as a central hub that integrates chromatin modifications with transcription factor activity19. As a core component of both the SET1/MLL histone methyltransferase complexes20,21 and the MOF acetyltransferase complex22,23, WDR5 mediates the establishment of active histone marks, including H3K4 methylation and H4K16 acetylation. WDR5 interacts with multiple transcription factors such as OCT419, MYC24–26, SOX227, and TOX328 to regulate transcriptional programs essential for stem cell identity and, under pathological conditions, contributes to oncogenic self-renewal and therapeutic resistance. WDR5 depletion perturbs active histone modifications, downregulates pluripotency genes, and compromises stem cell maintenance19,29. Furthermore, pharmacological inhibition of WDR5 has exhibited potent anti-tumor activity across diverse cancer types, further highlighting its therapeutic potential30–34. Through its scaffolding capacity, WDR5 coordinates transcription factors with chromatin-modifying complexes to establish a permissive epigenetic landscape critical for stem cell identity. Yet, how WDR5 engages transcription factors like NANOG at the molecular level, and whether WDR5 can actively modulate their LLPS behavior and downstream gene regulation, remain unclear.

In this study, we identify a direct interaction between NANOG and WDR5, mediated by a previously uncharacterized binding interface that is distinct from canonical WDR5-WIN motif interactions and involves a broad contact surface across NANOG’s structured homeodomain. Mechanistically, WDR5 remodels β-sheet-enriched, irregular NANOG aggregates into dynamic, liquid-like condensates, thereby enhancing NANOG’s chromatin recruitment and transcriptional activation at pluripotency-associated loci. NANOG R153A, a mutation which disrupts its interaction with WDR5, impairs LLPS formation, reduces active chromatin marks H3K4me3 and H4K16ac, and compromises the maintenance of pluripotency. Furthermore, pharmacological inhibition of the WDR5-NANOG interaction with the WDR5 WIN-motif inhibitor C16 significantly inhibits the expansion and self-renewal of leukemia stem cells (LSCs) in vivo, highlighting the therapeutic potential of targeting this axis in cancer.

Results
WDR5 binds NANOG and remodels its aggregation state
We identified an interaction between WDR5 and NANOG by co-immunoprecipitation (co-IP) in HEK293T cells co-transfected with both constructs (Fig. 1a) and confirmed their direct binding in vitro with purified proteins (Supplementary Fig. 1a-c). Notably, purified NANOG exhibited a propensity for aggregation on the size-exclusion chromatography (SEC) (Supplementary Fig. 1a, b), which is consistent with a previous report showing that its C-terminal domain (CTD) drives higher-order oligomerization15. We then asked whether WDR5 could modulate NANOG’s structural behavior and aggregation propensity. To address this, we utilized electron microscopy (EM) to visualize purified proteins. While NANOG alone formed irregular, amorphous aggregates, the supplement of WDR5 induced a structural reorganization of NANOG, resulting in the formation of droplet-like structures that displayed characteristic features of LLPS (Fig. 1b, c).

To explore the molecular basis underlying this transition, we performed protein structure predictions using AlphaFold335 and analyzed intrinsic disorder characteristics using PONDR36. These analyses identified two intrinsically disordered regions (IDRs) in NANOG: a prominent IDR at its NTD and a distinct IDR at its CTD, the latter of which contains tryptophan-repeat motifs implicated in NANOG self-assembly15 (Supplementary Fig. 1d, e). Notably, WDR5 also features a disordered NTD, a structural feature commonly observed in proteins with phase separation potential. These predictive analyses, combined with our EM observations, suggest that WDR5 may modulate NANOG’s structural dynamics by promoting LLPS.

To validate these findings in vivo, we generated a Flag-tagged WDR5 knock-in androgenetic haploid embryonic stem cell (AG-haESC) line using CRISPR-Cas9 technology37–39. These cells can undergo spontaneous diploidization, allowing the rapid generation of homozygous mutations (Supplementary Fig. 1f). There was a substantial overlap between endogenous NANOG and WDR5 within nuclear puncta, indicative of biomolecular condensate formation in mESCs (Fig. 1d). These condensates rapidly dissolved upon treatment with 1,6-hexanediol (1,6-HD), a well-characterized disruptor of LLPS (Fig. 1d). Chromatin immunoprecipitation sequencing (ChIP-seq) of WDR5 and NANOG further demonstrated a global reduction in peak intensities upon 1,6-HD treatment, reflecting a widespread decrease in their chromatin occupancy (Fig. 1e). Additionally, ChIP-seq signals at key pluripotency genes, including Npm1, Nanog, Ezh2, and Pou5f1, were markedly reduced, suggesting that WDR5-NANOG phase-separated condensates may be crucial for sustaining their occupancy at pluripotency genes (Fig. 1f).

We next performed fluorescence recovery after photobleaching (FRAP) experiments to further characterize the dynamic properties of WDR5-NANOG condensates. In HEK293A cells transiently transfected with green fluorescent protein (GFP)-tagged NANOG and red fluorescent protein (RFP)-tagged WDR5 constructs, the two proteins showed strong co-localization in nuclear puncta (Supplementary Fig. 1g). FRAP analysis showed that GFP-NANOG puncta in co-transfected cells displayed partial fluorescence recovery within minutes, indicative of dynamic, liquid-like properties (Fig. 1g, h). In contrast, GFP-NANOG expressed alone exhibited negligible fluorescence recovery, suggesting the formation of solid-like condensates with impaired molecular dynamics (Fig. 1g, h).

Given the complexity of intracellular environments, we conducted in vitro phase separation assays using purified GFP-NANOG and WDR5-RFP proteins. Consistent with our EM observations, GFP-NANOG alone formed irregular aggregates, while WDR5-RFP exhibited weak LLPS that became more pronounced at concentrations above 40 μM (Fig. 1i). WDR5-RFP droplets exhibited rapid fluorescence recovery, while GFP-NANOG aggregates remained static, indicating a lack of fluidity (Fig. 1j). Notably, the addition of WDR5 induced a concentration-dependent transition of NANOG from irregular aggregates to liquid-like droplets (Fig. 1k), and this transition was confirmed by FRAP (Fig. 1l). In addition, a truncated NANOG 1-155 construct lacking the C-terminal domain failed to form condensates even under crowding conditions, highlighting a critical role of the CTD in NANOG aggregation (Supplementary Fig. 1h).

To gain molecular insights into this phenomenon, we employed AlphaFold3-based structural modeling to predict the structural organization of NANOG alone and in complex with WDR5 at varying molecular stoichiometries. In multimeric NANOG models (4, 8, and 16 molecules), the C-terminal region progressively adopted β-sheet-enriched stacking structures characteristic of aggregation (Supplementary Fig. 2a). In contrast, structural modeling of WDR5-NANOG complexes (1, 4, and 8 molecules) indicated that WDR5 binding imposes steric constraints that hinder β-sheet formation at the NANOG C-terminus, thereby reducing the propensity for aggregated states (Supplementary Fig. 2b). This predicted disruption of β-sheet assembly is consistent with the experimentally observed shift toward dynamic, liquid-like condensates in the presence of WDR5, providing a structural rationale for its role in promoting phase separation.

WDR5 binds directly to the NANOG homeodomain
Next, we sought to identify the region within NANOG that was responsible for its interaction with WDR5. Domain mapping by co-IP in HEK293T cells showed that WDR5 specifically binds the N-terminal fragment of NANOG (residues 1-155), but not the C-terminal fragment (residues 155-305) (Fig. 2a). To further define the minimal binding region, we conducted isothermal titration calorimetry (ITC) assays using shorter NANOG fragments: residues 1-96, 91-104, 96-155, and 101-155 (Fig. 2b). Among them, only the 96-155 fragment exhibited significant affinity for WDR5, with a dissociation constant of 5.13±0.03 μM, whereas no detectable binding was observed for the other fragments (Fig. 2c). These results define residues 96-155, which contain the complete homeodomain (HD), as the minimal region mediating the WDR5-NANOG interaction.

The crystal structure of NANOG HD domain40 shows that the residues 96-155 of NANOG comprise an unstructured loop (96-104) followed by three structured α-helices (104-155) (Supplementary Fig. 3a). Interestingly, WDR5 is well-documented to interact with proteins such as MLL/SET methyltransferases, RBBP5, KANSL1, and KANSL2 via its WIN (WDR5-interacting) and WBM (WDR5-binding) pockets, typically engaging short loop sequences (Supplementary Fig. 3b, c). However, unlike these canonical interactions, the NANOG binding region encompasses both an unstructured loop and adjacent α-helices, indicating a previously unrecognized mechanism of WDR5 engagement.

To assess the specificity of the WDR5-NANOG interaction, we examined other NKL subclass homeodomain transcription factors, including HLX, DLX1, HHEX, and BARX141. Binding assays (ITC) demonstrated variable affinities: HLX showed relatively high-affinity binding (Kd = 4.13 μM), DLX1 exhibited a weaker interaction (Kd = 206.2 μM), whereas HHEX and BARX1 lacked detectable binding (Supplementary Fig. 3d). Sequence alignment showed that the homeodomain is relatively conserved between mouse and human orthologs but displays limited conservation across NKL subclass members (Fig. 2d). These findings highlight the specificity of the WDR5-NANOG interaction, suggesting that it represents a structurally distinct and sequence-dependent binding paradigm.

Molecular basis of the WDR5–NANOG interaction
To elucidate the molecular basis underlying the interaction between NANOG and WDR5, we determined the crystal structure of NANOG HD in complex with WDR5 using X-ray crystallography. Initial crystallization trials using various NANOG truncations bound to WDR5 failed to yield diffracting crystals; we therefore engineered a series of NANOG mutants designed to retain WDR5-binding activity while enhancing crystallization efficiency. Among these mutants, NANOG HDR100A successfully formed high-quality crystals in complex with WDR5, allowing for the determination of its structure at a 2.5 Å resolution (Fig. 3a, Supplementary Fig. 4a, Table 1). The NANOG HDR100A mutation did not significantly affect its binding affinity to WDR5, with a Kd value of approximately 5.78 μM, comparable to that of the wild-type NANOG homeodomain (Supplementary Fig. 4b). Additionally, the NANOG HDR100A also formed a stable heterodimeric complex with WDR5 in solution (Supplementary Fig. 4c).

Structural analysis showed that both the N-terminal arm and the C-terminal region of NANOG HD mediate the interaction with WDR5 (Fig. 3b, c). At the N terminus, the side-chain amine groups of NANOG K96 and K98 form electrostatic interactions and hydrogen bonds with WDR5 D213 and D192, respectively. Additionally, Q97 participates in a water-mediated hydrogen bond with WDR5 residue N214 (Fig. 3b). The C-terminal region of NANOG HD engages WDR5 through a network of extensive hydrophobic interactions and hydrogen bonds, thereby establishing a stable interaction interface (Fig. 3c, d).

Like previously characterized complexes such as WDR5-MLL1 and WDR5-KANSL1, the C-terminal region of NANOG HD interacts with WDR5 through the insertion of a key arginine residue, R153, into WDR5’s central channel (Fig. 3c, d, Supplementary Fig. 4d). R153 establishes extensive interactions with WDR5: the amine nitrogen of R153 forms hydrogen bonds with WDR5 residues S91, F133, and C261, while its guanidinium group engages in cation-π interactions with the aromatic side chains of WDR5 residues F133 and F263 (Fig. 3d). However, unlike the canonical WIN motif, which typically consists of a tetrapeptide sequence (A-R[position 0]-[A/C/S/T]-[E/K/R]) with conserved arginine (position 0) and alanine (position −1) residues critical for interaction42 (Supplementary Fig. 3c), the surrounding NANOG sequence is far less conserved. Notably, in NANOG, the residue at position −1 is K, which does not contribute to the interaction interface. Instead, residues at position −2 (C) and position +1 (W) form new interaction pockets with WDR5, thereby defining a noncanonical and previously unrecognized binding mode (Fig. 3c).

To validate whether the interaction observed in the crystal structure persists under physiological conditions, we employed nuclear magnetic resonance (NMR) spectroscopy experiments. For backbone resonance assignment, we prepared 15N/13C-labeled NANOG HD and 15N/13C/2H-labeled WDR5, enabling residue-specific peak mapping (Supplementary Fig. 4e, f). Subsequently, titration of unlabeled WDR5 into 15N-labeled NANOG HD, and vice versa, yielded 15N-1H heteronuclear single quantum coherence (HSQC) spectra that revealed distinct chemical shift perturbations upon complex formation (Supplementary Fig. 4g-i). Residue-level analysis indicated that WDR5’s central channel and adjacent surface regions are involved in binding NANOG HD, while both the N-terminal arm and C-terminal region of NANOG HD participate in the interaction with WDR5 (Fig. 3e). Key residues, including Q97, K98, and R153 in NANOG, and F133 and D192 in WDR5, exhibited pronounced chemical shifts, consistent with their involvement in the crystal interface (Supplementary Fig. 4h, i). The strong correlation between the chemical shift perturbation patterns and the crystallographic interface provides compelling evidence that the interaction observed in the crystal structure is accurately recapitulated in solution.

We then introduced site-directed mutations at key contact residues and assessed their effects on binding to functionally validate the structural interface. Mutations such as NANOG R153A, WDR5 F133A, and WDR5 F263A significantly impaired binding affinity, whereas NANOG K96A and NANOG K98A caused only a modest decrease (Fig. 3f). Consistent with these in vitro findings, the R153A mutation in full-length NANOG markedly impaired its interaction with WDR5 in HEK293T cells (Fig. 3g). These data highlight the essential roles of hydrophobic and electrostatic interactions in stabilizing the WDR5-NANOG complex and provide physiological support for the crystallographic model.

To assess how WDR5 binding influences the assembly behavior of NANOG, we performed Thioflavin-T (ThT) kinetic assays to monitor the formation and modulation of β-sheet-enriched aggregates. NANOG WT showed a time-dependent increase in ThT fluorescence, whereas the CTD mutant NANOG W8A displayed substantially lower ThT signal (Fig. 3h), consistent with previous studies indicating that the CTD promotes NANOG aggregation14,15. Addition of WDR5 WT suppressed the ThT signal of NANOG WT, whereas the binding-impaired mutant WDR5 F133A exhibited a weaker effect (Fig. 3h), consistent with our structural data and supporting a model in which WDR5 engages NANOG to remodel its β-sheet-enriched assemblies.

Given that the NANOG homeodomain also mediates DNA binding, we next asked whether WDR5 engagement is compatible with DNA binding. Structural superposition of the WDR5-NANOG HD complex with the DNA-bound NANOG HD43 revealed a steric clash between WDR5 and the DNA duplex at the canonical recognition helix of NANOG HD, indicating that WDR5 and DNA likely compete for an overlapping binding surface on NANOG HD (Supplementary Fig. 5a). Electrophoretic mobility shift (EMSA) assays further confirmed that NANOG HD bound the target DNA with high affinity and that WDR5 progressively disrupted NANOG-DNA complex formation in a concentration-dependent manner (Supplementary Fig. 5b). Interestingly, under molecular crowding conditions, WDR5, NANOG, and DNA can co-partition into tripartite phase-separated condensates (Supplementary Fig. 5c), suggesting that although WDR5 and DNA binding to an individual NANOG homeodomain are mutually exclusive in a strict structural sense, they can nonetheless coexist within higher-order condensates, likely through multivalent, dynamic interactions. This co-condensation is likely concentration-dependent and may be facilitated by the dynamic assembly properties of phase-separated condensates, which can accommodate partially competing binding events via multivalent interactions.

Disrupting WDR5-NANOG binding impairs mESC pluripotency
To elucidate the functional significance of the WDR5–NANOG interaction, we utilized the NANOG R153A mutant, which specifically disrupts WDR5 binding while preserving NANOG’s DNA-binding ability under physiological salt conditions (Supplementary Fig. 5d, e). This mutation served as an ideal model to dissect the specific contributions of the WDR5-NANOG interaction. The purified GFP-NANOG R153A, like GFP-NANOG WT, formed high-molecular-weight aggregates, indicating that this mutation does not intrinsically affect NANOG’s self-assembly propensity (Supplementary Fig. 5f, g). However, GFP-NANOG R153A retained its intrinsic fibrillar state and failed to transition into liquid-like condensates upon the addition of WDR5-RFP (Fig. 4a, Supplementary Fig. 5h), under conditions that readily induced LLPS in GFP-NANOG WT (Fig. 1k, l). When the concentration of WDR5-RFP was increased to 80 μM (a fourfold molar excess over GFP-NANOG R153A), phase separation was partially restored, albeit forming fewer and smaller condensates (Fig. 4a) compared to GFP-NANOG WT (Fig. 1k). Quantitative analysis of droplet condensate areas further confirmed that the LLPS capacity of GFP-NANOG R153A was markedly diminished, even at elevated WDR5-RFP concentrations, highlighting the pivotal role of the WDR5–NANOG interaction in reshaping NANOG’s conformational state (Fig. 4b).

We next investigated whether the WDR5-NANOG interaction is essential for the pluripotency of mESCs. We generated NANOG R153A knock-in mESC lines (Supplementary Fig. 6a) and initially cultured them under conditions that support pluripotency (2i medium). We then transferred the cells to serum/LIF medium, a relatively weaker condition for pluripotency maintenance, to evaluate the effect of disrupting the WDR5-NANOG interaction on mESC self-renewal. Immunofluorescence analysis revealed reduced co-localization between WDR5 and NANOG R153A compared to NANOG WT, as quantified by Pearson’s correlation coefficient (Fig. 4c). Meanwhile, NANOG R153A mESCs exhibited striking morphological changes, transitioning from compact, dome-shaped colonies to flattened, irregular colonies indicative of differentiation (Fig. 4d). These morphological changes were accompanied by reduced alkaline phosphatase (AP) staining, a classical marker of pluripotency, as well as a marked reduction in colony number, indicating an impaired self-renewal capacity of these NANOG R153A cells compared to NANOG WT mESCs (Fig. 4e). Given these defects in self-renewal, we next examined whether early lineage specification was altered using embryoid body (EB) differentiation assays. R153A mutant cells displayed increased expression of somatic lineage markers relative to WT cells, whereas the expression of primordial germ cell (PGC) markers was reduced, consistent with previous findings that NANOG is required not only to maintain pluripotency but also to promote PGC specification44 (Supplementary Fig. 6b).

To dissect how the WDR5-NANOG interaction regulates mESC pluripotency at the transcriptional level, we performed RNA sequencing (RNA-seq) analysis comparing NANOG WT and NANOG R153A mESCs under serum/LIF culture. Remarkably, disruption of the WDR5-NANOG interface induced widespread transcriptional reprogramming, affecting 3,710 genes, including 1,131 downregulated and 2579 upregulated genes (Fig. 4f, Supplementary Fig. 6c). Core pluripotency factors, such as Klf4, Esrrb, Sox2, and Nanog itself, were significantly repressed in NANOG R153A mESCs, establishing a link between the WDR5–NANOG interaction and the regulation of pluripotency genes. Conversely, genes associated with differentiation and lineage commitment, including Runx2, Hoxa7, and Shc4, were robustly upregulated, reflecting a pronounced shift towards differentiation (Fig. 4f, Supplementary Fig. 6c). Gene Ontology (GO) analysis further revealed that downregulated genes were enriched in pathways related to stem cell population maintenance, stem cell division, and cell fate commitment, while upregulated genes were associated with developmental processes, including embryonic forelimb morphogenesis, stem cell differentiation, and cellular adhesion (Fig. 4g).

Collectively, these results demonstrate that the WDR5-NANOG interaction is a critical molecular switch in safeguarding mESC identity by stabilizing the core pluripotency network and preventing premature activation of lineage-specific differentiation programs.

Loss of WDR5–NANOG interaction impairs active chromatin
We then used ChIP-seq to identify how the physical interaction between NANOG and WDR5 influences their genome-wide chromatin occupancy. The analyses were performed in both NANOG WT and NANOG R153A mESCs cultured in serum/LIF to compare the binding profiles of NANOG and WDR5. We found that both NANOG and WDR5 were predominantly enriched at promoter regions, rather than enhancers, although the signals were substantially reduced in the mutant relative to wild-type mESCs (Supplementary Fig. 7a). Motif enrichment analyses confirmed that NANOG WT- and NANOG R153A-bound regions were similarly enriched for its own canonical motifs as well as those of other core pluripotency transcription factors, including OCT4, SOX2, and KLF4. A similar motif pattern was observed in WDR5-bound regions in NANOG WT, supporting the coordinated recruitment of these two proteins to shared regulatory elements. In contrast, WDR5-bound regions in NANOG R153A mESCs lacked most pluripotency motifs, except for c-MYC, suggesting partial redirection of WDR5 by other transcription factors in the absence of NANOG interaction (Supplementary Fig. 7b). Importantly, genome-wide profiling data further demonstrated that both NANOG and WDR5 binding signals were significantly diminished at shared co-occupied loci in NANOG R153A mESCs (Fig. 5a, b). Specifically, there was a marked reduction in co-occupied peaks, from 7796 in NANOG WT mESCs to only 143 in NANOG R153A mESCs (Fig. 5c). Moderate signal reductions were also observed at their respective unique binding sites (Fig. 5a, b), likely reflecting secondary effects from transcriptional reprogramming and partial differentiation in the mutant background. Despite these changes in binding intensity, the overall genomic distribution of binding sites remained largely unchanged, with promoter and distal intergenic regions still representing the major binding compartments of WDR5 and NANOG in NANOG R153A mESCs (Supplementary Fig. 7c).

To identify target genes co-bound and co-regulated by NANOG and WDR5, we integrated ChIP-seq and RNA-seq datasets in mESCs. Specifically, we intersected 7796 genes co-bound by NANOG and WDR5 with 3710 differentially expressed genes (DEGs) identified in NANOG R153A mESCs, resulting in 2087 genes affected by the disruption of WDR5-NANOG interaction (Fig. 5d). Further analysis revealed that 113 genes exhibited increased binding and upregulated expression, while 96 genes showed decreased binding and downregulated expression. The remaining 1878 genes displayed no significant binding changes but still exhibited transcriptional changes, likely through indirect regulation. Enrichment analysis indicated that genes with significant binding changes also showed statistically significant expression differences, whereas DEGs without binding changes did not exhibit significant expression differences (Fig. 5e). Genes with reduced binding were enriched for pathways essential for stem cell maintenance and pluripotency, such as embryonic pattern specification and cellular response to leukemia inhibitory factor (LIF). In contrast, genes with increased binding were associated with differentiation-related processes, including apoptotic signaling and Wnt pathway activation (Fig. 5f).

Given that WDR5 is a core component of both the MLL/SET complex, which mediates H3K4 trimethylation (H3K4me3), and the MOF complex, responsible for H4K16 acetylation (H4K16ac), we investigated whether disruption of the WDR5-NANOG interaction affects these epigenetic marks at target loci. ChIP-seq profiling in NANOG R153A mESCs revealed global reductions in both H3K4me3 and H4K16ac, with the most marked losses at co-occupied WDR5-NANOG regions (Fig. 5g, h). Representative loci showed context-specific chromatin remodeling patterns. For example, at the Tcf3 locus, NANOG, WDR5, and H3K4me3 were co-enriched in wild-type cells but all diminished in the mutant, indicating that the WDR5-NANOG interaction is required to maintain H3K4me3 at this site (Fig. 5i, left). At Dnmt3a, NANOG, WDR5, H3K4me3, and H4K16ac were co-localized and reduced in the mutant (Fig. 5i, right), indicating that the WDR5-NANOG interaction contributes to maintaining both activating marks at this promoter. Similar patterns were observed at other pluripotency genes, such as Nr5a2 and Esrrb (Supplementary Fig. 7d).

These genome-wide analyses describe the steady-state chromatin changes associated with disruption of the WDR5-NANOG interaction. To relate these changes to the dynamics of differentiation, we used a time-resolved differentiation assay to place these changes into a temporal framework. NANOG WT and NANOG R153A ESCs were shifted from 2i/LIF to serum/LIF and harvested at 0, 6, and 24 h. Within the first 6 h after 2i withdrawal, H3K4me3 levels at WDR5-NANOG co-bound pluripotency promoters dropped significantly in R153A cells, and H4K16ac decreased at a subset of the same loci (Supplementary Fig. 8a, b). At this early stage, steady-state mRNA levels showed only modest, gene-specific changes (Supplementary Fig. 8c), whereas a parallel nascent RNA labeling experiment, performed immediately after transfer from 2i/LIF to serum/LIF, already revealed reduced transcription of the same gene set in NANOG R153A ESCs (Supplementary Fig. 8d). More uniform down-regulation of bulk mRNA became evident by 24 h (Supplementary Fig. 8c). These kinetics indicate that disruption of WDR5-NANOG condensates first destabilizes activating histone marks and attenuates nascent transcription at pluripotency-associated genes, with loss of steady-state expression arising as a downstream consequence of these epigenetic changes. Thus, the WDR5-NANOG interaction emerges as a critical determinant of active chromatin and transcriptional programs that sustain stem cell identity.

Targeting WDR5-NANOG suppresses LSC self-renewal
Building on our findings in mESCs, we next examined whether the WDR5-NANOG interaction is also functionally relevant in pathological contexts, particularly in the maintenance of CSCs. Leukemia stem cells represent a prototypical malignant stem cell population, characterized by sustained self-renewal, impaired differentiation, and resistance to chemotherapy. To evaluate the potential involvement of the WDR5-NANOG axis in LSC function, we first analyzed the expression levels of NANOG and WDR5 across human hematopoietic lineages using publicly available single-cell RNA-seq data. Both genes were preferentially expressed in hematopoietic stem and progenitor cells (HSPCs), with significantly lower expression in differentiated subsets (Supplementary Fig. 9a). Utilizing a murine acute myeloid leukemia (AML) model driven by the AML1-ETO9a (AE9a) fusion oncogene45, which mimics the human RUNX1::RUNX1T1 translocation and is known to generate a therapy-resistant LSC population, we found that NANOG expression was upregulated in AE9a HSPCs, while WDR5 levels remained comparable to those in WT HSPCs (Fig. 6a). These observations indicate that the self-renewal ability of AE9a HSPCs may depend on elevated NANOG expression and its interaction with WDR5.

To functionally interrogate this hypothesis, we targeted the WDR5-NANOG axis in AE9a LSCs using C1646, a selective small-molecule inhibitor that binds the WIN site of WDR5 (Supplementary Fig. 9b). Endogenous Co-IP experiments showed that C16 preferentially weakened the WDR5-NANOG interaction, whereas WDR5 complexes with MLL1 or KANSL1 were only modestly affected at the concentrations tested (Supplementary Fig. 9c). We further used revumenib (RE)47, a selective menin-MLL1 inhibitor, to differentiate potential effects arising from MLL1-related pathways. In AE9a leukemia cells, C16 treatments significantly reduced colony-forming units (CFUs) and decreased the number of viable AE9a cells by approximately 60% compared to the DMSO control group, whereas RE had minimal impact on either parameter (Fig. 6b). In WT HSPCs, both C16 and RE treatments reduced CFUs by about 15% compared to the DMSO control group. However, RE led to a sharp 75% decline in cell viability, while C16 only caused a modest ~10% decrease (Fig. 6b). Fluorescence-activated cell sorting (FACS) analysis further revealed that C16 treatment almost completely depleted the LSC population, while LSCs in the DMSO and RE groups remained at 6%. The flow cytometry gating strategies are shown in Supplementary Fig. 10a, b. Additionally, C16 treatment promoted the differentiation of AE9a leukemia cells into mature myeloid cells (Fig. 6c). Taken together, these results suggest that targeting the WDR5-NANOG axis specifically inhibits the self-renewal of LSCs in the AE9a leukemia model in vitro.

To gain insights into the molecular mechanisms, we performed RNA-seq analysis on C16- or RE-treated AE9a and WT c-Kit+ cells. Among all groups, C16-treated AE9a cells exhibited the most extensive transcriptional alterations (Fig. 6d, Supplementary Fig. 10c). A total of 3,323 genes were downregulated in the C16-treated AE9a group, among which 2,941 were specifically downregulated in AE9a cells compared to C16-treated WT cells (Fig. 6e). In contrast, only 259 genes were downregulated in RE-treated AE9a cells (Supplementary Fig. 10d). The number of upregulated genes in these groups was less dramatically different relative to WT cells, with 846 genes upregulated in C16-treated AE9a cells and 478 genes upregulated in RE-treated AE9a cells (Supplementary Fig. 10e, f). Next, we performed GO analysis to examine the functional impact of these transcriptional changes. The downregulated genes were associated with stemness-related processes, including embryonic hematopoiesis, stem cell proliferation, and the Notch signaling pathway, while the upregulated genes were predominantly involved in differentiation processes, such as cell differentiation and the Wnt signaling pathway (Fig. 6f). These results suggest that C16 exerts its unique effects on AE9a LSCs primarily through the suppression of transcriptional programs essential for stem cell maintenance. We further evaluated the in vivo effects of C16 treatment on AE9a LSCs using a retrovirus-transformed AE9a mouse leukemia model (Fig. 6g). Consistent with the in vitro results, C16-treated mice showed a ~50% reduction in the populations of lineage-Sca-1+c-Kit+ (LSK) cells, long-term hematopoietic stem cells (LT-HSCs), and multi-potent progenitor (MPP) cells compared to the DMSO control group, while RE treatment had a minimal effect on these populations in AE9a recipient mice (Fig. 6h and Supplementary Fig. 10g). Analysis of downstream progenitor cells revealed that RE treatments significantly expanded the populations of common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP), while C16 treatment showed comparable populations of these cells to the DMSO group (Supplementary Fig. 10g, h). Together, these findings indicate that C16 selectively impairs the expansion of stem cells in the AE9a mouse leukemia model, highlighting the therapeutic potential of targeting the WDR5-NANOG axis to eliminate leukemia stem cells.

Methods
Cell lines
Wdr5 C-terminal 3× Flag and Nanog R153A knock-in cell lines were generated by the CRISPR-Cas9 method. First, AG-haESC were transfected with a plasmid expressing Cas9/sgRNA along with a recombination donor DNA flanked by homology arms targeting either the Wdr5 C-terminal or the R153 site of Nanog. Three days after transfection, mCherry-positive cells were isolated by FACS, expanded, and validated via immunoblotting and Sanger sequencing. The sgRNA sequences for Wdr5−3×Flag and Nanog R153A knock-in are 5’- GAAGAGTGACTGCTAAGTCC-3’ and 5’- ACCAAAGGATGAAGTGCAAG-3’, respectively.

Cell culture
HEK293T and HEK293A cells were cultured in high-glucose DMEM (Gibco, 11995065) supplemented with L-glutamine and sodium pyruvate and 10% fetal bovine serum (ExCellBio, FSP500) at 37°C and 5% CO2.

For the maintenance of naïve pluripotency under 2i/LIF conditions, mouse embryonic stem cells (mESCs) were cultured on dishes coated with 0.1% gelatin, using a serum-free N2B27 medium composed of equal parts DMEM/F12 and Neurobasal media. This base was supplemented with 1× B27, 1× N2, 2 mM L-glutamine, 50 U/ml penicillin-streptomycin, and 0.1 mM 2-mercaptoethanol. To sustain the naïve state, the medium also included 3 μM CHIR99021 (a GSK3β inhibitor), 1 μM PD0325901 (a MEK inhibitor), and recombinant leukemia inhibitory factor (LIF).

For downstream applications, mESCs initially grown in 2i/LIF were passaged and transferred to serum/LIF medium, with a medium replacement 24 h post-transfer. Cells were collected 48 h later for RNA-seq and ChIP-seq analyses.

For serum/LIF culture, mESCs were maintained on 0.1% gelatin-coated plates in conventional ESC medium consisting of high-glucose DMEM supplemented with 15% fetal bovine serum (FBS), 100 μM non-essential amino acids (NEAA), 1% nucleoside mix, 2 mM L-glutamine, 50 U/ml penicillin–streptomycin, 0.1 mM 2-mercaptoethanol, and in-house produced recombinant LIF, which was functionally validated for supporting self-renewal. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Mouse cKit+ cells were cultured in SFEM medium (STEMCELL, Cat #09650) supplemented with 100 ng/mL murine SCF (R&D), 20 ng/mL murine IL-3 (R&D), and 20 ng/mL murine IL-6 (R&D). For RNA-seq analysis, cKit+ cells were treated for 72 h with either DMSO, 5 μM C16 (WDR5-IN-1, HY-133121, MedChemExpress) or 10 μM Revumenib (HY-136175, MedChemExpress) for 72 h.

Protein expression and purification
Mus musculus Nanog and Wdr5 fragments were amplified by G5 High-Fidelity DNA Polymerases (EnzyArtisan, China). Full-length WDR5 and NANOG, as well as their truncations or mutants, were cloned into a modified pET28b vector with an N-terminal 6× His-Sumo tag using One-Tube Cloning Kit (EnzyArtisan, China). The plasmids were then extracted with the SPARK easy Superpure Mini Plasmid Kit (Shandong Sparkjade Biotechnology Co., Ltd.) and transformed into Escherichia coli BL21 (DE3) competent cells (WEIDI bio, China, EC1002).

For WDR5 and soluble NANOG fragments, proteins were overexpressed with induction by 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16–18 h at 18°C. Harvested cells were resuspended in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 400–800 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and protease inhibitor cocktail. The cells were broken by sonication on ice and then centrifuged at 20,000 × g for 40 min. The supernatant was incubated with Ni-NTA beads (Qiagen, USA) for 2 h at 4°C, and the tag-free proteins were then eluted by on-beads digestion with ULP1 protease (for pET28b-Sumo vectors) added at a molar ratio of 1:200. Eluted proteins were further purified by size-exclusion chromatography (SEC) on Hiload Superdex 75 or Hiload Superdex 200 columns in the buffer of 25 mM Tris-HCl, pH 8.0, and 150–300 mM NaCl. Purified proteins were concentrated and stored at −80°C.

Full-length NANOG was expressed by induction with 1 mM IPTG at 37°C for 3–4 h and accumulated predominantly in inclusion bodies. Bacterial pellets were resuspended in denaturing buffer (50 mM Tris-HCl, pH 8.0, 800 mM NaCl, 8 M urea) containing protease inhibitors, lysed by sonication, and centrifuged to remove insoluble debris. The supernatant was applied to Ni-NTA resin in the same denaturing buffer, washed, and eluted with imidazole. Denatured NANOG was then refolded by stepwise dialysis into buffers with decreasing urea concentrations (4 M, 2 M, and 0 M) and finally into 50 mM Tris-HCl pH 8.0, 400 mM NaCl, 1 mM DTT, 10% glycerol. Precipitates were removed by centrifugation, the SUMO tag was cleaved with ULP1 where applicable, and refolded protein was further purified by SEC in 25 mM Tris-HCl pH 8.0, 300 mM NaCl and concentrated for downstream assays.

For NMR, uniformly 15N, 15N/13C and 15N/13C/2H-labeled proteins were produced in M9 minimal medium containing 15NH4Cl (and 13C-glucose and D2O where appropriate) and purified following the same protocol. The protein concentration was measured by UV absorbance at 280 nm or a BCA Protein Assay Kit (TIANGEN, PA115-01, China).

GST pull down
For pull-down assays, 100 μg of GST-tagged WDR5 and 100 μg NANOG full-length proteins were mixed with 10 μL of glutathione Sepharose 4B beads in 100 μL of binding buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM DTT). 10 μL reaction mixtures were taken out as input controls. After incubation at the indicated temperature for 3 h, the beads were washed four times with 500 μL of the binding buffer. The bound samples were eluted with 25 μL elution buffer (15 mM reduced glutathione, 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM DTT). The input and eluted samples were analyzed by SDS-PAGE.

Fluorescence polarization assay
NANOG HD WT and NANOG HD R153A proteins were serially diluted in buffer containing 25 mM HEPES, pH 7.8, 150 mM NaCl, 10% glycerol. An equal volume of 5’-FAM-labeled Tcf3 DNA (acctgttaatgggagcgc, synthesized by GenScript Biotech Corporation) was added to each tube to reach a final concentration of 25 nM. The reaction mixtures were incubated on ice for 30 min in the dark and transferred to a 384-well plate. Fluorescence polarization values were measured using a Synergy Neo Multi-Mode Reader (Bio-Tek, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

Electrophoretic mobility shift assay
NANOG HD WT, NANOG HD R153A and WDR5 proteins were serially diluted in either a low-salt binding buffer (10 mM HEPES pH 7.8, 50 mM KCl, 10 mM NaCl, 10% glycerol) or a physiological-salt buffer (25 mM HEPES pH 7.8, 150 mM NaCl, 10% glycerol). For NANOG-DNA binding assays, an equal volume of 5’-FAM-labeled Tcf3 DNA (acctgttaatgggagcgc; synthesized by GenScript Biotech Corporation) was added to each tube. For competition assays, NANOG HD and 5’-FAM-labeled Tcf3 DNA were pre-incubated in the low-salt buffer, and increasing amounts of WDR5 were then added while keeping NANOG and DNA concentrations constant. After incubation on ice for 30 min in the dark, the reaction mixtures were loaded onto 6% native polyacrylamide gels and separated by electrophoresis. Gels were imaged using an FLA-9000 scanner (STARION).

Co-immunoprecipitation
HEK293T cells were harvested and washed with PBS buffer. The cells were suspended in 500 μL lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl), and then broken by sonication. The cell lysates were centrifuged for 15 min at 12,000 × g at 4°C. The supernatant was mixed with 10 μL Anti-DYKDDDDK Affinity Beads (Smart-Lifesciences, China, SA042025) followed by incubation at 4°C for 3 h. Beads were washed three times with 1 ml wash buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40), and the bound samples were eluted by 40 μL 1× SDS loading buffer. Immunoprecipitation was conducted using anti-Flag (ABclonal, AE024, 1:2000) and anti-HA (Cell Signaling, 3724s, 1:1000).

For endogenous co-immunoprecipitation, WDR5-Flag knock-in mESCs were harvested and washed with PBS. The cells were suspended in 200 μL lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) and lysed by sonication. After centrifuging at 12,000 × g for 15 min at 4°C, the supernatants were collected and treated with 0 μM, 5 μM or 20 μM C16, respectively. Each sample was then incubated with 10 μL of Anti-DYKDDDDK Affinity Beads (Smart-Lifesciences, China, SA042025) for 3 h at 4°C with gentle mixing. Beads were washed three times with 200 μL lysis buffer, and bound proteins were eluted with 30 μL 1× SDS loading buffer. Immunoblotting was performed using the following antibodies: anti-Flag (ABclonal, AE024, 1:2000), anti-MLL1 (Cell Signaling Technology, 14197S, 1:1000), anti-NANOG (Cell Signaling Technology, 8822S, 1:1000) and anti-KANSL1 (Aviva Systems Biology, ARP55255_P050, 1:500).

Isothermal titration calorimetry (ITC)
ITC experiments were performed on a MicroCal ITC200 system (GE Healthcare, USA) at 20°C. Before measurement, proteins were dialyzed into the same buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl). For ITC experiments, WDR5 at 1.0 mM in the syringe was titrated into NANOG proteins (including truncations and mutation proteins) at 0.1 mM in the sample cell. Each titration consisted of 20 successive injections. The titration curves were processed using the Origin 7.0 software program (OriginLab) according to the “one binding site” fitting model. Each titration was repeated twice, and one representative plot was shown in the paper. The dissociation constants54, enthalpy changes (ΔH), and fitting errors were derived from the representative ITC plot.

Crystallization and structural determination
NANOG HD R100A-WDR5 complex was concentrated to 15 mg/mL for crystallization, and the crystal was obtained by the sitting-drop vapor diffusion method at 4°C by mixing 0.3 μL protein-protein complex solution and 0.3 μL crystal solution. The complex was crystallized in a solution composed of 100 mM BIS-TRIS pH 6.5, 2.0 M Ammonium sulfate. The crystal was cryoprotected in the same reservoir solution supplemented with 20% glycerol. The datasets were collected at the beamline BL19U1 of the Shanghai Synchrotron Radiation Facility and processed using HKL200055. The structures were solved by molecular replacement in PHASER56 by using the following template models: 2GNQ57 and 2VI640. The MR models were then refined by using PHENIX58 suite with manual building in COOT59, and the structural details were analyzed by PyMol (1.5.0).

Phase separation assays in vitro and in vivo
For in vitro experiments, purified GFP-NANOG WT, GFP-NANOG R153A, WDR5 1-334-RFP proteins were dialyzed into the protein buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl). Proteins and AMCA-labeled Tcf3 DNA (acctgttaatgggagcgc, synthesized by GENERAL BIOL, Chuzhou, China) were then diluted to the desired concentration into the droplet formation buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% PEG8000). For droplet visualization, 3–6 μL of the protein solution was deposited onto a microscope slide. Fluorescent images were acquired using a Zeiss LSM710 microscope.

For in vivo 293A imaging, 1×105 cells were grown on a cell culture plate (Cellvis, D29-14-1.5-N). Transfection was performed 18 h after plating when cell density reached ~30–40% confluency. For each well, 1 μg of each plasmid was transfected using 2 μL Lipo293TM transfection reagent (Beyotime, China, C0521) diluted in 125 μL opti-MEM (Gibco, 31985). Cells were cultured for ~48 h post-transfection, and then incubated with Hoechst 33342 (Beyotime, China, C1022) for 5 min. After washing twice with 1× PBS, fluorescent images were captured using a confocal laser scanning microscopy Olympus SpinSR10 under 100× oil immersion objective. Excitation lasers at 405 nm, 488 nm, and 561 nm were used to visualize Hoechst, GFP and RFP signals, respectively.

Fluorescence recovery after photobleaching (FRAP) measurements
Fluorescence recovery after photobleaching (FRAP) experiments were performed on in vitro droplets using a Zeiss LSM 710 confocal microscope equipped with 488 nm and 561 nm laser lines and a 63×/1.40 NA oil-immersion objective. Defined regions within individual droplets were photobleached for 100 iterations. After bleaching, fluorescence intensities in the bleached area were recorded every 10 s. The raw data (n=15) were processed and analyzed by GraphPad Prism 9.

For live-cell FRAP, 293A cells expressing GFP-NANOG and WDR5-RFP were imaged on a Leica TCS SP8 confocal microscope. Puncta were photobleached with a 488 nm laser (laser bleach 300 iterations; image acquisition interval, 5 s). The raw data (n=20) were processed and analyzed by GraphPad Prism 9.

NMR spectroscopy
The NMR experiments were carried out at 25°C on Agilent DD2 600 MHz or 800 MHz spectrometers equipped with a cryogenic probe. Chemical shifts were referenced to external DSS. For the assignment of backbone resonances of WDR5, the U-[2H, 13C, 15N] NMR samples were prepared in NMR buffer containing 40 mM HEPES (pH 7.5), 150 mM NaCl, 1× Protease Inhibitor Cocktail (MedChemExpress, HY-K0010), 1 mM PMSF, 0.03% NaN3, 3 mM DTT, and 90% H2O/10% D2O. The conventional TROSY versions of standard triple resonance experiments were acquired, including HNCO, HN(CA)CO, HNCA, HN(CO)CA, HN(COCA)CB and HNCACB60. In addition, a nuclear overhauser effect (NOE) mixing time of 150 ms was recorded for a 3D 15N-edited NOESY-HSQC experiment to validate backbone assignments. Spectra were processed with NMRPipe61 and analyzed with NMRFAM-SPARKY62.

For the assignment of backbone resonances of NANOG HD, the U-[13C, 15N] NMR samples were prepared in NMR buffer containing 25 mM HEPES (pH 7.0), 300 mM NaCl, 1× Protease Inhibitor Cocktail (MedChemExpress, HY-K0010), 0.03% NaN3 (w/v), 3 mM DTT, and 90% H2O/10% D2O. NMR spectra were collected at 25°C on an Agilent DD2 700 MHz spectrometer equipped with a HCN z-gradient room temperature probe. The backbone assignments were obtained using HNCO, HNCACO, CBCA(CO)NH, HNCACB, HNCA, HN(CO)CA and 15N-edited NOESY-HSQC spectra. All 3D spectra employed a non-uniform sampling scheme in the indirect dimensions and were reconstructed by multi-dimensional decomposition software MddNMR63,64, interfaced with NMRPipe.

To investigate the interaction between WDR5 and NANOG HD, 0.5 mM U-[15N] WDR5 was titrated with unlabeled NANOG HD at a 1:3 molar ratio or 0.5 mM U-[15N] NANOG HD was titrated with unlabeled WDR5 at a 1:1 molar ratio in a buffer containing 25 mM HEPES (pH 7.5), 300 mM NaCl, 3 mM DTT, 1× Protease Inhibitor Cocktail (MedChemExpress, HY-K0010), 0.03% NaN3 and 10% D2O. The 2D 1H-15N TROSY spectra were recorded at 25°C on an Agilent DD2 700 MHz spectrometer equipped with a HCN z-gradient room temperature probe.

RNA-seq
Total RNA was extracted using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions. RNA-seq libraries were prepared with the NuGEN RNA-seq kit (#0348–32) and sequenced on the Illumina HiSeq 4000 platform to generate paired-end reads with a length of 150 nucleotides.

ChIP-seq and ChIP-qPCR
ChIP-seq and Spike-in ChIP samples were prepared using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, #9005) following the manufacturer’s protocol. Briefly, 4×106 mESCs were fixed in 1% formaldehyde for 10 min at room temperature and quenched with 125 mM glycine for 5 min. Cells were rinsed three times with ice-cold PBS and stored at −80°C. For chromatin fragmentation, fixed cells were lysed, and nuclei were digested with micrococcal nuclease (MNase) to obtain mono-nucleosomal fragments. The reaction was stopped with EDTA, and the nuclei were collected by centrifugation. Chromatin extracts were incubated overnight at 4°C with rotation with the following primary antibodies: anti-NANOG (Bethyl Laboratories, A300-397A, 1:100), anti-H3K4me3 (Cell Signaling Technology, #9751S, 1:100), anti-H4K16ac (Abcam, ab109463, 1:100), or anti-FLAG (Sigma-Aldrich, F1804, 1:100). Immunoprecipitated complexes were captured using Protein G magnetic beads for 2 h at 4°C. After a series of low-salt and high-salt washes, the chromatin was eluted and reverse-crosslinked at 65°C in the presence of Proteinase K and NaCl. Purified ChIP DNA was used for library construction using the NEBNext Ultra II DNA Library Prep Kit (E7103S). For quantitative normalization, a calibrated amount of Spike-in DNA was introduced into each sample during the library preparation stage. Libraries were sequenced on an Illumina platform with paired-end 150-bp reads.

ChIP for qPCR analysis was performed using the same cross-linking, chromatin preparation, and immunoprecipitation procedures described above for ChIP-seq, following the manufacturer’s instructions for the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Immunoprecipitated chromatin and 1.25% input DNA were analyzed by quantitative PCR using SYBR Green Master Mix (Vazyme) on an Applied Biosystems QuantStudio real-time PCR system. Primer sequences targeting the promoters of Nanog, Nr5a2, Klf4, Tbx3, and Dnmt3a are listed in source data. ChIP-qPCR enrichment was calculated as percent input, and data are presented as mean ± SD from three independent replicates.

Embryoid body (EB) differentiation
To induce EB formation, ESCs were dissociated into single cells and seeded as 30 μL hanging drops containing approximately 1000 cells per drop in ESC medium lacking leukemia inhibitory factor (LIF) to allow spontaneous differentiation. The hanging drops were incubated for 4 days under standard culture conditions (37°C, 5% CO2), after which the formed EBs were collected for RT-qPCR analysis.

Thioflavin T assay
To monitor β-sheet-enriched protein assemblies, we utilized the Thioflavin T (ThT) assay. ThT is a fluorescent dye whose emission increases upon binding to β-sheet-rich structures, thereby reporting on the aggregation state of NANOG. A 1 mM Thioflavin T (ThT, MedChemExpress, HY-D0218) stock solution was prepared in 100% DMSO. The stock was diluted with PBS (pH 7.4) to achieve a final ThT concentration of 20 μM in each well. During the assay, the concentrations of full-length NANOG WT and NANOG W8A were maintained at 5 μM. Full-length WDR5 WT or WDR5 F133A was added at a 2:1 molar ratio relative to NANOG. The reaction mixtures were thoroughly mixed by pipetting up and down. ThT fluorescence was measured in 30 μL reactions using 384-well plates on a Synergy Neo Multi-Mode Reader (Bio-Tek) with excitation and emission wavelengths set at 450 nm and 488 nm, respectively. All experiments were performed in triplicate.

Immunofluorescence
Immunofluorescence was performed as previously described65. The following primary antibodies were applied: anti-FLAG (M2) (Sigma-Aldrich, F1804), anti-NANOG (Cell Signaling Technology, 8822S). Images were acquired using a Leica TCS SP8 confocal laser microscopy system.

Nascent RNA capture
The nascent RNA synthesis was detected using a STAR-Click Nascent RNA Capture Prep Kit (Foreverstar Biotech, Beijing, China) according to the manufacturer’s instructions. Briefly, cells were incubated with 1 μM 5-ethynyluridine (EU), an alkyne-modified nucleoside, for 30 min. Total RNA labeled with EU was then isolated and subjected to a copper-catalyzed click reaction with an azide-modified biotin. This reaction introduced a biotin tag, enabling the capture of nascent RNA transcripts using streptavidin magnetic beads. The captured transcripts were immediately reverse transcribed using TransScript First-Strand cDNA Synthesis SuperMix kit (TransGen Biotech, China) and followed by qPCR analysis. Primer sequences targeting the genes of Nanog, Nr5a2, Klf4, Tbx3, and Dnmt3a are listed in source data.

Data analyses
RNA-seq reads were aligned to the mouse genome (mm9) using STAR (v2.7.6a) with default parameters to ensure accurate mapping. Gene-level read counts were used for differential expression analysis with DESeq2. P values were calculated using the two-sided Wald test and adjusted by the Benjamini-Hochberg method (FDR). Differentially expressed genes (DEGs) were identified as those with |log2[fold change]| ≥ 1 and FDR < 0.05. Gene Ontology (GO) analysis was performed using the DAVID platform 6.8 (https://david.ncifcrf.gov/). Upregulated and downregulated genes sets were analyzed separately. GO terms were selected according to adjusted P values (Benjamini-Hochberg FDR < 0.05). P values were derived from one-sided Fisher’s exact test.

ChIP-seq reads were processed using Trim Galore (v0.6.3) for adapter removal and quality filtering, followed by alignment to the mm9 mouse genome with Bowtie2 (v2.3.4). The aligned reads were exported, sorted, and duplicates removed using the MarkDuplicates tool from PICARD (v2.14.0). The resulting BAM files were converted to TDF format for visualization in IGV (v2.7.2). Peak calling was conducted with MACS2 (v2.2.7) using default settings, and peaks were annotated against the mm9 genome using the annotatePeaks module in HOMER (v4.11). Heatmaps and average intensity profiles for ChIP-seq data at specific genomic regions were generated with the NGSplot program (v2.61). For spike-in samples, reads were mapped to both the mouse (mm9) and Drosophila (dm6) genomes. The ratio of Drosophila reads was used to calculate the exogenous normalization factor for each sample.

Colony formation assay
Mouse cKit+ cells were isolated from the bone marrow (BM) of WT or transgenic mice using a magnetic bead sorting system (Miltenyi Biotec) according to the manufacturer’s instructions. The isolated cells (3000 per well) were plated in methylcellulose medium (M3434, StemCell Technologies) in 6-well plates containing either DMSO, 2 μM C16, or 2 μM Revumenib. Cells were cultured at 37°C with 5% CO2 for 7 days before colony enumeration and immunophenotypic analysis.

Flow cytometric analysis
Single-cell suspensions from colonies and BM were stained with fluorochrome-conjugated antibodies. For colony immunophenotypic analysis, cells were stained with BV421-cKit, PE-cy7-Gr1 and APC-CD11b for 30 min at 4°C. For MPP cells, LT-HSCs, ST-HSCs and BM cells were stained with APC-conjugated lineage cocktail (B220, CD4, CD5, CD8, Gr1 and Ter119), Percp-cy5.5-Sca1, BV421-cKit, PE-CD150, APC-cy7-CD48. LSK were immunophenotypically defined as Lin-cKit+Sca1+, LT-HSC as Lin-cKit+Sca1+CD48-CD150+, ST-HSCs as Lin-cKit+Sca1+CD48-CD150-, MPP as Lin-cKit+Sca1+CD48+. For committed progenitor cells, BM cells were stained with APC-conjugated lineage cocktail, Percp-cy5.5-Sca1, BV421-cKit, PE-cy7-CD16/32, PE-CD34. Granulocyte-monocyte progenitors (GMP) were immunophenotypically defined as Lin-cKit+Sca1-CD34+CD16/32high, common myeloid progenitors (CMP) as Lin-cKit+Sca1-CD34int/+CD16/32int, and megakaryocyte-erythrocyte progenitors (MEP) as Lin-cKit+Sca1-CD34-CD16/32low. All analyses were performed using a BD FACSAria™ III cell sorter, with data processed in FlowJo software (v10).

Mouse model and bone marrow transplantation
AE9a-transformed mouse BM cells were generously provided by Prof. Yuheng Shi. The recipient mice, at 8 weeks old, underwent sublethal irradiation (4.5 Gy) and were then transplanted with 1×105 BM cells via tail vein injection. The WDR5 inhibitor, C16 (WDR5-IN-1, HY-133121, MedChemExpress) was prepared as a 2 mg/mL solution in 20% 2-(hydroxypropyl)-β-cyclodextrin (HP-β-CD, HY-101103, MedChemExpress) in ddH2O. The Menin inhibitor, Revumenib was dissolved in PBS to 2 mg/mL. Mice were injected intraperitoneally at a maximum dosing volume of 5 mL/kg to give a final 10 mg/kg body weight dose. Recipient mice were maintained in an SPF facility with a 12 h light/12 h dark cycle; the room was controlled at 22 ± 2°C and 50 ± 10% relative humidity, and standard chow and water were provided ad libitum. All animal experiments described in this study were conducted in accordance with the ethical guidelines of the Department of Laboratory Animal Science, Fudan University, China (No. 202307021S).

Statistics and reproducibility
Western blotting and electron microscopy experiments were independently repeated twice with similar results, and representative data are shown. Phase separation assays, fluorescence recovery after photobleaching, and immunofluorescence experiments were independently repeated three times, with similar results. The cellular phenotype of the NANOG R153A mutant was confirmed in three independent experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Wang et al described a direct and previously unrecognized interaction between the pluripotency transcription factor NANOG and the chromatin adaptor WDR5. The authors demonstrate that WDR5 remodels NANOG's prion-like aggregates into dynamic, phase-separated condensates, facilitating its chromatin recruitment and activation of pluripotency-related genes. This interaction is structurally resolved through crystallography and NMR, and its disruption leads to loss of stem cell identity and suppression of leukemia stem cell self-renewal. However, several issues must be addressed before the work can be considered for publication. Therefore, I recommend major revision.\n1. The manuscript presents compelling evidence that disruption of the WDR5-NANOG interaction impairs leukemia stem cell self-renewal, primarily based on inhibition using the WIN-site antagonist C16. However, WDR5 is a multifunctional scaffold protein that interacts with numerous partners (e.g., MLL complex and MOF complex), and the specificity of C16 toward disrupting the WDR5-NANOG axis remains uncertain.\n2. While ChIP-seq shows a loss of H3K4me3 and H4K16ac upon disruption of NANOG-WDR5 interaction, it remains unclear whether these epigenetic changes are a direct consequence of impaired condensate formation or secondary to the loss of pluripotency.\n3. Although the manuscript establishes a correlation between the presence of NANOG/WDR5 condensates and transcriptional activation, it does not directly demonstrate that disruption of these condensates leads to acute suppression of transcription at target loci. Without such evidence, the causal link between condensate formation and transcriptional output remains speculative.\n4. The manuscript proposes that WDR5 remodels NANOG condensates by acting on its prion-like C-terminal domain (CTD), yet no domain-specific or loss-of-function analyses have been conducted to support this model.\n5. The reported Rmerge value for the X-ray diffraction data appears high. The authors should verify the accuracy of the data processing and clarify whether this value reflects issues in data quality or resolution-dependent artifacts.", "reviewer_2": "This study reports a direct interaction between the Nanog homeodomain, otherwise involved in DNA binding, with the WDR5 co-regulator using X-ray crystallography further verified via NMR. This interaction affects formation of LLPS, genome-wide binding profiles, and cancer stemness as shown with a structure-informed R153A mutation. They propose that WDR5 induced a structural switch of Nanog aggregates from inactive prion-like forms towards active liquid-like forms. This is a bold and intriguing claim with a profound impact on the field of transcription factor biology. This reviewer is not fully convinced that the data presented in past and present papers support such a far-reaching claim. I find this is an important study with high-quality data, but I feel some of the interpretations go too far and require a major revision and down-toning of the proposed amyloid to LLPS switch unless very compelling new data are added.\nMajor Strengths:\nLinking the molecular details of how chromatin regulators work together at atomic resolution with their operation in the nucleus to regulate genes and cell behavior is of paramount importance, and I applaud the authors for taking this holistic approach. The experimental content and the breadth of methods (NMR, X-ray, EM, LLPS, and several cell-based and in vivo analyses) are impressive.\nKey Limitations and Recommendations for Revision:\nThe authors claim that WDR5 switches Nanog’s functionally inert prion-like state to a transactivating LLPS state. However, their cryo-EM analysis does not appear to clearly reveal a prion-like state. The previous NCB paper that first claimed the prion character of Nanog is very interesting, but it did not contain a structure or data for prion-like seeding or templating. Protease resistance was also not shown there. Could authors visualise the amyloids via cryo-EM? As of now, it seems the prion-like behaviour of Nanog is a hypothesis from with indirect support from fibrous aggregate morphology and beta-richness alone? Is there any evidence that the fibrillar state authors report in Figures 1 and 4 represent bona fide amyloids with beta structures (refer to work by Ruben Hervas and Ryskeldi-Falcon labs and others for structural work on amyloids to provide ultimate evidence)?\nAuthors fail to elaborate on how WDR5 affects Nanog's DNA binding. Questions that come to mind: Can DNA/WDR/Nanog form a ternary complex? Or does the Nanog-WDR5 interaction block DNA binding? Does a mutation that disrupts WDR5 interaction not also impede DNA binding? The proposed mechanism is not clear and should, in my opinion, be resolved before publication in a high-visibility journal. Authors are experts in quantitative molecular binding assays and have a battery of proteins at their disposal, so I do not see why they couldn’t, with reasonable effort, study how WDR5 affects Nanog’s DNA interaction.\nThe Nanog R153A ESC lines provide a powerful tool that I felt was underused. Why was stemness and pluripotency not further examined using this line (rather than with cancer stem cells)? It would be interesting to study differentiation potential, EB formation, and (optionally) developmental potential in vivo.\nMinor comments:\nHow does 1,6-hexanediol treatment affect ESC viability, morphology and self-renewal?\nFor easier cross-referencing, I recommend adding conventional HD numbering to key residues (i.e. Q50, N51)\nIn Figure 2 or 3, please include the sequence of the Nanog HD with annotated helices, DNA binding residues (Q50,N51), and mark those subject to mutational analysis. There is an alignment in the supplement that could be modified and moved to the main text.\nThe x-ray structure of Nanog was resolved in apo and DNA-bound forms. Why did authors use structural modelling rather than these experimental structures for some of their analysis?\nPlease compare DNA motifs bound by Nanog and the R153A mutant. Does it affect preferences for the TAATGG like HD motif. Does the mutant alter the otherwise often seen co-occupancy of loci by Nanog, Sox2 and Oct4?\nThe Nanog-DNA Kd’s in Figure S5 are a lot lower (1000x?) thn previously reported. Whilst many parameters can affect binding affinity this difference looks very drastic. Please ensure titrations are not performed with [DNA]>=[Kd] when reliable Kd’s are aspired. I recommend following these very useful guidelines: https://elifesciences.org/articles/57264\nFigure S6: Does the motif utilization change in shared and Nanog/mutant unique peaks?\nSimilar amorphous aggregates were reported for other pluripotency factors; this could be discussed.\nThey also tested the disruption of NANOG and WDR5 interaction in LSCs, drug group (C16) and DMSO control. I wonder if they treat healthy stem cells (ESCs, MSCs, et al) with C16. The switch to LSCs looks a bit abrupt and inconsistent with the rest of the study. Please comment/explain.\nThe LLPS shown in Figures 1 and 4 had very high protein concentrations, which may not reflect the real physiological concentrations experienced in the cell. Would phenomena persist if changed to use a more physiological concentration of NANOG and WDR5 in vitro ?\nThe FRAP of Figure 1H does not seem to recover too much after adding WDR5. Please comment/explain.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-Point Responses to Reviewers' Comments\nDear Reviewers,\nWe appreciate your careful evaluation and constructive feedback, which have\nsignificantly helped us improve our manuscript. In response, we have substantially\nrevised the manuscript to address the key conceptual and technical concerns raised.\nAll revisions are addressed in a point-by-point response below and have been\nincorporated into the revised manuscript as indicated.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Wang et al described a direct and previously unrecognized\ninteraction between the pluripotency transcription factor NANOG and the chromatin\nadaptor WDR5. The authors demonstrate that WDR5 remodels NANOG's prion-like\naggregates into dynamic, phase-separated condensates, facilitating its chromatin\nrecruitment and activation of pluripotency-related genes. This interaction is structurally\nresolved through crystallography and NMR, and its disruption leads to loss of stem cell\nidentity and suppression of leukemia stem cell self-renewal. However, several issues\nmust be addressed before the work can be considered for publication. Therefore, I\nrecommend major revision.\nWe thank the reviewer for their constructive comments, which have helped us\nimprove the quality of this work. We address each point in detail below and have revised\nthe manuscript accordingly.\n1. The manuscript presents compelling evidence that disruption of the WDR5-NANOG\ninteraction impairs leukemia stem cell self-renewal, primarily based on inhibition using\nthe WIN-site antagonist C16. However, WDR5 is a multifunctional scaffold protein that\ninteracts with numerous partners (e.g., MLL complex and MOF complex), and the\nspecificity of C16 toward disrupting the WDR5-NANOG axis remains uncertain.\nWe thank the reviewer for raising this point. We fully agree that WDR5 is a\nmultifunctional scaffold protein, and its interaction with multiple partners may raise\nconcerns about the specificity of C16. In response, we have provided additional data to\nclarify the specificity of C16 toward disrupting the WDR5-NANOG axis.\nTo investigate the effects of C16 on the WDR5-NANOG interaction, we\nconducted endogenous co-immunoprecipitation (Co-IP) assays in mESCs, as well as\non other key WDR5-associated complexes, including WDR5-MLL1 and WDR5-\nKANSL1. Our results indicate that increasing concentrations of C16 weaken the\nWDR5-NANOG interaction in a dose-dependent manner. Within the same\nconcentration range, the effects on WDR5-MLL1 and WDR5-KANSL1 interactions\nwere modest, suggesting that the WDR5-NANOG interface is more sensitive to WIN-\nsite blockade, possibly reflecting a distinct mode of interaction (Revised Figure 1A and\nSupplementary Fig. 9c).\nWe re-analyzed RNA-seq data from mESCs (NANOGWT vs NANOGR153A) and\nAE9a leukemia cells (C16 vs DMSO) to assess the transcriptional impact and\nspecificity of C16 treatment. Our goal was to explore the overall effects of disrupting\nthe WDR5-NANOG interface genetically and inhibiting WDR5 pharmacologically\nwith C16. Differentially expressed genes (DEGs) were independently identified in\nESCs and AE9a cells (|log₂FC| > 1), and shared genes were selected based on common\nidentity. This resulted in 181 genes with consistent regulation. GO analysis of this\nshared gene set showed significant enrichment for modules involved in maintaining\nleukemia stem cells, including leukocyte cell-cell adhesion, leukocyte migration,\npositive regulation of leukocyte activation, and chemotaxis/taxis (Revised Figure 1B).\nThese findings suggest that disrupting the WDR5-NANOG interaction in mESCs (via\nNANOG R153A mutation) and inhibiting WDR5 with C16 in leukemia stem cells\nactivate a shared, WDR5/NANOG-dependent transcriptional program. Despite\nbaseline differences between mESCs and leukemia cells, the consistent responses imply\nthat C16’s anti-leukemic effects mainly result from disrupting the WDR5-NANOG axis.\nConsistent with this axis-level specificity, as shown in our manuscript (Fig. 6b-h\nand Supplementary Fig. 9d-i), an independent comparison with a menin-MLL1\ninhibitor (revumenib, RE) revealed divergent transcriptional and phenotypic outcomes.\nGiven WDR5’s role in the MLL1 complex, we used RE as a control to test whether\nC16 specifically disrupts the WDR5-NANOG axis in the original version of the\nmanuscript. While C16 treatment reduced AE9a colony-forming units (CFUs), depleted\nthe leukemia stem cell (LSC) population, and downregulated a broad spectrum of\nstemness-related genes, RE had minimal effects on AE9a CFUs or LSCs. Moreover,\nRE induced far fewer transcriptional changes and exhibited toxicity toward WT HSPCs\n(Fig. 6b-h and Supplementary Fig. 9d-i). These results argue against a nonspecific effect\nof MLL1 pathway inhibition and instead support the model that C16 exerts its\nleukemic-suppressive effects via targeted disruption of the WDR5-NANOG axis rather\nthan through general perturbation of WDR5/MLL1-dependent transcription.\nWe also attempted to generate NANOG-deficient AE9a leukemia cells and rescue\nthem with NANOG WT or NANOG R153A to directly evaluate the functional\nrelevance of the WDR5-NANOG interaction in this context. Although gene editing was\nsuccessful, as confirmed by T7E1 assays (Revised Figure 1C), the Nanog knockout\ncells exhibited poor viability and a significant reduction in colony-forming units (CFUs)\n(Revised Figure 1D), precluding further rescue experiments. These findings underscore\nthe essential role of NANOG in maintaining AE9a cells. We also initially considered\ngenerating a NANOG R153A knock‑in line to establish a genetic model. However, due\nto the inherent technical challenges of base editing and the low transfection efficiency\nin primary AE9a c-Kit+ cells, this approach has proven particularly challenging. We\nfully acknowledge this limitation and are continuing to optimize strategies to achieve it\nin future work. In the meantime, the combined results from the knockout experiments\nand transcriptional analyses still provide insight into how disruption of the WDR5-\nNANOG axis affects leukemia stem cell self‑renewal.\nWe hope these additional data help address the reviewer’s concerns. While some\ntechnical challenges remain, we are actively refining our system and deepening our\nunderstanding of the WDR5-NANOG axis in leukemia stem cells. These data give us\nconfidence that the observed anti-LSC effects primarily reflect disruption of the\nWDR5-NANOG interaction, rather than broader inhibition of other WIN-motif partners.\nWe sincerely appreciate the reviewer’s insights, which have significantly strengthened\nboth the study and its presentation.\nRevised Figure 1. Selective disruption of the WDR5-NANOG interaction by C16.\n(A) Endogenous Co-IP of WDR5-binding partners in mESCs treated with increasing\nconcentrations of C16. Flag-tagged WDR5 was immunoprecipitated, and associated\nproteins were detected by immunoblotting.\n(B) GO enrichment analysis of biological processes for differentially expressed genes\nshared between mESCs expressing NANOGR153A versus NANOGWT and AE9a\nleukemia cells treated with C16 versus DMSO.\n(C) T7 endonuclease I (T7E1) mismatch-cleavage assay confirming CRISPR/Cas9-\nmediated editing at the Nanog locus in AE9a cells.\n(D) Colony-forming units (CFUs) for AE9a sgCtrl (Mock) versus AE9a sgNanog cells.\nData represent mean ± SD (n = 3). *P < 0.05 by unpaired t-test.\n2. While ChIP-seq shows a loss of H3K4me3 and H4K16ac upon disruption of\nNANOG-WDR5 interaction, it remains unclear whether these epigenetic changes are a\ndirect consequence of impaired condensate formation or secondary to the loss of\npluripotency.\nWe appreciate the reviewer’s concern. To determine whether the observed\nreductions in H3K4me3 and H4K16ac result directly from condensate disruption rather\nthan representing secondary effects of pluripotency loss, we conducted a time-resolved\ndifferentiation assay under defined conditions. Specifically, NANOG WT/R153A\nmESCs were transitioned from 2i/LIF to serum/LIF and harvested at 0, 6, and 24 hours.\nAs shown in Revised Figure 2A, 2 h after 2i/LIF withdrawal, WDR5 and NANOG\nalready display reduced co-localization in R153A cells compared with WT. At the\nindicated time points, we then measured H3K4me3 and H4K16ac levels at\nrepresentative WDR5-NANOG co-bound promoters by ChIP-qPCR (Revised Fig. 2B,\nC) and quantified the corresponding mRNA levels by RT-qPCR (Revised Fig. 2D).\nIn this time course, R153A cells displayed an early reduction of H3K4me3 at all\nexamined pluripotency-associated promoters (Nanog, Nr5a2, Klf4, Tbx3, Dnmt3a) and\na decrease in H4K16ac at a subset of these loci by 6 h, whereas transcript levels of the\ncorresponding genes showed only modest, gene-specific changes at 6 h and more\nconsistent down-regulation by 24 h (Revised Fig. 2D). These findings suggest that the\nloss of activating histone marks precedes detectable transcriptional down-regulation\nand is unlikely to be a downstream consequence of pluripotency exit.\nAdditionally, we previously conducted an integrative analysis of our ChIP-seq and\nRNA-seq datasets (original Fig. 5d-f), which identified a set of 1,026 genes that are\nboth co-bound by NANOG and WDR5 and transcriptionally affected by condensate\ndisruption. Among these, 113 genes were upregulated and 96 were downregulated in\nthe R153A mutant. Notably, genes showing simultaneous loss of binding and\ndownregulation were significantly enriched for core pluripotency pathways, including\nembryonic pattern specification, cellular response to leukemia inhibitory factor (LIF),\nand positive regulation of transcription by RNA polymerase II (Fig. 5f, upper panel),\nsuggesting that WDR5-NANOG condensates directly support the transcriptional\nmaintenance of pluripotency-related genes. This orthogonal genome-wide evidence\nfurther supports a direct mechanistic link between condensate disruption and epigenetic\nrepression. Conversely, genes whose expression changed without corresponding loss of\nbinding likely represent secondary or compensatory responses rather than direct\nregulation by condensates.\nTaken together, these time-course experiments and genome-wide integrative\nanalyses strongly support that the observed reductions in H3K4me3 and H4K16ac are\na direct consequence of impaired WDR5-NANOG condensate formation, rather than a\ndownstream effect of pluripotency exit. These findings have been incorporated into the\nrevised Results (Supplementary Fig. 8a-c). We thank the reviewer for this insightful\nsuggestion, which helped clarify the temporal and mechanistic relationship between\ncondensate integrity and epigenetic regulation.\nRevised Figure 2. Early loss of activating chromatin marks upon disruption of the\nWDR5-NANOG interface.\n(A) Representative immunofluorescence images of NANOG (green) and WDR5 (red)\nin NANOGWT and NANOGR153A mESCs 2 h after withdrawal from 2i/LIF. Right,\nquantification of NANOG-WDR5 co-localization by Pearson’s correlation coefficient\n(PCC). 272 WT cells and 344 R153A cells were analyzed across three independent\nexperiments. Data represent mean ± SD (n = 3). *P < 0.05 by unpaired t-test.\n(B) ChIP-qPCR analysis of H3K4me3 at the promoters of Nanog, Nr5a2, Klf4, Tbx3\nand Dnmt3a in NANOGWT and NANOGR153A mESCs at 0, 6 and 24 h after transfer\nfrom 2i/LIF to serum/LIF. Data are presented as mean ± SD (n = 3). Statistical\nsignificance was determined using a two-tailed t-test (ns, not significant; *P < 0.05; **P\n< 0.01; ***P < 0.001).\n(C) ChIP-qPCR analysis of H4K16ac at the same promoters, showing reduced\nH4K16ac enrichment on Klf4 and Dnmt3a in NANOGR153A mESCs at 6 h after 2i/LIF\nwithdrawal.\n(D) Quantitative RT-PCR analysis of Nanog, Nr5a2, Klf4, Tbx3 and Dnmt3a mRNA\nlevels in NANOGWT and NANOGR153A mESCs at the indicated time points after\ntransfer from 2i/LIF to serum/LIF. Data are presented as mean ± SD (n = 3). Statistical\nsignificance was determined using a two-tailed t-test (ns, not significant; *P < 0.05; **P\n< 0.01; ***P < 0.001, ****P < 0.0001).\n3. Although the manuscript establishes a correlation between the presence of\nNANOG/WDR5 condensates and transcriptional activation, it does not directly\ndemonstrate that disruption of these condensates leads to acute suppression of\ntranscription at target loci. Without such evidence, the causal link between condensate\nformation and transcriptional output remains speculative.\nWe appreciate this insightful comment and have performed additional analyses\nand experiments to directly link condensate disruption to acute suppression of nascent\ntranscription.\nFirst, we re-analysed published nascent RNA-seq data from human SEM leukemia\ncells treated with 1,6-hexanediol, generated in the study by Crump et al.1. In this dataset,\nwe used the nascent RNA samples GSM4141680-GSM4141685 to compare untreated\ncells with cells exposed to 1.5% 1,6-hexanediol for 30 min. Differential gene expression\nanalysis of nascent RNA (edgeR, FDR < 0.05) identified 2,995 up-regulated and 3,454\ndown-regulated transcripts (Revised Fig. 3A). GO enrichment analysis on the down-\nregulated gene set revealed significant enrichment for transcription- and chromatin-\nassociated terms, including \"transcription by RNA polymerase I\", \"rRNA transcription\",\n\"transcription elongation by RNA polymerase II\", and \"regulation of chromatin\nbinding\" (Revised Fig. 3B), indicating that short-term 1,6-hexanediol treatment\nglobally suppresses nascent transcription driven by both RNA polymerases, and\nperturbs factors that regulate chromatin binding. In the same gene set, we also observed\nsignificant enrichment for early developmental processes: “in utero embryonic\ndevelopment”, “blastocyst formation”, and “blastocyst development”. These terms\nreflect genes with well-established roles in early embryogenesis and pluripotency-\nassociated programmes. Together, this independent dataset shows that acute dissolution\nof phase-separated nuclear condensates by 1,6-hexanediol rapidly reduces nascent\ntranscription, with a preferential impact on the transcriptional machinery and early\ndevelopment /stemness-linked gene programmes.\nSecond, to test this concept directly in our system, specifically for the WDR5-\nNANOG condensates, we used our NANOG R153A knock-in mESCs, in which the\nR153A substitution selectively disrupts the WDR5-NANOG interaction and associated\ncondensate formation. Following a short period of 2i withdrawal, we pulse-labelled\ncells with EU and quantified nascent RNA by qPCR at a panel of WDR5/NANOG co-\nbound pluripotency and epigenetic regulators (Nanog, Nr5a2, Klf4, Tbx3, Dnmt3a). As\nshown in Revised Fig. 3C, nascent transcript levels of all five genes are reduced in\nNANOG-R153A cells compared with wild-type controls.\nThese data provide locus-specific, short-term measurements of nascent\ntranscription in a genetic background that selectively disrupts WDR5-NANOG\ncondensate formation. Together with the global nascent RNA-seq analysis of 1,6-\nhexanediol-treated SEM cells, they indicate that disruption of phase-separated\ncondensates, either globally by 1,6-hexanediol or specifically via the WDR5-NANOG\ninterface, results in rapid repression of nascent transcription, encompassing both\nstemness-associated transcriptional programmes and direct WDR5-NANOG target\ngenes. These findings support a causal link between condensate integrity and\ntranscriptional output in our system.\nRevised Figure 3. Nascent transcription upon condensate disruption.\n(A) Volcano plot of nascent RNA-seq from human SEM leukemia cells treated with\n1.5% 1,6-hexanediol for 30 min versus untreated controls (GSM4141680-\nGSM4141685). Differential expression was assessed with edgeR (FDR < 0.05); red,\nup-regulated; blue, down-regulated transcripts, with gene numbers indicated.\n(B) GO enrichment of down-regulated nascent transcripts in (A). Bubble plot shows\nselected significant terms.\n(C) Nascent RNA levels of WDR5/NANOG co-bound pluripotency-related genes in\nNANOG WT/R153A mESCs shortly after shifting from 2i/LIF to serum/LIF, measured\nby 5-ethynyl uridine (EU) labelling followed by RT-qPCR. Data are shown as fold\nchange relative to wild-type (mean ± SD, n=3).\n4. The manuscript proposes that WDR5 remodels NANOG condensates by acting on\nits prion-like C-terminal domain (CTD), yet no domain-specific or loss-of-function\nanalyses have been conducted to support this model.\nTo directly address the reviewer’s concern regarding the absence of domain-\nspecific and loss-of-function evidence supporting our model, we performed two\nadditional experiments:\nFirst, we purified the N-terminal region of NANOG (GFP-NANOG 1-155), which\nlacks the CTD, and tested its ability to phase separate in vitro. This truncated construct\nfailed to form detectable condensates even under crowding conditions (10% PEG8000),\nsupporting the necessity of the CTD for condensate formation (Revised Fig. 4A and\nSupplementary Fig. 1h).\nSecond, we performed Thioflavin-T (ThT) kinetic assays to assess the formation\nof β-sheet-enriched aggregates, comparing wild-type NANOG and a CTD mutant\n(W8A) previously shown to impair self-association2. As shown in Revised Fig. 4B,\nNANOGW8A markedly attenuated ThT fluorescence relative to NANOGWT, indicating\nthat the CTD contributes to the formation of β-sheet-enriched aggregates. Addition of\nWDR5WT suppressed the ThT signal of NANOGWT, whereas the WIN-site mutant\nWDR5F133A exhibited a weaker effect, consistent with our structural data showing that\nthe WIN motif serves as a key interface mediating the WDR5-NANOG interaction.\nTogether, these domain-specific and loss-of-function data, including CTD\ntruncation, CTD point mutation, and differential response to WDR5WT versus\nWDR5F133A, provide direct experimental support for the model in which WDR5\nmodulates the biophysical state of NANOG condensates by engaging the homeodomain\nand sterically constraining the CTD, thereby limiting its assembly into β-sheet-enriched\naggregates. We have added these new results to the revised manuscript (Fig. 3h and\nSupplementary Fig. 1h).\nRevised Figure 4. Mechanistic assays dissecting NANOG assembly and its\nmodulation by WDR5.\n(A) In vitro droplet formation of 20 μM GFP-NANOG 1-155 under the indicated\nPEG8000 concentrations (0, 5, 10%). Assays were performed in 25 mM Tris-HCl (pH\n8.0) and 150 mM NaCl. Scale bars, 10 μm.\n(B) ThT aggregation kinetics of full length NANOGWT alone, NANOGWT + WDR5WT,\nNANOGWT + WDR5F133A, and NANOGW8A; Buffer + ThT serves as control. Traces\nshow normalized ThT fluorescence over time (mean ± SD, n = 3).\n5. The reported Rmerge value for the X-ray diffraction data appears high. The authors\nshould verify the accuracy of the data processing and clarify whether this value reflects\nissues in data quality or resolution-dependent artifacts.\nIn the original “Data collection” line, we inadvertently reported the integration\nrange (50.00-1.78 Å). During data processing, we applied a resolution cutoff at 2.50 Å,\nbecause the outer-shell I/σI fell below 2.0, which is the classical signal-to-noise cut-off\nfor usable reflections3. The structure was therefore refined using the 50.0-2.50 Å dataset.\nWe have corrected Table 1 to accurately reflect 50.0-2.50 Å. We thank the reviewer for\nprompting us to re-examine the processing parameters and present the limits\nunambiguously.\nFor the same dataset, HKL2000 (truncated at 2.50 Å) scaling yielded: overall\nR =0.179, R =0.193, R =0.073 (outer shell: R =0.650, R =0.703,\nmerge meas pim merge meas\nR =0.266 and CC =0.803). An overall R of 0.179 is typical for 2.5 Å data\npim 1/2 merge\ncollected with ~7-fold redundancy3. The higher value in the last shell reflects the well-\nknown resolution dependence of this metric. Since R inflates with redundancy,\nmerge\ncurrent guidelines recommend the redundancy-corrected R and CC as more robust\npim 1/2\nindicators4,5. Our outer-shell CC (0.803) and I/σI (2.1) confirm that the highest-\n1/2\nresolution reflections carry a reliable signal. Together with the refined model statistics\n(R /R (%) = 17.36/22.14, 0.3% Ramachandran outliers), these numbers\nwork free\ndemonstrate that the data are of acceptable quality.\nReviewer #2 (Remarks to the Author):\nThis study reports a direct interaction between the Nanog homeodomain, otherwise\ninvolved in DNA binding, with the WDR5 co-regulator using X-ray crystallography\nfurther verified via NMR. This interaction affects formation of LLPS, genome-wide\nbinding profiles, and cancer stemness as shown with a structure-informed R153A\nmutation. They propose that WDR5 induced a structural switch of Nanog aggregates\nfrom inactive prion-like forms towards active liquid-like forms. This is a bold and\nintriguing claim with a profound impact on the field of transcription factor biology. This\nreviewer is not fully convinced that the data presented in past and present papers\nsupport such a far-reaching claim. I find this is an important study with high-quality\ndata, but I feel some of the interpretations go too far and require a major revision and\ndown-toning of the proposed amyloid to LLPS switch unless very compelling new data\nare added.\nWe sincerely thank the reviewer for his/her thoughtful and constructive evaluation.\nIn response to these concerns, we have implemented a series of focused revisions.\n(1) We removed the phrase “prion-like to LLPS switch” and replaced all references\nto “prion-like” with a more accurate description of NANOG-alone assemblies as β-\nsheet-enriched, irregular aggregates.\n(2) We incorporated an orthogonal ThT kinetics assay comparing NANOGWT,\nNANOGW8A, NANOGWT + WDR5WT, and NANOGWT + WDR5F133A (see our Response\nto Reviewer 1.4). This experiment further validates the role of NANOG’s C-terminal\ndomain (CTD) in the formation of β-sheet-enriched aggregates, while also revealing\nhow WDR5 modulates the condensate state.\n(3) We performed additional EMSA and phase separation assays to clarify how\nWDR5 influences NANOG-DNA engagement, showing that WDR5 modulates\nNANOG’s DNA-binding behavior in a context- and concentration-dependent manner.\n(4) We performed additional EB differentiation assays using R153A ESC lines and\nclarified our rationale for incorporating leukemia stem cells (LSCs) to assess whether\nthe WDR5-NANOG regulatory axis operates in a transcriptionally plastic, disease-\nrelevant context.\nTogether, we address the reviewer’s concerns by refining structural terminology,\nintroducing orthogonal experimental support, and sharpening the mechanistic narrative.\nThe point-by-point revision response is listed as follows.\nMajor Strengths:\nLinking the molecular details of how chromatin regulators work together at atomic\nresolution with their operation in the nucleus to regulate genes and cell behavior is of\nparamount importance, and I applaud the authors for taking this holistic approach. The\nexperimental content and the breadth of methods (NMR, X-ray, EM, LLPS, and several\ncell-based and in vivo analyses) are impressive.\nWe sincerely thank the reviewer for the thoughtful and encouraging comments on\nour study.\nKey Limitations and Recommendations for Revision:\n1. The authors claim that WDR5 switches Nanog’s functionally inert prion-like state to\na transactivating LLPS state. However, their cryo-EM analysis does not appear to\nclearly reveal a prion-like state. The previous NCB paper that first claimed the prion\ncharacter of Nanog is very interesting, but it did not contain a structure or data for prion-\nlike seeding or templating. Protease resistance was also not shown there. Could authors\nvisualise the amyloids via cryo-EM? As of now, it seems the prion-like behaviour of\nNanog is a hypothesis from with indirect support from fibrous aggregate morphology\nand beta-richness alone? Is there any evidence that the fibrillar state authors report in\nFigures 1 and 4 represent bona fide amyloids with beta structures (refer to work by\nRuben Hervas and Ryskeldi-Falcon labs and others for structural work on amyloids to\nprovide ultimate evidence)?\nWe thank the reviewer for raising this point. We agree that our current data do not\ndemonstrate bona fide amyloid fibrils in the strict structural sense. Accordingly, we\nhave revised the manuscript to adopt more conservative terminology (e.g., “β-sheet-\nenriched, irregular aggregates”) and removed the phrase “prion-like to LLPS switch”\nthroughout the text to avoid overinterpretation.\nOur original use of the term “prion-like” was based on Choi et al.2, who proposed\nthat NANOG undergoes prion-like assembly and gelation. However, as the reviewer\nastutely noted, that study did not provide direct evidence of templated seeding, protease\nresistance, or a cross-β ultrastructure. With this important clarification in mind, and\nupon re-evaluating both the literature and our own data, we now describe NANOG-\nalone assemblies as β-sheet-enriched, irregular aggregates rather than prion-like or\namyloid in nature. As noted in the manuscript, we originally employed negative-stain\nEM to visualize the morphological transition of NANOG upon WDR5 binding.\nWhile\nNANOG alone formed irregular, amorphous aggregates, co-incubation with WDR5\ninduced droplet-like structures exhibiting features consistent with LLPS (Fig. 1b, c).\nHowever, we have not conducted cryo-EM under optimized fibrillization conditions to\ncharacterize the molecular architecture of these assemblies. We are interested in this\ndirection and view it as an important future extension.\nConsistent with the scope of this study, our mechanistic focus is not on defining\nthe ultrastructure of the aggregates, but rather on demonstrating that WDR5 binding\nsuppresses aggregation and promotes a dynamic, liquid-like condensate state, which in\nturn enhances chromatin association and transcriptional activity. To strengthen this\ninterpretation, we have incorporated an orthogonal readout of β-sheet assembly using\nThT kinetics (Revised Fig. 4B, see our Response to Reviewer 1.4). Comparing\nNANOGWT, NANOGW8A, and NANOGWT in the presence of WDR5WT or the WIN-\ndeficient F133A mutant, we observed that ThT signal increases for NANOG alone but\nis significantly reduced upon WDR5WT binding; this effect is attenuated with the F133A\nmutant. These findings directly support our model without overinterpreting the\naggregates as bona fide amyloids.\nIn summary, we refined the terminology, added orthogonal ThT-based evidence\nthat WDR5 suppresses β-sheet-enriched aggregation, and explicitly bounded our claims.\nThe revised manuscript now frames WDR5 as a modulator of the NANOG condensate\nstate, rather than as the driver of an amyloid-to-LLPS switch.\n2. Authors fail to elaborate on how WDR5 affects Nanog's DNA binding. Questions\nthat come to mind: Can DNA/WDR/Nanog form a ternary complex? Or does the\nNanog-WDR5 interaction block DNA binding? Does a mutation that disrupts WDR5\ninteraction not also impede DNA binding? The proposed mechanism is not clear and\nshould, in my opinion, be resolved before publication in a high-visibility journal.\nAuthors are experts in quantitative molecular binding assays and have a battery of\nproteins at their disposal, so I do not see why they couldn’t, with reasonable effort,\nstudy how WDR5 affects Nanog’s DNA interaction.\nWe thank the reviewer for this comment. We agree that understanding how WDR5\nmodulates NANOG’s DNA-binding behavior is mechanistically relevant to our study.\nIn response, we have performed additional experiments, now included as\nSupplementary Fig. 5a-c, to assess whether WDR5 affects NANOG-DNA interactions.\nTo assess potential structural compatibility between WDR5 and DNA on NANOG,\nwe aligned the crystal structure of the WDR5-NANOG HD complex with the model of\nNANOG HD bound to DNA, based on the published NANOG-DNA structure (PDB:\n4RBO). The alignment revealed a steric clash between WDR5 and the DNA duplex at\nthe canonical recognition helix of NANOG HD, indicating that WDR5 binding and\nDNA binding likely compete for the same interface (Revised Fig. 5A).\nTo test this hypothesis experimentally, we performed electrophoretic mobility shift\nassays (EMSA) using fluorescently labeled NANOG target DNA (Tcf3-DNA) and\nincreasing concentrations of WDR5. These assays were carried out under low-salt\nconditions to enhance NANOG-DNA binding affinity and enable detection at lower\nprotein concentrations (Revised Fig. 5B). This helped minimize aggregation and well\nretention artifacts often observed at high concentrations, especially in the presence of\nexcess WDR5. We observed that WDR5 progressively disrupted NANOG-DNA\ncomplex formation in a dose-dependent manner (Revised Fig. 5B), supporting a\ncompetitive binding model in which WDR5 modulates NANOG’s DNA association by\noccupying overlapping surfaces on the homeodomain.\nInterestingly, under molecular crowding conditions, we observed that WDR5,\nNANOG, and DNA co-partition into tripartite phase-separated condensates (Revised\nFig. 5C), despite the apparent steric conflict at the atomic interface between WDR5 and\nDNA that prevents NANOG binding. This observation suggests that while WDR5\nbinding and DNA binding to NANOG are mutually exclusive in a strict structural\ncontext, they can nonetheless coexist within higher-order condensates, likely through\nmultivalent, dynamic interactions. This co-condensation is likely concentration-\ndependent and may be facilitated by the dynamic assembly properties of phase-\nseparated condensates, which can accommodate partial competition between individual\nbinding events through multivalent interactions. Such behavior is consistent with recent\nfindings that transcription factors, coactivators, and DNA can co-enrich within the same\ntranscriptional condensates6,7.\nMoreover, as shown in our original Supplementary Fig. 5d-e, the R153A mutation,\nwhich disrupts the WDR5-NANOG interaction, does not impair NANOG’s DNA-\nbinding capability, as confirmed by both EMSA and fluorescence polarization assays.\nThis finding further supports the specificity of the WDR5 effect and excludes the\npossibility that loss of DNA binding accounts for the observed phenotypes in the mutant.\nTaken together, these data indicate that WDR5 modulates NANOG’s DNA-\nbinding behavior in a context- and concentration-dependent manner, likely by shifting\nthe equilibrium between WDR5-bound and DNA-bound states of NANOG. We have\nincorporated these new data into Supplementary Fig. 5a-c and added a corresponding\nexplanation in the Results section.\nRevised Figure 5. WDR5 modulates NANOG-DNA binding.\n(A) Structural superimposition of the NANOG HD-WDR5 complex and the NANOG\nHD-Tcf3 DNA complex (PDB: 4RBO) reveals overlapping binding interfaces on the\nNANOG homeodomain.\n(B) EMSA showing the concentration of NANOG HD required for complete binding\nto Tcf3 DNA (left, 1 nM DNA with increasing NANOG HD). Based on these results,\nthe right panel assesses competitive inhibition by titrating WDR5 (0-320 nM) into pre-\nmixed samples containing 40 nM NANOG HD and 1 nM DNA. All assays were\nperformed in buffer containing 10 mM HEPES (pH 7.8), 50 mM KCl, 10 mM NaCl,\nand 10% glycerol.\n(C) Representative fluorescence microscopy images showing the formation of phase-\nseparated condensates in ternary mixtures of GFP-NANOG, WDR5-RFP, and AMCA-\nlabeled Tcf3 DNA at defined molar ratios of 1:3:1 and 1:4:1. Droplet assays were\nperformed in buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5%\nPEG8000. Scale bars, 10 μm.\n3. The Nanog R153A ESC lines provide a powerful tool that I felt was underused. Why\nwas stemness and pluripotency not further examined using this line (rather than with\ncancer stem cells)? It would be interesting to study differentiation potential, EB\nformation, and (optionally) developmental potential in vivo.\nWe appreciate the reviewer’s suggestion. In the revised manuscript, we expanded\nour analyses using embryoid body (EB) differentiation assays, while retaining the\nleukemia stem-cell (LSC) model to assess whether the WDR5-NANOG regulatory axis\nextends to a disease-relevant, pathological stem-like state.\nWe examined LSCs alongside ESCs to test whether the WDR5-NANOG\nregulatory mechanism is conserved beyond early development and functions in\npathological stemness. This extension is grounded in both biological and translational\nrationale. Biologically, NANOG and WDR5 are aberrantly expressed or functionally\nrepurposed in multiple malignancies, where they support plastic, stem-like\ntranscriptional programmes; Thus, a condensate-based regulatory mechanism defined\nin ESCs could plausibly apply in cancer. Translationally, C16 is a WDR5 WIN-site\nprobe developed and characterized in leukemic systems, making LSCs the most\nrelevant context to assess whether pharmacologic disruption of the WDR5-NANOG\ninterface can blunt malignant self-renewal. In this framework, ESCs offer precise\nmechanistic dissection, while LSCs provide pathophysiological context and therapeutic\nrelevance.\nAs suggested by the reviewer, we performed EB differentiation assays to further\nexamine the impact of the WDR5-NANOG interaction on early lineage specification.\nAs shown in Revised Fig. 6, during EB differentiation, R153A mutant cells displayed\nincreased expression of somatic lineage markers (Fgf5, Gata4, Gata6, Nestin) relative\nto WT cells. Conversely, the expression of primordial germ cells (PGC) markers\n(Prdm14, Prdm1, Nanos) is reduced, aligning with previous findings that NANOG is\nrequired for maintaining pluripotency while also facilitating PGC specification8. We\nhave incorporated these new data into Supplementary Fig. 6b.\nWe agree that in vivo developmental assays would further strengthen our\nconclusions and represent an important direction for future investigation. However, due\nto current limitations in time and resources, such experiments are beyond the scope of\nthe present study. We view these studies as a key direction for future research and are\ncommitted to pursuing them using appropriately designed models.\nRevised Figure6. EB differentiation of NANOG WT and R153A lines.\nQuantitative RT-PCR analysis of pluripotency/PGC markers (A) and somatic lineage\nmarkers (B) in ESCs and EBs derived from isogenic NANOG WT and R153A lines.\nData are mean ± SD (n = 3), normalized to actin and plotted relative to ESC WT.\nStatistical significance was determined using a two-tailed t-test (*P < 0.05, **P < 0.01,\n***P < 0.001). T is also known as Brachyury.\nMinor comments:\n4. How does 1,6-hexanediol treatment affect ESC viability, morphology and self-\nrenewal?\nUnder our experimental conditions (3% 1,6-hexanediol for 15 minutes), we\nobserved no appreciable changes in ESC morphology or viability, as shown in the\nRevised Fig. 7. These results indicate that the treatment is acutely well tolerated and\ndoes not induce overt cytotoxicity.\nWe did not assess long-term self-renewal, as the purpose of this perturbation was\nto probe the biophysical sensitivity of WDR5-NANOG condensates rather than their\nfunctional consequences on self-renewal. Nonetheless, the absence of acute toxicity\nsupports the validity of our interpretation. Similar concentrations and exposure\ndurations of 1,6-hexanediol have been widely used in ESCs and other mammalian\nsystems to transiently disrupt phase-separated compartments without compromising\ncell integrity6,9.\nRevised Figure 7. Short-term 1,6-hexanediol treatment in ESCs.\n(A) Phase-contrast images of ESC colonies under untreated control conditions (MOCK)\nor after treatment with 3% 1,6-hexanediol (1,6-HD) for 15 min.\n(B) Quantification of ESC viability after MOCK or 3% 1,6-HD treatment for 15 min,\nexpressed as percentage of control. Data represent mean ± SD (n = 3); ns, not significant\n(unpaired t-test).\n5. For easier cross-referencing, I recommend adding conventional HD numbering to\nkey residues (i.e. Q50, N51)\nIn Figure 2 or 3, please include the sequence of the Nanog HD with annotated helices,\nDNA binding residues (Q50, N51), and mark those subject to mutational analysis. There\nis an alignment in the supplement that could be modified and moved to the main text.\nTo address this comment, we have updated Figure 2 in the revised manuscript to\nshow the sequence of the NANOG HD with annotated helices and conventional\nhomeodomain numbering, and highlights conserved base-specific DNA-contacting\nresidues, including Q50 and N51, as blue dots (Fig. 2d). The multiple sequence\nalignment that was previously presented in Supplementary Fig. 3e has been moved to\nFig. 2d to improve accessibility. The residues subjected to mutational analysis in this\nstudy were designed later, based on structural insights from the WDR5-NANOG\ncomplex, and are first introduced in the corresponding structural figures. Nevertheless,\nthis alignment provides a useful reference for mapping these mutants onto the\nhomeodomain.\n6. The x-ray structure of Nanog was resolved in apo and DNA-bound forms. Why did\nauthors use structural modelling rather than these experimental structures for some of\ntheir analysis?\nWe agree that the available apo and DNA-bound crystal structures of the NANOG\nhomeodomain provide valuable structural information. Our original use of a predicted\nmodel in Supplementary Fig. 3a was intended to visualize the flexible loop region\n(residues 96-104), which is not resolved in the available experimental structures due to\ndisorder but is predicted to contribute to the WDR5-binding surface.\nIn accordance with the reviewer’s suggestion, we have now replaced the model in\nSupplementary Fig. 3a with the experimentally determined structure of the NANOG\nhomeodomain (PDB: 2VI6). The flexible loop spanning residues 96-104, which is not\nresolved in the crystal structure, is indicated by a dashed line to represent its disordered\nconformation.\nWe also performed a structural alignment between the DNA-bound NANOG\nhomeodomain (PDB: 4RBO) and our WDR5-NANOG complex. This alignment is now\nincluded in Supplementary Fig. 5a to illustrate the spatial relationship between the\nDNA-binding surface and the WDR5-binding region.\n7. Please compare DNA motifs bound by Nanog and the R153A mutant. Does it affect\npreferences for the TAATGG like HD motif. Does the mutant alter the otherwise often\nseen co-occupancy of loci by Nanog, Sox2 and Oct4?\nFigure S6: Does the motif utilization change in shared and Nanog/mutant unique\npeaks?\nWe thank the reviewer for the insightful question. We performed motif enrichment\nanalysis using HOMER (q-value ≤ 1×10⁻³) on NANOGWT and NANOGR153A ChIP-seq\ndatasets, and functionally categorized all significant motifs based on established\ntranscription factor families. As shown in Revised Fig. 8A, most motifs (208) are shared\nbetween the two datasets, while 56 and 12 motifs are uniquely identified in NANOGWT-\nonly and NANOGR153A-only regions, respectively.\nMotif composition analysis reveals that shared binding sites are predominantly\nenriched for NANOG-associated transcriptional programmes, including the TAAT-\ncontaining HD motif and the pluripotency core module characteristic of NANOG-\nSOX2-OCT4 co-occupancy (Revised Fig. 8B). In contrast, NANOGWT-unique and\nNANOGR153A-unique regions contain relatively fewer motifs, most of which fall into\nuncharacterized or noncanonical categories (Revised Fig. 8B). These results suggest\nthat the R153A mutation does not redirect NANOG binding toward alternative\ntranscriptional modules.\nRevised Figure 8. Motif utilization in NANOG WT and R153A peaks.\n(A) Number of significant motifs identified in NANOGWT-unique, shared, and\nNANOGR153A-unique peaks (HOMER known-motif enrichment, Benjamini-Hochberg\nq ≤ 1×10⁻³).\n(B) Heatmap showing the distribution of enriched motifs grouped by functional\ncategories. Color scale represents motif counts; numbers within cells indicate the count\nper category.\n8. The Nanog-DNA Kd’s in Figure S5 are a lot lower (1000x?) thn previously reported.\nWhilst many parameters can affect binding affinity this difference looks very drastic.\nPlease ensure titrations are not performed with [DNA]>=[Kd] when reliable Kd’s are\naspired. I recommend following these very useful guidelines:\nhttps://elifesciences.org/articles/57264\nWe thank the reviewer for the expert comment regarding Kd determination. As\nstated in the original Methods section, our fluorescence polarization (FP) experiments\nwere performed using 25 mM HEPES (pH 7.8), 150 mM NaCl, 10% glycerol, and\n25 nM 5′-FAM-labeled Tcf3 DNA (acctgttaatgggagcgc). Under these physiological-salt\nconditions, the fitted Kd for wild type NANOG HD was approximately 1.9 µM\n(Supplementary Fig. 5d), and consistent results were obtained from parallel EMSA\nassays (Supplementary Fig. 5e).\nPrior reports span a broad range of affinities owing to differences in construct\ndesign, DNA sequence, and buffer composition. For example, Jauch et al.10reported an\napparent Kd of ~26 nM under low-salt conditions (10 mM HEPES, 50 mM KCl,\n10 mM NaCl), whereas Hayashi et al.11 reported a Kd of ~5.9 µM for wild-type human\nNANOG HD binding to a biotinylated OCT4 promoter sequence under higher salt and\nmore stringent conditions. Thus, our measured Kd values in the low micromolar range\nare consistent with these prior studies, considering differences in buffer composition,\nDNA sequence, and assay format.\nTo explicitly evaluate the impact of ionic strength, we additionally performed a\nlow-salt EMSA (10 mM HEPES, 50 mM KCl, 10 mM NaCl; 1 nM Tcf3 DNA) in the\nRevised Fig. 5B. Titration of NANOG HD under these conditions yielded an apparent\nKd of ~20-40 nM, closely matching the magnitude reported by Jauch et al. This direct\ncomparison confirms that the large shift relative to our FP value primarily reflects the\nexpected ionic-strength dependence of electrostatic DNA binding, rather than\ndifferences in assay quality or analysis.\nImportantly, as the reviewer noted, accurate Kd determination requires the\ncondition [DNA] ≪ Kd. Our assays used 25 nM DNA, more than 75-fold below the\nfitted Kd (~1.9 µM) under physiological-salt conditions, and the low-salt EMSA used\n1 nM DNA versus a 20-40 nM Kd (≥20-fold lower), both within recommended bounds.\nWe have reiterated the relevant concentration ranges in the revised figure legends and\nMethods to ensure transparency and reproducibility.\n9. Similar amorphous aggregates were reported for other pluripotency factors; this\ncould be discussed.\nWe thank the reviewer for this insightful suggestion. Indeed, in addition to\nclassical liquid-like condensates, recent studies have reported that several pluripotency\ntranscription factors can also adopt less dynamic or amorphous assemblies under\ndefined conditions. For example, SOX2 has been shown to form amorphous aggregates\nover time in vitro rather than maintaining a spherical droplet12. Similarly, MYC has\nbeen reported to form stress-induced multimers13 and undergo sequence-dependent\nreductions in condensate dynamics, culminating in irreversible aggregation14.\nThese findings raise the possibility that transcription factors involved in\npluripotency regulation may share a common capacity to transition between distinct\nmaterial states, ranging from dynamic condensates to less fluid or aggregated\nassemblies. Such regulated modulation of material properties may serve as a general\nmechanism to coordinate genome organization and transcriptional output.\nIn this context, the β-sheet-enriched aggregates we observe for NANOG and their\nremodeling by WDR5 may reflect a more generalizable strategy by which cofactor\ninteractions modulate the material properties of transcription factors to affect chromatin\noccupancy and gene regulation. We have expanded the Discussion to include this\nperspective, proposing that regulated transitions in transcription factor condensate state\nmay be a broadly conserved mechanism for pluripotency maintenance and genome\ncontrol.\n10. They also tested the disruption of NANOG and WDR5 interaction in LSCs,\ndrug group (C16) and DMSO control. I wonder if they treat healthy stem cells (ESCs,\nMSCs, et al) with C16. The switch to LSCs looks a bit abrupt and inconsistent with the\nrest of the study. Please comment/explain.\nWe thank the reviewer for the opportunity to clarify the scope of our\npharmacological experiments. As noted in our response to Comment 3, we used ESCs\nto mechanistically dissect the WDR5-NANOG interaction through a targeted genetic\nperturbation (NANOG R153A). This approach allowed us to isolate the effects of\ndisrupting this specific interface without introducing the broader network-level\nperturbations that small molecules might cause in pluripotent cells.\nIn contrast, we evaluated pharmacologic inhibition using C16, a WIN site\nantagonist of WDR5 originally developed and validated in AML models. Accordingly,\nwe performed these experiments in LSCs, which provide a disease-relevant context to\nassess on-target activity and therapeutic relevance. For this reason, we did not apply\nC16 to ESCs or MSCs in the present study. We did, however, include normal\nhematopoietic progenitors (HSPCs) as a functional control. Notably, C16 induced only\nmodest changes in CFU output from HSPCs at concentrations that robustly suppressed\nAE9a LSC colony formation (Fig. 6b), suggesting a degree of selectivity in a\ntherapeutically relevant context.\n11. The LLPS shown in Figures 1 and 4 had very high protein concentrations, which\nmay not reflect the real physiological concentrations experienced in the cell. Would\nphenomena persist if changed to use a more physiological concentration of NANOG\nand WDR5 in vitro?\nWe appreciate the reviewer’s insightful comment. It is a well-established and\nbroadly accepted principle in LLPS research that protein concentrations used in in vitro\nassays often exceed average physiological levels. This methodological choice is not\nonly widely recognized but also practically necessary, as simplified reconstitution\nsystems lack critical cellular components such as macromolecular crowding, chromatin\nscaffolds, and multivalent cofactors like RNA and transcriptional coactivators, all of\nwhich are known to significantly lower the saturation concentration (C ) required for\nsat\ncondensate formation. As discussed by Alberti et al15, increasing protein concentrations\nin vitro can help compensate for the absence of these phase-promoting factors.\nConsequently, the use of micromolar concentrations of proteins is a standard practice\nin LLPS studies.\nIn the context of transcription factors, Morin et al.16 reported that KLF4 forms\nliquid-like condensates at physiological salt and pH, but only above ~1.0 µM in the\nabsence of DNA, a concentration that exceeds its estimated nuclear abundance. At the\nsame time, the addition of λ-DNA triggers foci formation below the bulk C ,\nsat\nillustrating how DNA scaffolds can significantly lower the condensation threshold.\nSimilarly, Boija et al.7 demonstrated that a range of transcription factors, including\nOCT4, MYC, SOX2, and RARα, form condensates in vitro at concentrations of 10-40\nµM. Sabari et al6 observed BRD4-IDR and MED1-IDR condensates at concentrations\nof 5-20 µM. These examples collectively support that micromolar concentrations are\ntypical for in vitro LLPS studies of transcriptional regulators. Our use of 20 µM\nNANOG and 20-80 µM WDR5 is therefore well within the established experimental\nrange and methodologically consistent with prior work. Importantly, our core\nconclusions do not depend on the absolute concentration thresholds required to initiate\nLLPS, but rather on the consistent and reproducible effect of WDR5 in modulating the\nmaterial properties of NANOG condensates under matched experimental conditions.\nIn addition to the in vitro evidence, our in vivo experiments further support the\nbiological relevance of WDR5-NANOG condensates. We observed that endogenous\nNANOG and WDR5 colocalize within nuclear puncta displaying hallmarks of phase-\nseparated condensates, including sensitivity to 1,6-hexanediol and dynamic molecular\nexchange. These condensates showed WDR5-dependent changes in biophysical\nproperties and chromatin occupancy, consistent with the modulatory effects\ncharacterized in vitro. Together, these results provide complementary in vitro and in\nvivo evidence that WDR5 regulates NANOG's phase behavior in a physiologically\nmeaningful manner.\n12. The FRAP of Figure 1H does not seem to recover too much after adding WDR5.\nPlease comment/explain.\nWe appreciate the reviewer’s observation. While FRAP recovery in Figure 1h is\nincreased when NANOG is co-expressed with WDR5, we acknowledge that the extent\nof this increase may appear modest by visual inspection.\nBiologically, partial and relatively slow FRAP recovery is often observed in vivo\nfor transcription-related condensates, even when they are functional. This phenomenon\nis well documented and reflects the presence of a substantial immobile fraction arising\nfrom chromatin tethering and the viscoelastic nature of the nuclear environment17.\nIn our case, although the absolute extent of recovery is not dramatic, it is\nconsistently higher with WDR5 co-expression than with NANOG alone. Together with\nour in vitro FRAP results (Fig. 1j and 1l), which show more pronounced recovery under\ncontrolled conditions, these findings support the conclusion that WDR5 enhances the\ndynamic exchange of NANOG molecules, even though a chromatin-tethered fraction\nlikely remains immobile in vivo. We believe this shift is mechanistically meaningful\nand consistent with the behavior of other transcriptional condensates.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe sincerely appreciate your time and effort in reviewing our manuscript and\nthank you for your thoughtful comments and constructive contribution to the review\nprocess.\nREFERENCES\npromoter contact. 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EMBO\nreports 23, e54458, doi:10.15252/embr.202154458 (2022).\nstructure and DNA binding of the homeodomain of the stem cell transcription\nfactor Nanog. Journal of molecular biology 376, 758-770,\ndoi:10.1016/j.jmb.2007.11.091 (2008).\nproperties to promote self-renewal and reprogramming of pluripotent stem cells.\nProceedings of the National Academy of Sciences of the United States of\nAmerica 112, 4666-4671, doi:10.1073/pnas.1502855112 (2015).\ninduction and maintenance by engineered SOX17. Nucleic acids research 51,\n8934-8956, doi:10.1093/nar/gkad597 (2023).\npolymerase. Nature 612, 148-155, doi:10.1038/s41586-022-05469-4 (2022).\nand tumor growth by providing negative charges. The Journal of biological\nchemistry 300, 107836, doi:10.1016/j.jbc.2024.107836 (2024).\nStudying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell\n176, 419-434, doi:10.1016/j.cell.2018.12.035 (2019).\ntranscription factor on DNA. Nature Physics 18, 271-276, doi:10.1038/s41567-\n021-01462-2 (2022).\nchromatin. Epigenetics & chromatin 14, 50, doi:10.1186/s13072-021-00424-5\n(2021)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All issues have been addressed. I recommend acceptance for publication.", "reviewer_2": "I co-reviewed this paper with an ECR in my team. A regulated switch between prion and LLPS state driven by WDR would be fascinating but requires more structural evidence, which the authors acknowledge. The authors have added assays to support the claim that WDR5 can nudge NANOG into undergo liquid-liquid phase separation and to regulate stem cell transcription. The newly added assays, such as Co-IP, ChIP-qPCR, a sequential differentiation assay, and re-analysis of RNA/ChIP-seq data, support the rational use of C16, the direct cause of epigenetic changes, and transcriptional activation by the WDR5/NANOG LLPS. The newly added EMSA/LLPS show that WDR5 competes with DNA for NANOG interactions.\nI remain a bit puzzled as to the main mechanistic model and would encourage the authors to further clarify the graphical summary and the key narrative. I note that a WDR5/NANOG appears – at least in 1-to-1 stoichiometry, incompatible with DNA binding – yet, WDR5 binding to NANOG facilitates its binding to target genes. It would be nice if this could be reconciled/explained. We recommend acceptance of this fascinating study with only minor remaining suggestions:\n• In Figure 1g, please circle where you bleached to make it more straightforward.\n• ChIP-qPCR only has 3 technical analyses. Any possible independent replicates?\n• Please ensure statistical analysis is clarified in all legends and relevant methods sections\n• Please clarify the independent replication (not just repeat recordings) of the new THT fluorescence assay and briefly explain its principle (possibly with a small cartoon next to the data figure).\n• I am still confused as to the main model how WDR5 helps promote the binding of NANOG to its target genes when EMSAs suggest WDR5 prevents the HD from binding DNA. I cannot follow the explanations the authors provide for these observations in the main text. Along these lines: If the WDR5 competes with NANOG for binding DNA, why would a mutation (R153A), which reduces WDR but not DNA binding, result in a lot less binding intensity (Figure 5A)?\n• I find the graphical summary in Fig. hard to read and struggle to link the legend to the displayed items. Could this be less convoluted and more concise?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-Point Responses to Reviewers' Comments\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nAll issues have been addressed. I recommend acceptance for publication.\nWe sincerely thank the reviewer for the positive comments and support.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this paper with an ECR in my team. A regulated switch between prion\nand LLPS state driven by WDR would be fascinating but requires more structural\nevidence, which the authors acknowledge. The authors have added assays to support\nthe claim that WDR5 can nudge NANOG into undergo liquid-liquid phase separation\nand to regulate stem cell transcription. The newly added assays, such as Co-IP, ChIP-\nqPCR, a sequential differentiation assay, and re-analysis of RNA/ChIP-seq data,\nsupport the rational use of C16, the direct cause of epigenetic changes, and\ntranscriptional activation by the WDR5/NANOG LLPS. The newly added\nEMSA/LLPS show that WDR5 competes with DNA for NANOG interactions. I remain\na bit puzzled as to the main mechanistic model and would encourage the authors to\nfurther clarify the graphical summary and the key narrative. I note that a\nWDR5/NANOG appears – at least in 1-to-1 stoichiometry, incompatible with DNA\nbinding – yet, WDR5 binding to NANOG facilitates its binding to target genes. It would\nbe nice if this could be reconciled/explained. We recommend acceptance of this\nfascinating study with only minor remaining suggestions:\nWe thank the reviewer and the co-reviewer for their careful evaluation and are\ngrateful for their positive recommendation. Regarding the reviewer’s inquiry about the\nmechanistic model and the compatibility of WDR5/DNA binding, we have provided a\ndetailed discussion and reconciliation of these observations in our response to the\nspecific comment below. We have also revised the graphical summary (Fig. 7) to clarify\nthis dynamic mechanism as suggested.\n• In Figure 1g, please circle where you bleached to make it more straightforward.\nWe thank the reviewer for this suggestion. We have now more clearly marked the\nbleached regions in Fig. 1g by using thicker white dashed circles.\n• ChIP-qPCR only has 3 technical analyses. Any possible independent replicates?\nWe thank the reviewer for this question and apologize for any ambiguity in our\nprevious description. We would like to clarify that the ChIP-qPCR experiments were\nperformed using three independent replicates. Specifically, for each genotype\n(NANOGWT and NANOGR153A), we carried out the time-resolved differentiation assay\nindependently in three separate mESC cultures, and the samples at each time point were\nharvested and processed independently through chromatin preparation. Thus, the data\npoints represent independent replicates, not technical qPCR repeats from a single\nextraction, and statistical testing was performed across these independent replicates.\nWe have updated the figure legend to state this explicitly.\n• Please ensure statistical analysis is clarified in all legends and relevant methods\nsections.\nWe thank the reviewer for this helpful comment. In line with Nature\nCommunications’ statistical reporting guidance, we have systematically revised all\nfigure legends and the relevant methods sections to provide complete information on\nthe statistical analyses.\n• Please clarify the independent replication (not just repeat recordings) of the new THT\nfluorescence assay and briefly explain its principle (possibly with a small cartoon next\nto the data figure).\nWe thank the reviewer for raising this point. We have now clarified in the legend\nto Fig. 3h that the ThT fluorescence assay was performed as three independent ThT\nexperiments. In addition, we have expanded the methods section to briefly describe the\nassay principle, stating that ThT is a fluorescent dye whose emission increases upon\nbinding to β-sheet-enriched protein assemblies and therefore reports on the aggregation\nstate of NANOG. Given the well-established nature of this assay, we believe this textual\ndescription effectively clarifies the principle.\n• I am still confused as to the main model how WDR5 helps promote the binding of\nNANOG to its target genes when EMSAs suggest WDR5 prevents the HD from binding\nDNA. I cannot follow the explanations the authors provide for these observations in the\nmain text. Along these lines: If the WDR5 competes with NANOG for binding DNA,\nwhy would a mutation (R153A), which reduces WDR but not DNA binding, result in a\nlot less binding intensity (Figure 5A)?\nWe thank the reviewer for the opportunity to clarify this point. The EMSA assay\nisolates a minimal system with purified NANOG HD and short DNA probes to directly\nassess NANOG HD-DNA binding. This setup addresses two specific questions:\nwhether the R153A substitution alters the intrinsic NANOG HD-DNA interaction, and\nwhether WDR5 binding is compatible with NANOG HD-DNA binding in this\nsimplified in vitro context.\nIn the nucleus, however, the functional outcome depends critically on the assembly\nstate of NANOG within the complex chromatin environment. Our data indicate that, in\nthe absence of WDR5, NANOG tends to form β-sheet-enriched, slowly exchanging\nassemblies driven by its C-terminal region. We therefore infer that the R153A mutant,\nwhich impairs WDR5 binding, functionally mimics this \"NANOG-alone\" state and is\nprone to forming non-productive aggregates that are less available for productive\nchromatin engagement. This can explain why R153A exhibits reduced ChIP-seq signals\ndespite intact HD-DNA binding.\nIn the presence of WDR5, we propose that NANOG is remodeled into dynamic,\nliquid-like condensates. Within these condensates, the high local concentration of\nsoluble NANOG and the multivalent nature of the assembly are expected to overcome\nthe direct steric competition observed under dilute EMSA conditions. Instead of a rigid,\npermanent WDR5-NANOG-DNA ternary complex, NANOG-WDR5 association and\nNANOG-DNA engagement can proceed via a dynamic exchange mechanism in which\nNANOG transiently dissociates from WDR5 to engage high-affinity DNA motifs and\nthen re-engages WDR5 and other cofactors. Furthermore, WDR5 acts as a scaffold to\nco-recruit MOF and MLL complexes, establishing a permissive chromatin environment\nthat further stabilizes NANOG occupancy at pluripotency-associated promoters.\nConsistent with this view, our ternary LLPS experiment shows that NANOG,\nWDR5,\nand DNA can co-partition into the same phase-separated droplets. We have revised the\ngraphical summary and figure legend of Fig. 7 to present this mechanism more clearly.\n• I find the graphical summary in Fig. hard to read and struggle to link the legend to the\ndisplayed items. Could this be less convoluted and more concise?\nWe appreciate the reviewer’s comment. In response, we have revised Fig. 7 to\nmake the graphical summary less convoluted and easier to follow.\n1. We simplified the visual design by removing distracting details and focusing\nthe schematic on the two key NANOG assembly states highlighted in our study—an\naggregate-like, less dynamic state (upper/lower panels) versus a WDR5-associated,\nfunctional condensate state (middle panel).\n2. We made the proposed mechanism more explicit by adding a “dynamic\nexchange” arrow in the WDR5 panel and by strengthening the visual contrast between\nthe NANOG-alone assemblies and the WDR5-remodeled condensates.\n3. We revised the figure legend to improve the correspondence between the text\nand the schematic, making it easier to link the legend to the visual elements.\nWe hope that these changes improve the readability of Fig. 7 and clarify the\nproposed model.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe sincerely appreciate your time and effort in reviewing our manuscript and\nthank you for your thoughtful comments and constructive contribution to the review\nprocess."}]

Decision letter