Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index

Abstract

Background: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent.

Results: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment.

Conclusions: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

Fig. 1

Hi-MS effectively enrich chromatin-associated proteins. A Schematic of Hi-MS. B The examples of GGCC distribution in the gene promoter region. Top, RUNX1; bottom, POLE4. These two regions were selected as represents of super enhancer and typical enhancer [23]. C The distribution of human genome sequence GATC and GGCC proximate to H3K27ac and EZH2 binding peaks. D Chromatin-associated proteins are effectively enriched by Hi-MS compared with undigested control. E MS/control fold enrichment of NUPs agreed with their spatial location. NPC model was adapted from Ref [24]

Fig. 2

AICAP consists with known physicochemical properties involved in phase separation. A Schematic of 1,6-HD treatment and definition of AICAP. B Scatter plot of proteins with AICAP values 0~2 captured by Hi-MS. C The disorder compositions of 6 groups of proteins. IDR, intrinsically disorder region; LCD, low complexity domain. D The Spearman correlation between AICAP and corresponding residue percentage in proteins with AICAP < 1. Each column panel refers to residue percentage in different regions (whole sequence, whole sequence minus IDR, IDR, and PLD). PLD, prion-like domain. E The LLPS predictor scores of 6 groups of proteins

Fig. 3

Comparison between AICAP and proteins in biomolecular condensates. A Distribution of AICAP values of proteins in different condensates. B GSEA enrichment of “Nuclear puncta” [35] using AICAP ranked proteins. C Distribution of AICAP values of proteins annotated to be of different material states. D Distribution of AICAP values of self-assembling (Self) and partner-dependent (Partner) proteins

Fig. 4

Experimental validation of AICAP. A Immunofluorescence of MED1, RNAPII, and BRD4 before (−) and after (+) 1,6-HD treatment. Scale bar indicates 5 μm. B ChIP-seq signal enrichment of BRD4, MED1, and RNAPII at regions defined as super enhancer in mESC before (1,6-HD−) and after (1,6-HD+) 1,6-HD treatment. The decreased percentage of signal is noted. Data resource [6]. C FRAP experiments on 7 proteins in HeLa cells. Scale bar indicates 5 μm. D FRAP curves of proteins in C. E Calculated parameters of FRAP experiments using chemical interaction model (top). The mobile fraction was fitted using linear regression (bottom)

Fig. 5

Active transcriptional chromatin regions and associated proteins are more sensitive to 1,6-HD treatment. A Intra-chromosome interactions of 15 types epigenome chromatin states after 1,6-HD treatment. B The AICAP values of hallmark proteins in different functional categories. C Gene ontology biological process enrichment analysis of proteins. Proteins were divided into 6 groups based on AICAP (Y-axis). Number of proteins was noted in the corresponding cell

Fig. 6

A/B compartment of chromatin exhibited different sensitivities to 1,6-HD treatment. A A (red) and B (blue) compartments can be classified into 4 compartment change-types as strengthened, stable, weakened based on their PC1 value ratio (1,6-HD+/−). 20% was chosen as the threshold for distinguishing stability or not. B The fraction of four kinds of compartment change-types. C Contact probability between compartments along genomic distance. D Examples of strengthened/stable compartments and corresponding nuclear speckle/lamina TSA-seq [39] plots. The plotting of log2 ratio of TSA read density versus input read density was used to measure the distance from a chromatin region to a specific nuclear condensate. Chr2, 0–80 M. E AICAP values of nuclear speckle and lamina associated proteins. Data resource [40, 41]

Fig. 7

Active chromatin TADs/loops are more sensitive to 1,6 HD treatment. A Examples of stable/lost TAD boundaries. example “a, b” lost, example “c” stable. B Aggregation analysis of CTCF/SMC3 ChIP-seq peaks at stable/lost TAD boundaries. C Intra-/inter-TAD interaction changes (1,6-HD(+)/(−) ratio) in different subcompartments. Interactions between different subcompartments were skipped. D Examples of loop domain "b", and DNA stripe "a" after 1,6-HD treatment. E Schematic illustration of loop anchor interaction, loop domain, DNA stripe, and stripe anchor. F Left and right stripe signal aggregation at 10 kb resolution. G Aggregation analysis of loop domain (left) and loop anchor interaction (right) signal changes after 1,6-HD treatment. “both/no stripe” indicates both or no overlap between loop anchor and stripe anchor