FAIRE‐MS reveals mitotic retention of transcriptional regulators on a proteome‐wide scale

Mitosis entails global and dramatic alterations, such as higher‐order chromatin organization disruption, concomitant with global transcription downregulation. Cells reliably re‐establishing gene expression patterns upon mitotic exit and maintaining cellular identities remain poorly understood. Previous studies indicated that certain transcription factors (TFs) remain associated with individual loci during mitosis and serve as mitotic bookmarkers. However, it is unclear which regulatory factors remain bound to the compacted mitotic chromosomes. We developed formaldehyde‐assisted isolation of regulatory elements‐coupled mass spectrometry (FAIRE‐MS) that combines FAIRE‐based open chromatin‐associated protein pull‐down and mass spectrometry (MS) to quantify the open chromatin‐associated proteome during the interphase and mitosis. We identified 189 interphase and mitosis maintained (IM) regulatory factors using FAIRE‐MS and found intrinsically disordered proteins and regions (IDP(R)s) are highly enriched, which plays a crucial role in liquid–liquid phase separation (LLPS) and chromatin organization during the cell cycle. Notably, in these IDP(R)s, we identified mitotic bookmarkers, such as CEBPB, HMGB1, and TFAP2A, and several factors, including MAX, HMGB3, hnRNP A2/B1, FUS, hnRNP D, and TIAL1, which are at least partially bound to the mitotic chromosome. Furthermore, it will be essential to study whether these IDP(R)s through LLPS helps cells transit from mitosis to the G1 phase during the cell cycle.


| INTRODUCTION
During mitosis, cells undergo profound changes in their chromosome organization and nuclear architecture, including nuclear envelope breakdown, chromosome condensation, and loss of long-range interactions between distal enhancers and proximal promoters. 1,2 Furthermore, mitotic chromosomes are characterized by the eviction of numerous proteins, including RNA polymerase II (RNA Pol II) (through phosphorylation of its subunit Gdown1), components of the general transcription machinery, and sequence-specific transcription factors (TFs). 3 Hence, the transcription of mitosis is typically silenced. However, how daughter cells can accurately reestablish parental transcriptional memory to maintain cell identity and function remains unclear.
A highly sensitive technique called EU-RNA-seq was developed to determine and quantify mitotic transcription globally. Approximately 30% of transcripts (8000/28 000) displayed measurable expression in the mitotic population compared with the asynchronous cell population. 4,5 This result indicates that mitotic transcription is largely reduced but not strictly transiently silenced. Although many gene transcription patterns are mostly retained at a low level through mitosis, they may be essential for transcriptional memory propagation from mother to daughter cells. 1 This new model also showed that the mitotic chromosome does not exhibit extreme condensation, as previously reported. 4 Further studies on many different cell types have indicated that the heritability of defined gene expression programs might rely on mitotic bookmarking, which enables proper re-expression of genes postmitosis. [6][7][8][9][10][11] The two mechanisms involved in mitotic bookmarking are TFs and histone modifications, retained at a subset of their locations during mitosis. 1 Collectively, these results suggest that mitotic bookmarking, especially TFs, may contribute to the low level of mitotic RNA synthesis and faithful propagation of cell identity after cell division.
More recently, genome-wide chromatin accessibility profiles using DNase I sensitivity assay coupled with high-throughput sequencing (DNase-seq) and an assay for transposase-accessible chromatin with sequencing (ATAC-seq) comparing asynchronous and mitotic cells indicated that chromatin accessibility is globally preserved and is only locally modulated during mitosis. 3,12 These studies also demonstrate that maintaining local chromatin accessibility is possible with the basal expression of mitotic genes.
Formaldehyde-assisted isolation of regulatory elements (FAIRE) is an alternative method for identifying genomic open chromatin regions and has proven successful in many eukaryotic cells and tissue types. [24][25][26][27][28][29] By integrating FAIRE-seq and Hi-C, researchers recently developed a method called open chromatin enrichment and network Hi-C (OCEAN-C) to detect global open chromatin interactions. 30 This technique has been successfully used to reveal functional regulatory elements and chromatin architecture variations during wheat evolution. 31 In this study, we developed a new in vitro method called FAIRE-MS that combines FAIRE-based chromatin-associated protein pull-down and highresolution mass spectrometry (MS). We utilized it to quantify changes in chromatin-binding transcriptional regulators across the interphase and mitotic phases of the A549 human cell cycle. Our results provide a rich data set of transcriptional regulators retained on mitotic chromosomes, including the widespread retention of TFs.

| FAIRE-seq
FAIRE-seq was performed according to the protocol described by Simon et al. 24 with some modifications to the method. Briefly, A549 cells were fixed for 5 min with 1% formaldehyde at room temperature (RT) and then quenched with 2.5 M glycine (final concentration 0.125 M). The fixed cells were scraped, collected, and washed twice with cold PBS. Fixed cells (1 × 10 7 ) were resuspended in 1 ml cold lysis buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.1% NP-40, 5% sucrose, and 1× protease inhibitors) and incubated on ice for 10 min. The cells were homogenized using a Dounce homogenizer with 10 smooth strokes using a tight pestle (30 strokes) on ice, then centrifuged to collect cell nuclei. The nuclear pellet was resuspended in 2 ml lysis buffer A (10 mM Tris-HCl pH 8.0, 2% Triton X-100, 1% SDS, 100 mM NaCl, and 1 mM EDTA). Chromatin DNA was sheared to approximately 200-400 bp fragments using a Covaris sonicator. Next, the samples were extracted with phenol/chloroform, precipitated with ethanol, and treated with RNase A. The samples were de-crosslinked using proteinase K, incubated overnight at 65°C, and the DNA was purified using a Qiagen Mini Elute kit. Finally, FAIRE DNA was amplified to 100-200 ng of starting materials, which was prepared according to the TruSeq Illumina protocol, enriched by PCR amplification (15 cycles), and sequenced on Illumina HiSeq2000.

| FAIRE-seq data processing and analysis
Data processing and analysis were performed according to the protocol described by Simon et al. 24 with minor modifications to the method. Briefly, sequences were mapped to hg38 using an algorithm such as Bowtie with parameters "--local --very-sensitive-local-no-unusualno-mixed-no-discordant -I 10 -× 700." Peak detection was performed using the MACS2 software suite with the added parameters "-g dm -nomodel -shiftsize 50 -q 0.01." Gene ontology annotation analysis was performed using DAVID (ver. 6.7). De novo motif identification was performed using MEME ver. 4.3.0. Next, we used TOMTOM in the MEME suite to compare a database of TFs with motifs.

| Nuclear extract (NE) and whole-cell extract (WCE) preparation
NE was prepared from A549 cells as previously described, with minor modifications. 32 Briefly, cells were suspended in 10X volumes of hypotonic buffer (10 mM Tris-Cl pH 7.3, 1.5 mM MgCl 2 , 10 mM KCl, and 1 × PMSF) for 10 min on ice. The samples were centrifuged at 1000g for 15 min at 4°C, and pellets were dounced 15 times to break the cell membranes further. Then, all samples were centrifuged at 4000g for 5 min to separate the nuclear and nonnuclear fractions. The nuclear pellets were resuspended in 0.5× volumes of low salt buffer (20 mM Tris-Cl pH 7.3, 1.5 mM MgCl 2 , 20 mM KCl, 0.2 mM EDTA, and 1 × PMSF) and dounced another 10 strokes to resuspend the nuclei. Another half volume of high salt buffer (20 mM Tris-Cl pH 7.3, 1.5 mM MgCl 2 , 1.2 M KCl, 0.2 mM EDTA, and 1 × PMSF) was added dropwise while the solutions were stirred to extract nuclear proteins. The solutions were stirred at 4°C for an additional 30 min after high salt addition and then centrifuged at 25 000g for 20 min at 4°C. The supernatant containing NE was dialyzed against BC-150 (20 mM Tris-Cl pH 7.3, 150 mM KCl, 0.2 mM EDTA, 20% glycerol, and 1 × PMSF) for 1 h at 4°C. WCE was extracted using cell lysis buffer for western blotting and an IP kit (Beyotime). Finally, the resulting NE and WCE were stored at −80°C.

| FAIRE DNA-protein pull-down assay
Biotin pull-down was performed using the purified DNA probes. Dynabeads® M-280 Streptavidin (Invitrogen) was mixed with a DNA probe for 15-20 min at RT with rotation. As negative controls, beads without FAIRE DNA probes (negative control amplification using Biotin-M13F and M13R as primers and pMD19-T vector as a template) were added in the same amount. The sequences of the CTCF binding site primers used were as follows 33,34 : H19-ICR-F: 5′-GTCCACGAGGTACCAGCCTA-3′, H19-ICR-R: 5′-AAGTGGGAGTTGTGGTGAGG-3′; CBS-6-F: 5′-CCA CATCCACCTGTCACTT-3′, CBS-6-R: 5′-CTGTTT CACATCCATCGCA-3′. Then beads-biotinylated DNA complexes were washed with BC-150 buffer once and blocked with BC-150 buffer containing 1.0% BSA and 0.5 mg/ml sperm DNA overnight at 4°C with rotation. Before NE (interphase) or WCE (mitotic phase) was added to the complexes, NE or WCE was spun down at 100 000g in a Beckman Optima ultracentrifuge (TLA100 rotor) for 20 min at 4°C. The supernatant was then transferred to a new 1.5 ml tube and kept on ice. The concentration of the supernatant was measured using a bicinchoninic acid protein assay kit (TIANGEN). NE (from interphase) or WCE (from mitosis) was added to the prepared beadbiotinylated DNA complex and incubated for 2 h at 4°C with rotation. The supernatant was discarded, and the beads were washed twice with NETN (20 mM Tris-Cl pH 8.0, 50 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and then three times with PBS.

| MS sample preparation
For samples subjected to SDS-PAGE analysis, beads were resuspended in 20 μl 1× SDS sample buffer and boiled for 10 min. The samples were then loaded onto a 12% SDS-PAGE gel and run to a 1/3 length. The gel was minimally stained with Coomassie Brilliant Blue or silver and briefly washed in 5% ethanol/10% acetic acid solution. The protein bands of interest were excised using SDS-PAGE. Next, bands were washed with 25 mM NH 4 HCO 3 /50% acetonitrile followed by dehydration with acetonitrile and reduction with 0.1 M NH 4 HCO 3 /10 mM TCEP solution at 56°C for 30 min and finally alkylated in the dark by 0.1 M NH 4 HCO 3 /30 mM iodoacetamide (IAA) at RT for 30 min followed by washing, dehydrating, and drying by a vacuum centrifuge. For digestion, gel fragments were rehydrated by incubating with 20 ng/μl sequencing grade trypsin in 0.1 M NH 4 HCO 3 on ice for 45 min followed by overnight incubation at 37°C. The following day, the digested peptides were extracted, desalted, and resuspended in a buffer (1% formic acid and 1% acetonitrile solution).

| Nano-UPLC-MS/MS analysis
A linear gradient from 98% A (0.1% formic acid and 2% acetonitrile solution) to 35% B (0.1% formic acid in acetonitrile) over a 75-min period at a flow rate of 300 nl/min was applied. For identification, peptides were fragmented via collision-induced dissociation and analyzed using an LTQ-Orbitrap Velos (Thermo Fisher) with a full MS spectrum (m/z = 300-1600) using an automatic gain control (AGC) target of 1 × 10 6 . The top 20 most intense ions were selected for higher-energy collisional dissociation (HCD) fragmentation, and MS/MS spectra were generated with an AGC target of 1 × 10 4 at a resolution of 30 000 and a dynamic exclusion time of 50 s. The data were analyzed using MaxQuant software and label-free quantification (LFQ). The mass tolerance of the precursor ions was set to 20 parts per million (ppm). The product ion tolerance was set to 50 milli mass unit (mmu) on the Q Exactive instrument and ±0.5 Da on the Fusion instrument. Up to two missed cleavages are allowed for protease digestion. N-terminal protein acetylation and methionine oxidation were used as alternative modifications. The cutoff ion score for peptide identification was 20, and proteins with more than one peptide (1% false discovery rate at the peptide level) were selected for further analysis. The quantities of identified proteins were estimated using a label-free and intensity-based absolute quantification (iBAQ) approach.
First, chromatin-enriched proteins in each individual phase were defined as proteins with a p value of ≤ .05 and a log2 fold change (fc) of >1.0, compared with the control. Then, we used a limma-trend approach to calculate the significance of the changes between the different components. 35 Fold changes were estimated by fitting a linear model on the log2-transformed values for each protein using the lmFit function and then smoothed using empirical Bayes with the function eBayes. The resulting p values were adjusted using the FDR approach. We calculated adjusted p values and fold changes between mitosis and interphase. Mitosis (M)-enriched proteins were defined with an adjusted p value of < .1 and a log2(fc) of > 1.5; interphase (I) enriched proteins were defined with an adjusted p value of < .1 and a log2(fc) of <−3; IM-common proteins were defined with adjusted p-value > .01, and −2 < log2(fc) < 1. Gene ontology (GO) enrichment analysis was performed using the clusterProfiler package. IDPs were identified by using the following URL: https:// metap redict.net.

| Workflow of FAIRE-MS and experimental strategy for performing FAIRE-seq on cells in mitosis versus interphase
Although many transcriptional regulatory factors are still associated with mitotic chromosomes, proteome-wide profiling of transcriptional regulators by open chromatin DNA binding during mitosis is lacking. FAIRE allows unbiased identification of potential regulatory elements without requiring prior knowledge of binding factors. 27 In our workflow, we first isolated FAIRE DNA, cloned it into a pMD19-T vector, and prepared FAIRE DNA probes by PCR amplification with biotinylated primers. Biotinylated PCR products were enriched with streptavidin beads and then incubated with nuclear extracts (NE) or whole-cell extracts (WCE). The pulled-down FAIRE DNA-bound proteins were digested with trypsin and identified using MS ( Figure 1A). Studying mitotic transcriptional machinery requires the preparation of mitotic cells at high purity because contaminating interphase cells do not reflect the exact mitotic chromosome configuration. Therefore, we applied a previously established protocol combining synchronization and shaking off to collect pure mitotic A549 cells 22 (Figure 1B). To determine the mitotic cell purity, we used a combination of four different methods; flow cytometry, DAPI staining, western blotting, and immunofluorescence (IF) using an antibody against the mitosis-specific H3Ser10ph epitope to obtain a mitotic population at approximately 98% purity 22 (Figures 2A,B and S1). Next, we performed FAIRE-seq as previously described. 24 FAIRE-seq libraries were generated in two independent biological replicates for asynchronous (containing approximately 95% interphase cells, hereafter referred to as "interphase") and synchronized mitotic cells, yielding approximately 132 million interphase total mapped reads and approximately 85.5 million mitosis total mapped reads (Table S1). In addition, the pairwise Pearson's correlation analysis of FAIRE-seq data sets showed a strong correlation between two biological replicates (Pearson's correlation coefficient in interphase was 0.901 and mitosis was 0.789) ( Figure 2C). Further, we compared the data of FAIRE-seq and ATAC-seq, 36 and according to the results, they are consistent in the changes of global and individual signal patterns ( Figures 2D and S2). These results provide a solid basis for further analysis using these methods.

| Distal cis-regulatory elements preserve accessibility more than promoters
To determine the degree of genomic cis-regulatory elements that remain accessible to transcription regulators during mitosis, we analyzed the genome-wide peaks using MACS2 software. We identified 73 051 peaks in the interphase cells and 25 594 peaks in the mitotic cells. The results showed that a subset of DNA accessibility was maintained during mitosis. In addition, we found that only 5% corresponded to promoters, while 17% in interphase were maintained during mitosis, and intergenic regions changed from 44% (interphase) to 63% (mitosis) ( Figure 3A). Previous studies have indicated that DNaseseq is preferred to identify promoters, whereas FAIRE-seq may detect some distal regulatory elements, such as enhancers. 24 These results may be related to the preference for the method used to some extent. To further examine the retention of mitotic cis-regulatory elements, we considered the intersection of all peaks across the two biological replicates as the basis for the following analysis. We obtained three distinct categories: interphase-occupied sites (I-OS), interphase and mitosis-occupied sites (IM-OS), and mitosis-occupied sites (M-OS) ( Figure 3B). In these three categories, we found that promoter regions sequentially decreased from I-OS to IM-OS to M-OS, whereas intergenic regions significantly increased, especially in the IM category, which accounted for approximately 70% ( Figure 3C). However, the individual regulatory locus dynamics during the transition from interphase to mitosis remain unclear. Thus, the results shown in Figure 3D show that the peaks display diverse patterns of interphase to mitosis dynamics in DNA accessibility.
Motif search in the three categories confirmed that some ubiquitously expressed TFs, such as CTCF, BORIS, and ZNF143, and some TFs related to the survival status of A549 cells, such as Fra1/AP-1 and BATF, were significantly enriched in FAIRE-seq peaks ( Figure 3E). Moreover, many TF motifs were enriched among these three categories (Table S3). Gene ontology (GO) annotation analysis indicated that I-OS and IM-OS are related to DNA binding and regulation, strongly consistent with the function of FAIRE cis-regulatory elements ( Figure 3F).

| Widespread retention of transcription regulatory factors on mitotic chromosome
We performed a FAIRE DNA pull-down assay to capture FAIRE DNA-associated proteins. We first used CTCF binding sites (H19-ICR and CBS6) and FAIRE DNA to pull down the CTCF protein by western blotting to evaluate the efficiency of our approach. As results, both CTCF binding sites and FAIRE DNA probes could effectively pull down CTCF protein ( Figure S3).
Next, we independently performed FAIRE-MS on A549 cells in interphase and mitosis for three biological replicates (Table S4). Overall, our FAIRE-MS assay identified a total of 1745 proteins in interphase, 1401 proteins in mitosis, and 1454 proteins in negative control, respectively ( Figure S4A). Data quality control in the three groups showed that our FAIRE-MS experiments were highly reproducible between replicates ( Figure S4B-D). Furthermore, the heatmap showed that the protein fold-enrichment of the three groups was different ( Figure S5A). The result of differential expressed proteins (DEPs) analysis showed the proteins were significantly enriched in different phase during the cell cycle ( Figure S5B,C).
In order to investigate the proteins retained on the mitotic chromosome, we used a limma-trend approach to calculate the significance of changes between interphase and mitosis. 35 After determining the significant changes, we found that 189 interphase regulatory factors were enriched in the mitotic chromosome (IM) (adjusted p value > .01, and −2 < log2(fc) < 1) ( Figure 4A and Table S5). In addition, GO analysis indicated that these candidate proteins were enriched in DNA and/or RNA binding ( Figure 4B). We first focused on TFs, and transcriptional coregulators maintained during mitosis because they are vital for controlling cellular identity. Previous studies have shown that several sequence-specific TFs remain mitotic bookmarkers for subsequent rapid gene activation. In this list, we identified a total of 15 TFs and transcriptional coregulators that are enriched in chromatin during mitosis, such as TATA-box binding protein associated factor 15(TAF15), CCAAT enhancer-binding protein beta (CEBPB), high mobility groups (HMGA2, HMGB1, and HMGB3), transcription factor AP-2 alpha (TFAP2A), and MYC-associated factor X (MAX) ( Table S5). When comparing previous results, we found that three mitotic bookmarking factors, CEBPB, 37 HMGB1, 14 and TFAP2A, 38 were identified in our FAIRE-MS data sets. We further identified significant enrichment of a large group of RNA or DNA-binding proteins, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), which bind together with several TFs and other RNA-binding proteins (RBPs) to promoter and enhancer sequences to direct transcription, and DEAD-box proteins (DDX) and damage-specific DNA-binding proteins (DDB), which play a critical role in transcription regulation and DNA repair. We also identified two proteins, HIST1H2AJ and HLTF, that can remodel chromatin accessibility. We collectively identified several groups of transcriptional regulators retained on the mitotic chromosome.

| IDP(R)s are widely enriched
during the cell cycle IDP(R)s have received much attention in recent years. Generally, IDPs tend to be involved in protein and DNA/ RNA binding and play vital roles in gene expression regulation, chromatin organization maintenance, and LLPS initiation. 39,40 GO enrichment analysis indicated that the proteins identified by FAIRE-MS were significantly associated with DNA/RNA binding ( Figure 4B). Previous studies also indicated that IDPs are commonly distributed in human-encoded proteins. 41,42 We next investigated the enrichment degree of IDP(R)s in our protein list (Table S6). Interestingly, IDP(R)s were significantly enriched in I, IM, and M groups compared with control and others (significantly enriched in chromatin, but no cell cycle stage-specific feature) groups ( Figure S6A). We also found that the proportion of long IDRs (≥30 amino acids (aa), class III, and class IV) in I, IM, and M groups were significantly higher than that in the control group ( Figure 5). The sequences of IDPs are often repetitive and are enriched in glycine, polar sidechains (Gln, Asp, and Ser), positively charged sidechains (Arg and Lys), negatively charged sidechains (Asp and Glu), and aromatic sidechains (Phe and Tyr). 43 Meanwhile, we found that there were differences in repetitive sequence and amphipathic properties among them ( Figure S6B,C). These results suggest that proteins in I, IM, and M groups are more likely to belong to IDP(s) and involve in LLPS and further possibly with changes in chromatin state during the cell cycle (compared with "others group"). Additionally, further analysis also found that the proportion of IDP(R)s in I and M is higher than that in IM ( Figure S6A). However, TFs/transcription factories are significantly enriched in IM 44 ( Figure S7 and Table S7). Thus, relative to participating in LLPS, TFs/transcription factories in the IM group, as potential mitotic bookmarking, may play a vital role in transcription regulation, maintenance, and timely reactivating gene expression as cells re-enter the G1 phase of the cell cycle. Taken together, IDP(R)s are more likely to bound to mitotic chromosomes, a property that suggests that they may contribute to epigenetic inheritance. However, their role in the transition between cell cycle phases needs further investigation.

| IF analysis demonstrates that several transcription regulatory factors remain bound to mitotic chromosomes
To further investigate whether IM proteins obtained from our FAIRE-MS datasets were truly retained and bound to chromatin during mitosis, we first randomly detected the contents of six proteins by western blotting, including MAX, HMGB3, hnRNP A2/B1, FUS, hnRNP D, and TIAL1. Our results showed that some proteins were largely retained in mitoses, such as MAX, FUS, hnRNP D, and TIAL1, and some proteins were reduced during mitoses, such as hnRNP A2/B1, and some were increased in mitosis, such as HMGB3 ( Figures 6A and S8A). However, previous studies have shown that some transcription regulatory factors, especially TFs, may not be truly bound to DNA but remain in the vicinity of DNA. 11 Therefore, studying further and verifying these results using IF is necessary. After IF staining, we found that MAX was still tightly bound to the mitotic chromosome, and the other five regulatory factors were partially bound to mitotic chromosome ( Figures 6B and S8B). Considering these and our findings, we conclude that an increasing number of proteins in our IM list may be retained and bound to the mitotic chromosome during mitoses, such as SUB1, TFE3, and YBX1.

| DISCUSSION
Developing a technology that can efficiently enrich proteome-wide DNA-binding transcriptional regulators, especially the low abundance of TFs, remains a challenge. Several methods have been developed in recent years to use a concatenated tandem array of transcription factor response elements (catTFRE) or hormone response elements (HREs) as affinity reagents to enrich TFs and transcriptional coregulators in vitro that show more efficiency than the control. 32,45 However, their limitation remains their inability to capture a wider range of TFs genome-wide.
FAIRE does not require enzymes, such as DNase or Tn5 transposase, which reliably identify active regulatory elements. In this study, based on FAIRE, we developed FAIRE-MS to investigate DNA-binding protein complexes in the open chromatin region. We used biotin primers to amplify the FAIRE DNA probe and then incubated it with NE or WCE to pull down FAIRE DNA-binding protein complexes, which were detected by MS measurement. A subset of chromatin accessibility is maintained during mitosis, indicating that many transcription regulatory factors are expected to remain associated with mitotic chromosomes. Importantly, we show that FAIRE-MS performs well in identifying proteome-wide transcriptional regulators. Our protein list indicating mitotic bookmarking includes CEBPB, HMGB1, and TFAP2A, as previously reported, and MAX, HMGB3, SUB1, TFE3, and YBX1 were significantly enriched in our FAIRE-MS experiments. We also detected the retention of six factors, MAX, HMGB3, hnRNP A2/B1, FUS, hnRNP D, and TIAL1, and demonstrated that they are at least partially bound to the mitotic chromosome. Among these IDPs, for example, HMG domain TFs mediate LLPS that regulate the chromatin architecture by affecting their DNA-binding abilities. 39 FUS-dependent LLPS is necessary for the initiation of the DNA damage response (DDR). 46 FUS and hnRNP A2/B1 containing disease-associated IDRs have been implicated in LLPS. 47 LLPS, driven by collective interactions between multivalent and IDPs, is thought to mediate the formation of membrane-less organelles (MLOs) in cells. Several studies suggest that LLPS preferentially involves IDPs because of their peculiar conformational properties. 43 During the cell cycle, studies also revealed that LLPS plays a crucial role in regulating chromatin organization and chromatin behavior. 48 In this study, our results indicated that IDP(R) s are widely enriched during the cell cycle. So far, however, the role and molecular mechanism of IDP(R) in the transmission and maintenance of epigenetic information during the cell cycle remains unclear. We hypothesized that a large number of IDP(R)s (transcription regulatory factors) that remain bound to mitotic chromosomes could regulate chromatin organization when cells transit from mitosis to the G1 phase.
Recently, researchers have investigated various approaches to identify nonhistone and histone proteins bound to chromatin. For example, multiclassifier combinatorial proteomics (MCCP), density-based enrichment for mass spectrometry analysis of chromatin (DEMAC), and Hi-MS require harsh conditions and may not be broadly generalizable. 35,49,50 In addition, these methods quantify all proteins that bind to chromatin with no specificity. Thus, developing a concise and time-efficient method for capturing DNA-associated transcriptional regulators is urgently required. In this study, we tested the efficiency of FAIRE-MS in enriching both interphase and mitotic phase transcriptional regulators. After capture, we obtained more than 1000 regulatory proteins in each assay. Furthermore, we showed that FAIRE-MS is highly reproducible and can accurately describe the dynamic binding changes across the cell cycle.
During mitosis, chromosomal structural features of high-order conformations, including compartments, topologically associating domains (TADs), and chromatin loops temporarily disappear. 2,51 How is chromatin organization gradually reestablished after mitosis? The current view is that mitotic bookmarkers help it to reestablish, and the formation of contact domains follows a "bottom-up" model. 2 Further studies have indicated that mitotic chromosomes remain accessible using DNaseseq. 12 We also found that the open state of mitotic chromosomes was maintained using FAIRE-seq. However, the chromatin accessible regions were different between DNase-seq and FAIRE-seq. DNase I hypersensitivity prefers some promoters, whereas FAIRE tends to identify distal regulatory elements, such as enhancers. 24 These features are consistent with those previously described. In our study, the percentage of promoters in interphase was 17%, whereas mitosis was 5%; the intergenic region was 44% in interphase and 63% in mitosis. Specifically, only a subset of bookmarking factors retained on mitotic chromosomes may suggest that they should be tightly and specifically bound to the conserved promoters, ensuring the recruitment of other transcriptional regulators to bind during mitotic exit. In addition, the maintenance of intergenic regions, especially enhancers, may be responsible for the re-establishment of enhancer-promoter loops during mitotic exit to reinforce the expression of cell type-specific genes.
Although the compaction of mitotic chromosomes represents a dramatic change in chromosome organization and nuclear architecture, some TFs and histone modifications are thought to be partially retained, as most regulatory regions remain accessible. 52,53 These findings are essential for the transcriptional activation of genes upon mitotic exit and maintenance of cell identity. However, to date, there have been only sporadic reports of TFs, such as GATA1, FOXA1, HNF1β, OCT4, SOX2, ESRRB, KLF4, and CTCF, which are partially bound to mitotic chromosomes and act as mitotic bookmarkers to help cells reenter a normal cell cycle. [13][14][15][16][17][18][19][20][21] In this study, we implemented a genome-wide DNA-protein interaction method to capture as many transcriptional regulators as possible using FAIRE-MS. It can capture large-scale regulatory proteins using genome-wide regulatory elements (FAIRE DNA library). However, several mitotic bookmarking factors, such as RBPJ, 54 MLL, 55 and RUNX2, 56 were not measured by MS. It is possible that the abundance of these proteins is extremely low, or they are unstable in A549 cells, such that current methods are unable to detect them. Recently, two new ATAC-seqbased technologies, ATAC-MS and iDAPT-MS, have been reported. 57,58 Thus, these technologies could help overcome the above limitations.
In conclusion, FAIRE-MS relies on a first step of cross-linking protein-DNA complexes in cells in vivo with formaldehyde treatment, and on the second step of hybridization capture of target DNA for the chromatin-associated proteome. Both steps are well described. 24,32,45 In this study, we provide data showing that FAIRE-MS is an efficient and potentially useful method for capturing open chromatin-binding proteins. We also acknowledge that there are differences between FAIRE-MS and ATAC-MS. Specifically, we noted that FAIRE-seq has a lower signal-to-noise ratio than ATACseq and DNase-seq 24 ( Figure S2). Therefore, FAIRE-MS requires more stringent experimental and data quality control. Simultaneously, we found that ATAC-MS may introduce a large number of cytoplasmic proteins, resulting in high background protein contamination. 57 However, FAIRE-seq and ATAC-seq show similar signal patterns both in interphase and mitotic phase cells ( Figure 2D). Although considerable accessibility can still be maintained during mitosis, the in vivo crosslink-based FAIRE-MS and ATAC-MS can be used to capture transcriptional regulatory proteins that are retained during mitosis.

AUTHOR CONTRIBUTIONS
Bingyu Ye, Wenlong Shen, and Yanchang Li contributed to the experimental operation, data analysis, and manuscript draft writing. Dong Wang, Ping Li, Man Yin, Yahao Wang, Dejian Xie, Shu Shi, Tao Yao, and Juncai Chen contributed to data analysis and the experimental operation. Yan