Modulation of DNA rheology by a transcription factor that forms aging microgels

TL;DR

This work addresses how a transcription factor can regulate genome dynamics by forming aging, gel-like condensates rather than inducing large-scale chromatin remodeling. The authors combine microrheology, cryo-EM, mass photometry, DLS, and Mpipi coarse-grained simulations to characterize NANOG fluids. They show that WT NANOG forms self-limited micelle-like clusters (~$30$ proteins) with the WR domain driving oligomerization; in the presence of entangled $\lambda$DNA, these clusters bridge DNA and drive rheology, with gel-like behavior emerging around $6\, \mathrm{h}$ and viscosity increasing by ~$10^4$ over $12\, \mathrm{h}$. W10A cannot oligomerize and lacks aging, while N51A binds DNA more weakly and shows reduced aging, highlighting the complementary roles of WR and DNA binding. This mechanism suggests a physical basis for transcriptional regulation via stabilization of genome dynamics and potential memory in gene networks, without requiring large-scale chromatin remodeling.

Abstract

Proteins and nucleic acids form non-Newtonian liquids with complex rheological properties that contribute to their function in vivo. Here we investigate the rheology of the transcription factor NANOG, a key protein in sustaining embryonic stem cell self-renewal. We discover that at high concentrations NANOG forms macroscopic aging gels through its intrinsically disordered tryptophan-rich domain. By combining molecular dynamics simulations, mass photometry and Cryo-EM, we also discover that NANOG forms self-limiting micelle-like clusters which expose their DNA-binding domains. In dense solutions of DNA, NANOG micelle-like structures stabilize intermolecular entanglements and crosslinks, forming microgel-like structures. Our findings suggest that NANOG may contribute to regulate gene expression in a unconventional way: by restricting and stabilizing genome dynamics at key transcriptional sites through the formation of an aging microgel-like structure, potentially enabling mechanical memory in the gene network.

Figures (4)

• Figure 1: NANOG solutions form macroscopic aging gels through the intrinsically disordered WR domain.a. Mouse NANOG primary structure: N-terminal domain (ND), homeodomain (HD), C-terminal domains (CD1,2), tryptophan repeat (WR). Amino acid sequences of HD and WR mutants are shown. WT residues are shown in black; mutated residues are shown in red. b. EMSA of NANOG mutants at different DNA:protein ratios. Each lane contains 5 nM of a 26-bp dsDNA oligomer. (M=monomer, C=DNA-NANOG complex, O=oligomer). c. Photographs of $\sim$mL samples of purified NANOG mutants aged at $\sim$1 mM overnight at 37$^\circ$C. WT NANOG and N51A form a non-pipettable gel, whereas W10A remains liquid. Scale bar is 2 mm. d-e-f. Microrheology of the 3 mutants over the course of 12h at 37$^\circ$C. g. The samples' viscosity showing a 10'000-fold increase for WT and N51A and absence of aging for the W10A mutant lacking the tryptophan residues. h. Elastic ($G^\prime$) and viscous ($G^{\prime \prime}$) moduli for 3 ageing times. i. Elasticity of the sample taken as $G_p = G^\prime(\omega=10$ Hz) showing a significant gelation for the WT and N51A proteins over the course of 12 hours, while W10A displays no increase in elasticity.
• Figure 2: NANOG forms micelle-like self-limited clusters through their WR. a. Snapshot from equilibrated simulations of NANOG made of $M = 50$ proteins, each $N = 305$ amino acids long at $T = 300~\mathrm{K}$ and $\rho = 0.1~\mathrm{g/cm^3}$. Green, orange, pink and blue represent the different NANOG domains as in Fig. \ref{['main_v2:fig:aging_protein']}a. b. Reconstruction of the system in (a) through periodic boundary conditions showing two disjoint clusters. c. Time evolution of the number of molecules forming the two main clusters and the third smaller cluster of molecules. d. Distribution of distances between the cluster COM to the residues in the different domains: N-terminal domain (ND, orange), homeodomain (HD, blue) and tryptophan repeat (WR, pink). e. Simulated forced mixing of clusters (colored red and blue) and re-equilibration leading to the re-establishment of two disjoint clusters made of a different combination of molecules. f. Representative cryo-EM motion corrected micrographs (scale bar 50 nm). g. 2D classes of objects showing highly heterogeneous and "disordered" assembly. Box size is 364 Å, mask is 240 Å, approximately the size of $\sim$20-30 NANOG molecules. g. Mass photometry showing peaks at weight corresponding to $\sim$22-25 NANOGs per cluster. h. DLS quantifying the hydrodynamic size of clusters in samples of NANOG incubated at 37$^\circ$C and different times.
• Figure 3: NANOG micelle-like clusters act as crosslinkers of entangled DNA. a-c. MSD of tracers at varying DNA:protein stoichiometries in a solution of $\lambda$DNA at 7.9 nM and (a) WT, (b) W10A and (c) N51A, at different concentrations. Insets depict schematics of NANOG structures in the presence of $\lambda$DNA . d. Viscosity of DNA–protein solutions as a function of protein concentration.
• Figure 4: Aging of NANOG-DNA solutions suggest a rheological gene regulation. a. Time resolved MSDs of tracers' embedded in solutions of NANOG (7.9 $\mu$M) and $\lambda$DNA (7.9 nM) for 12 hours at 37$^{\circ}$C. b. Normalized viscosity of $\lambda$DNA -NANOG solutions obtained for different mutants. c. Time resolved elastic modulus $G^\prime$ obtained from the MSD via the GSER. d. Solution's elasticity $G_p = G^\prime (\omega=10 \textrm{Hz})$ as a function of ageing time.