Interplay of condensation and chromatin binding underlies BRD4 targeting

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4–chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

Representative segmentations of nuclear outlines (blue) and puncta (black), with marked Yes PS (Y) / No PS (N) calls from a field of BRD4 FL Corelet cells in -JQ1 (top) and +JQ1 (bottom) conditions, before light activation (Pre-act, left), and during light activation (Act., right).Y* is a nucleus called as 'Yes PS' in -JQ1 that entered mitosis before the +JQ1 images were taken and so was excluded from the analysis.See also Movie S1.F. Simulation Corelet BRD4FL phase diagrams with chromatin at three acetylation levels; 20%, 30% and 40% of tails acetylated.The critical Corelet valence for the formation of finite-size chromatin-associated condensates depends on the fraction of acetylated histone tails in the coarsegrained simulations.Error is SD.ns = not significant by one-way ANOVA.D. Count of condensates per nucleus as a function of BRD4 FL -mCh expression level in living cells.An expression level gate is used to bound the expression level of cells for calculating average count in Fig. 6E, the same as was used in Fig. 1.E. Nucleation rate as a function of expression level is shown, with the expression level gate shaded.

Supplementary Information: Coarse-grained simulation parameters
In this section, we describe the coarse-grained force field and the simulation environment.Parameter values are provided in the tables below.
Non-bonded interactions between particles/blobs of types 1 and 2 are modeled via a combination of a repulsive WCA potential S1 and an attractive Gaussian potential.For the WCA interactions, the length scale is σ12 = (σ1 + σ2) / 2, where σ1 and σ2 are the diameters of the hard cores of the particles, and the energy scale is set to 1 kT.For the Gaussian interactions between polymer blobs, the potential takes the functional form Ugauss(r) = -A exp(-Br 2 ) for r < rcut, where the length-scale parameter is B = 0.8 / [(Rg,1 + Rg,2) / 2] 2 and Rg,1 and Rg,2 are the radii of gyration of the blobs in dilute solution S2 .The cutoff distance is set to rcut = 1.5 (Rg,1 + Rg,2).The interaction-strength parameter, A, is chosen to be a constant value of 8 kT for all interactions among N-terminal and C-terminal BRD4 blobs; however, because of the differing σ and Rg values of these species, the resulting well depths for the summed WCA and Gaussian potentials differ as well (Umin,NN/kT = -0.75,Umin,CN/kT = -1.2,Umin,CC/kT = -1.9).These choices lead to the reproduction of the experimentally determined phase diagram as demonstrated in the main text.The interaction-strength parameter, A, between acetylated histone tails and the N-terminal BRD4 blob is varied to represent either weakly attractive interactions with A = -25 kT (resulting in a second virial coefficient for the blob-blob interactions of b2 = B2/Vendog ~ -0.5, where Vendog =  (4/3 ) ⟨Rg, endog⟩ 3 ) or strongly attractive interactions with A = -50 kT (resulting in b2 ~ -25).

Figure S1 .
Figure S1.JQ1 does not affect BRD4 ∆N puncta, related to Figure 2 A. Schematic diagram of the Corelet system, light-induced oligomerization platform that triggers droplet formation of phase-separation-prone protein regions in living cell nuclei.B. Representative images of BRD4 ∆N light-induced Coreletcondensates +/-JQ1 treatment.Quantification of count (C) and area (D) of BRD4 ∆N Corelet condensates +/-JQ1 in 3 trials of 25 cells each.Student's t-test.E. Single-cell BRD4 FL Corelet-condensate count per nucleus (pink) before JQ1 treatment (X-axis) and after JQ1 treatment (Y axis).Each point is one nucleus.Number of BRD4 ΔN Corelet condensates per nucleus (black) are unaffected by JQ1 and lie on the diagonal.Quantification of count (F) and area (G) of BRD4 FL condensates in cells expressing Corelet components but without light activation.Error bars represent SEM across four trials of 25 cells each, **** p = 0.0001 by Student's t-test.

Figure S2 .
Figure S2.Valence-dependence of BRD4 Corelet condensate volume, related to Figure 3 A-B.Experimental phase diagrams -/+ JQ1 recolored by average volume of condensed phase per nucleus.C. Phase diagram condensate volume data from -/

Figure S3 .
Figure S3.The ratio of homogeneous to heterogeneous nucleation is dependent on supersaturation, related to Figure 5 A. Pie charts representing the probability of on-(orange) and off-chromatin (blue) nucleation in Endogenous and Corelet simulation systems at three supersaturation levels (highest at top).Supersaturation, S, is defined as the ratio of the applied pressure to the equilibrium coexistence pressure.B. Nucleation rates and C. delay times as a function of the supersaturation.Error bars represent the standard error.

Figure S4 .
Figure S4.Epigenetic modifying drugs alter acetylation but not BRD4 expression level, related to Figure6A.Representative images of immunofluorescence of U2OS cells treated with DMSO (control), 1 uM A485, or 100 nM TSA for 24 hours, stained with antibodies that recognize BRD4 (green) and H3K27Ac (magenta).B.Quantification of H3K27 Acetylation intensity across 3 biological replicates of 25