CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively  or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells.Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including Video AbstractThe online version contains supplementary material available at 10.1186/s12964-022-01029-5. Grh gene was discovered in Drosophila and its mammalian homologs have three members  [The d GRHL3) . GRHLs sd GRHL3) , 8–15. Cd GRHL3) , 14, 15.d GRHL3) , 17.GRHL2 is located on chromosome 8q22 that is frequently amplified in many cancers, including breast cancer, colorectal cancer and oral squamous cell carcinoma [arcinoma –20. GRHLarcinoma , 18, 19.arcinoma , 22. By arcinoma , 24.Loss of GRHL2 was reported in gastric cancer and GRHL2 was found downregulated at the invasive front of breast cancers and loss of GRHL2 expression in primary breast cancers correlated with lymph node metastasis , 25. A kBreast cancer represents a heterogeneous disease with multiple clinically relevant subtypes appearing to originate from luminal or basal epithelial cells in the duct –31. The In this study, we identify genomic binding sites of GRHL2 shared among 3 luminal breast cancer cell lines and find that only a small subset of these GRHL2 peaks is associated with ER binding sites. We integrate this ChIP-seq data with Bru-seq analysis of genes showing transcriptional responses at different time points after conditional GRHL2 knockout in MCF7 cells. For genes showing sustained up- or downregulation in response to GRHL2 deletion, we explore correlations with GRHL2 expression in breast cancer patients. Our findings reveal gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer that partly overlap but also appear markedly distinct from targets identified in other tissues.2). For production of lentiviral particles, VSV, GAG, REV and Cas9 or single guide (sg) RNA plasmids were transfected into HEK293 cells using Polyethylenimine (PEI). After 2 days, lentiviral particles were harvested and filtered. Conditional Cas9 cells were generated by infecting parental cells with lentiviral particles expressing the Edit-R Tre3G promotor-driven Cas9 (Dharmacon) and selected by blasticidin. Limited dilution was used to generate Cas9 monoclonal cells. Subsequently, Cas9-monoclonal cells were transduced with U6-gRNA:hPGK-puro-2A-tBFP control non-targeting sgRNAs or GRHL2-specific sgRNAs (Sigma) and selected by puromycin. The EHF plasmid was kindly provided by Dr. Giuseppina Carbone, Institute of Oncology Research, Bellinzona, Switzerland and described previously [Human breast cancer cell lines representing the luminal subtype  were obtained from the American Type Culture Collection. The Hs578T human basal-B breast cancer cell line served as a GRHL2-negative control. Cells were cultured in RPMI1640 medium with 10% fetal bovine serum, 25 U/mL penicillin and 25 µg/mL streptomycin in the incubator  buffer ). Lysates were sonicated and protein concentration was determined by bicinchoninic acid (BCA) assay. Cell lysates were mixed with protein loading buffer, separated by SDS-PAGE, and transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane . The membrane was blocked with 5% bovine serum albumin  for 1 h at room temperature (RT). Next, membranes were stained with primary antibody overnight at 4 °C and HRP-conjugated secondary antibodies for half hour at room temperature (RT). After staining with Prime ECL Detection Reagent , chemoluminescence was detected with an Amersham Imager 600 . The following antibodies were used: GRHL2  Cas9 , and GAPDH .Cells were grown in RPMI-1640 complete, serum-containing medium. Cross-linking was performed by 1% formaldehyde for 10 min at room temperature (RT). Then 1 M glycine (141 µl of 1 M glycine for 1 ml of medium) was used to quench for 5 min at RT. Cells were washed twice with ice-cold PBS containing 5 µl/ml phenylmethylsulfonyl fluoride (PMSF). Cells were harvested by centrifugation (2095 g for 5 min at 4 °C) and lysed with NP40 buffer  containing 0.1% SDS, 0.5% sodium deoxycholate and protease inhibitor cocktail . Chromatin was sonicated to an average size of 300 bp , a known direct target gene of GRHL2 [CLDN4 promoter  forward: atgggtgccactggggatct, Control reverse: tgccaaagcctaggggaaga, ZEB1 promoter# forward: cggtccctagcaacaaggtt, ZEB1 promoter# reverse: tcgcttgtgtctaaatgctcg. ZEB1## forward: gccgccgagcctccaacttt, ZEB1## reverse: tgctagggaccgggcggttt, OVOL2 exon forward: ccttaaatcgcgagtgagacc, OVOL2 exon reverse: gtagcgagcttgttgacacc, CDH1 intron forward: gtatgaacggcaagcctctg, CDH1 intron reverse: caagggagccaggaagagaa. ChIP-qPCR data were collected and analyzed using the 2−ΔΔCt method [In order to examine the quality of our samples before sequencing, ChIP-qPCR (quantitative polymerase chain reaction) was performed to validate interaction of GRHL2 with the promoter region of Claudin-4  sequencing were performed by GenomeScan . MCF7, T47D and BT474 had 87,393,758, 84,633,440, and 82,080,866 pair-end reads, respectively.Less than 5% of adapter sequences were present, and the mean per base sequence quality was > 30, indicating high quality reads and no requirement for adapter-trimming  and Bru-labelled nascent RNA was isolated using an anti-BrdU antibody conjugated to magnetic beads . Subsequp values. Genes with p < 0.05 and FC > 2 or FC < 0.5 in any of the doxycycline-treated samples relative to untreated cells were filtered. Subsequently, genes responding to Cas9 induction in the context of both GRHL2 sgRNAs were selected and genes responding also in the context of control sgRNA were eliminated from this list. A heatmap was generated by R. The function “fviz_nbclust” from the R package “factorextra” was used to determine and visualize the optimal numbers of clusters using the method “within cluster sums of square”. The STRING database (version 11.5) was used to assign protein interaction networks to Bru-seq data [To identify GRHL2-regulated genes, an inter-sample comparison analysis was performed comparing RPKM (reads per kilobase per million mapped reads) for each gene in the doxycycline-treated samples compared to the untreated sample, to obtain fold-change (FC) and seq data .A compendium microarray dataset, all Affymetrix U133a, was used, containing RNA expression data of primary tumors of 867 untreated, lymph node negative patients .RNAseq data retrieved from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data set , 55 was For Sulforhodamine B (SRB) assays, cells were seeded into 96-well plates. At indicated time points, cells were fixed with 50% Trichloroacetic acid  for 1 h at 4 °C and then plates were washed with demineralized water four times and air-dried at RT. Subsequently, 0.4% SRB (60 µl/well) was added and kept for at least 2 h at RT. The plates were washed five times with 1% acetic acid and air-dried. 10 mM (150 µl/well) Tris was added and kept for half hour at RT with gentle shaking. The absorbance value was measured by a plate-reader Fluostar OPTIMA.CLDN4, a known direct target gene of GRHL2 [To identify GRHL2 binding sites, ChIP-seq was performed in the human luminal breast cancer cell lines, MCF7, T47D and BT474. As a quality control of the ChIP samples, ChIP-qPCR confirmed the interaction of GRHL2 with the promoter region of of GRHL2 . In eachGRHL2 binding sites were mainly located in intergenic regions and introns, with ~ 3–5% of the peaks located in − 1000 to + 100 bp promoter regions  but not in the sgCTR samples  and sgGRHL2(2), respectively). At 0, 2, 4, 8, or 16 days after GRHL2 knockout, cells were incubated with bromouridine (BrU) for 30 min to label nascent RNA  in adherens junctions are regulated by this balance . It has egulator , 24, 65.CDH1 that was validated by ChIP-qPCR  while GRHL2 binding was conserved in the promoter regions of CLDN4 and OVOL2 .In our ChIP-seq data, a conserved intronic GRHL2 binding site was observed in PCR Fig. a, b. How reports , 21, 33 ZEB1 or ZEB2 as opposed to findings in mammary epithelial cells [ZEB1 promoter DNA sequences bound by GRHL2 in human mammary epithelial cells and in PEO1 but not OVCA429 human ovarian cancer cells [ZEB1 in luminal breast cancer cells. In agreement, no significant changes in transcription of ZEB1 and ZEB2 genes were observed after GRHL2 loss in MCF7 (data not shown). Together, these results indicated that CLDN4 is a direct GRHL2 target while CDH1, ZEB1, or ZEB2 are unlikely to represent direct GRHL2 target genes in luminal breast cancer cells. These latter genes may be regulated at later timepoints indirectly through other transcriptional regulators [No GRHL2 binding was observed at the promoter or other regions of al cells   epigenetic regulation of gene expression , (ii) translation initiation, and (iii) mitosis were clearly visible Fig.  and earlWe addressed to what extent GRHL2 regulated gene clusters identified in our conditional MCF7 KO model predicted associations with GRHL2 gene expression in breast cancer patients. We focused on all genes where the control sgRNA gave 0.75 < FC < 1.5 at each time point after GRHL2 KO while both GRHL2 sgRNAs triggered either FC < 0.75 in at least 3 time points (positive correlation with GRHL2) or FC > 1.5 in at least 3 time points (negative correlation with GRHL2). We made use of a cohort of 867 untreated breast cancer patients (MA-867 dataset ) and ranEHF and CLDN4 that were subject to promoter binding by GRHL2 and belonged to the positively correlated gene cluster  or IgG control Ab (grey bars). Detection was performed by qPCR using primers targeting the promoter region of CLDN4 or targeting the intergenic region upstream of the GAPDH locus (Control). Results are shown for 3 GRHL2-positive luminal cell lines  and 1 GRHL2-negative basal-B cell line (Hs578T).Additional file 3: Fig. S3. Cumulative presence of adapter sequences. Results show that cumulative presence of adapter sequences is less than 5% in each cell sample, indicating that the data sets could be further analyzed without adapter-trimming.Additional file 4: Fig. S4. Per base sequence quality for all sequencing data sets. Y axis is divided into high quality calls (green), reasonable quality calls (orange) and poor-quality calls (red). Analysis shows that the mean quality of base calls, indicated by the blue line, consistently remained in the green area, indicating that sequencing data sets were of high quality.Additional file 5: Fig. S5. Coverage of peak regions across chromosomes. Graphs represent the coverage of GRHL2 binding sites across all chromosomes in the indicated cell lines.Additional file 6: Table. S1. Candidate GRHL2 target genes in luminal breast cancer cells displaying promoter interaction. GRHL2 promotor interactions identified by ChIP-seq in 3 luminal breast cancer cell lines are listed. Genes also identified by Bru-seq in MCF7 conditional KO model showing up- or downregulation at one or more timepoints in response to GRHL2 loss are indicated in bold.Additional file 7: Fig. S6. Complete Western blots for Figure 3b.Additional file 8: Table S2. GRHL2-regulated genes identified by Bru-seq in MCF7 conditional KO model. AFC for indicated genes at the indicated timepoints (days) post induction of GRHL2 KO identified by Bru-seq in MCF7 conditional KO model and assignment to clusters is shown. Genes also displaying promoter interaction identified by ChIP-seq in MCF7cells are indicated in bold.


Recent studies with single-particle tracking in live cells have revealed that chromatin dynamics are directly affected by transcription. However, how transcription alters the chromatin movements followed by changes in the physical properties of chromatin has not been elucidated. Here, we measured diffusion characteristics of chromatin by targeting telomeric DNA repeats with CRISPR-labeling. We found that transcription inhibitors that directly block transcription factors globally increased the movements of chromatin, while the other inhibitor that blocks transcription by DNA intercalating showed an opposite effect. We hypothesized that the increased mobility of chromatin by transcription inhibition and the decreased chromatin movement by a DNA intercalating inhibitor is due to alterations in chromatin rigidity. We also tested how volume confinement of nuclear space affects chromatin movements. We observed decreased chromatin movements under osmotic pressure and with overexpressed chromatin architectural proteins that compact chromatin. The molecular genetic material, DNA, exists in a form of chromatin in eukaryotes throughout most of the cell cycle. Chromatin is a higher-order fiber structure of DNA formed by nucleosomes composed of histones. It is located inside a nucleus separated from cytoplasm by a physical barrier, known as a nuclear membrane. Transcription and many other gene regulatory processes occur within the chromatin context in the compartmentalized nuclear space.Recently established chromatin conformation capture (3C)-based sequencing and advanced live-cell imaging techniques have suggested that cell-type specific intrinsic structures and dynamics of chromatin are responsible for regulating gene expression . A serieAccumulated evidence with real-time tracking of chromatin loci in live cells indicates that dynamics of chromatin are related to gene expression regulation, rather than mere thermal motions in the cell nucleus . Given tHere, we investigated how alterations in physical properties of chromatin, rigidity, and volume confinement, influence chromosome dynamics in human cells. We found chromosome movements are directly affected by changes in physical properties of chromatin by calculating diffusion constants with single locus tracking in live-cell nuclei. Because the movement of chromosomes is heterogenous in the living cell nucleus, we statistically measured the motions of each chromosome by labeling telomere loci with a CRISPR/dCas9-based labeling system .To label specific chromatin loci within a single cell nucleus, we adopted CRISPR/dCas9-based labeling system which allows us to stably label specific DNA sequences by using nuclease-deactivated Cas9 (dCas9) with a small guide (sg) RNA . To visuWe expressed the sgRNA targeting human telomere regions along with a fluorescent protein, mCherry, fused dCas9 in HeLa cells . FluoresWe plotted averaged mean square displacement (MSD) over time from trajectories for diffusion analysis -is normally cultured Hela cells . We noteDtα, for two-dimensional motions. We found an apparent diffusion constant from all over the trajectories, Dapp was 3.33  x 10–3 μm2/s with an anomalous coefficient α = 0.292   . We coulDapp from individual trajectories under the normal condition, however, showed a multimodal distribution compared to a distribution from fixed cells (Dapp is due to mixed trajectories from 1) a global long-range movement of chromatin (Dfast), 2) a local movement of chromatin loci (Dslow), and 3) an immobilized portion with thermal fluctuation under the imaging condition (Dfixed).  × 10−3 μm2/s obtained from fixed cells, since we performed all imaging under the same condition (A distribution of fixed). . For fitondition .We next sought to test the effects of transcription activity on chromatin dynamics with known transcription inhibitors; triptolide, 5,6-Dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), flavopiridol, alpha-amanitin, and actinomycin D, which act at different steps during transcription. Triptolide is an inhibitor for blocking transcription initiation, which covalently binds to X-box binding protein 1 (XBP1), one of the two helicase subunits of an initiation transcription factor Transcription factor II Human (TFIIH) . FlavopiUnder treatment of each transcription inhibitor, we measured the apparent diffusion constant . InteresDfixed, then we fitted the distribution with double Gaussian peaks without the Dfixed peak  for their formations . RNA PolEspecially, the transcriptional condensates and nuclear speckles, which are comparably abundant subnuclear organelles, are known to be functional for gene expression regulation on active genes in the processes of transcription and splicing, respectively. Given that chromatin is where genes are located, those organelles also could be physical constrains for chromatin mobility. The condensates are thought to be formed by cooperative interactions of nucleic acids and proteins that contain intrinsically disordered regions (IDRs). Hydrophobic interaction is considered one of the key driving forces for condensation .To perturb the condensates, we tested 1,6-hexanediol which is known to disrupt hydrophobic interactions among biomolecules . AlthougDfixed becomes dominant  molecules. We observed a drastic decrease in chromatin motions proportional to the concentration of PEG . Checkindominant . ReducedRegarding our previous observation, we hypothesized that increasing nuclear volume may promote chromatin motion by allowing more space for movements. Rather than changing the volume of the nucleus itself, we decided to induce chromatin packing with chromatin architectural proteins, CCCTC-binding factor (CTCF), and Cohesin, which can indirectly increase the nuclear space for the chromatin mobility.in vitro real-time single-molecule assays have shown that CTCF and Cohesin indeed create chromatin loop structures, through the loop-extrusion mechanism. In detail, the ring-shaped Cohesin entraps and extrudes chromatin until it encounters a convergently oriented chromatin-bound CTCF pair (With advanced 3C-based genome-wide sequencing techniques combined with chromatin immunoprecipitation (ChIP), it has been found that CTCF and Cohesin are enriched at the most  of TAD boundaries, indicating that the chromatin loops are formed by those mechanical units . Recent TCF pair . Based oTo test the effects of chromatin architectural proteins on chromatin movements, we overexpressed CTCF and RAD21, a subunit of the Cohesin complex, independently in the cells. As we expected, CTCF did not show a significant change in the mobility of chromatin, meaning that the boundary element CTCF itself does not influence the chromatin packing . On the Dfast has increased for the overexpression of RAD21, indicating that the long-range movement of chromatin becomes more frequent.The multimodal distributions of diffusion constants also showed the aspect . The porWe further performed knockdown experiments on CTCF and Rad21. As expected, knockdown of CTCF did not significantly alter chromatin mobility. However, the knockdown of Rad21 still increased chromatin mobility. We speculate this result might be due to that knockdown of Rad21 seems to have a greater effect on reducing rigidity and making chromatin more flexible than the effect of reducing effective volume, similar to the results of transcription inhibitors .The genetic material, DNA which is wrapped into chromatin fibers, is a physical entity in a finite space, the nucleus. The physical properties of chromatin, therefore, are linked to gene expression regulation . We inveWe tested various chemicals that can inhibit transcription or that can intercalate into the DNA double helical structure inducing increased chromatin stiffness. We observed that transcription inhibitors increased chromatin mobility while DNA intercalators drastically reduced chromatin motions. These opposite responses of the chromatin mobility to different chemicals may indicate the same physical consequence; transcription machinery affected by the transcription inhibitors apply physical constraints by forming chromatin loops and repressing the movements of chromatin. Thus, inhibiting transcription should induce increased flexibility in chromatin structures. On the other hand, DNA intercalators stuck between bases in DNA make the chromatin more rigid.Upon the treatment of osmotic agents to the cells, we found the declined motions of chromatin. Osmotic pressure with a hypertonic solution to the cell causes an outward flow of water molecules across the nuclear membrane, with shrinking volume to adjust concentrations in and out of the nucleus . The redTaken together, our experiments perturb the physical properties of chromosomes and showed alterations in the physical environment directly influence the chromosome motions, which is also related to transcriptional activities. Since we measured the motions of chromosomes with telomere regions, which may not fully represent chromosome movements, additional measurements from the movements of whole chromosomes should be required to confirm our conclusions in a further study.Mycoplasma contamination using the e-MycoTM Mycoplasma PCR Detection kit (iNtRON).The human cervical cancer cell line HeLa (Korean Cell Line Bank # 10002) was cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose, devoid of phenol red , supplemented with 10% fetal bovine serum (FBS) , 1% antibiotic-antimycotic , 1% GlutaMAX™ supplement , and 1% sodium pyruvate . Cells were cultured at 37°C and 5% CO2 in a humidified incubator. Cell lines were periodically tested for ® HD Transfection Reagent  in a ratio of 1:5  until 80% confluency was reached. For telomere labeling, cells were transfected with 300 ng of SpydCas9-3XmCherry-SgRNA/Telomere-All-in-one vector  and 1,500 ng of SpySgRNA/DTS13-Telomere  using FuGENEo of 1:5 . Cells wFor fixed cell imaging, the culture medium was exchanged with 4% paraformaldehyde (PFA)  after washing with Dulbecco’s phosphate buffered saline (DPBS) . After 10 min incubation at room temperature, PFA was exchanged with 1x PBS. To completely remove PFA, cells were incubated in DPBS for 5 min, and this process was repeated three times.Cells were cultured in 35-mm glass-bottom dishes before treatment with transcription inhibitors. For each transcription inhibitor, cells were incubated for 2 h under actinomycin D , 100 μM 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) , 5 μM α-amanitin , 10 μM flavopiridol , and 125 nM triptolide  diluted in dimethyl sulfoxide , respectively, before imaging.Cells were cultured in 35-mm glass-bottom dishes before treatment of DNA intercalators. For each DNA intercalator, cells were incubated for 2 h under 2 μM actinomycin D , for 10 min under 5 μM DRAQ5  and 9 μM Hoechst .Cells were cultured in 35-mm glass-bottom dishes before treatment with osmotic agents. Cells were treated with 1% (wt/vol) 1,6-hexanediol  for 5 min, with 10%, 30% (wt/vol) Polyethylene glycol (PEG200)  for 5 min, before imaging on the microscope stage.For expression of chromatin architectural proteins, CTCF or RAD21 (a subunit of cohesion complex), 1 µg of pKS070-pCAGGS-3XFLAG-(human)CTCF-eGFP , and 1 µg of Dendra2-Rad21-C-18  expression vectors were transfected to the cells for each experiment. We confirmed the expression of each protein in cell nuclei, with GFP signals for CTCF and Dendra2 signals for RAD21, respectively. We imaged and collected data from fluorescence positive cells.Cell imaging was performed with highly inclined and laminated optical (HILO) illumination from a Nikon TIRF microscope based on the Nikon Eclipse Ti microscope body. For detections of telomere loci, mCherry was excited with a built-in 561 nm laser in the microscope. Images were acquired through a 100X objective lens of 1.49 numerical aperture  with the electron multiplying charge coupled device (EMCCD) , and processed using the NIS-Elements software (Nikon). For streaming image acquisition, snapshots were taken every 100 ms for 2 min. Image analysis was performed using ImageJ scripts and custom-built MATLAB codes for mean square displacement calculation.Image analysis was performed using ImageJ scripts and custom-built MATLAB codes for mean square displacement calculation. Single locus tracking was performed by an ImageJ plugin-in software, TrackMate. Each fluorescent locus per frame was identified by Laplacian Gaussian fitting with an LoG detector in the TrackMate. Background noise was minimized by the LoG detector which is based on applying an LoG filter on each image and detecting local maxima. 2D trajectories of X-Y coordinates over time for each particle were obtained.For telomere movement analysis, single particle analysis was performed using a custom-built MATLAB code. MSD equation was used to measure the characteristic of diffusion with time-dependent trajectory information of each particle. The mean square displacement (MSD) was obtained through this formula if we know the X-Y coordinates and time interval in 2D for each frame in the trajectory we obtained and the number of total frames. We calculated MSD-values for each individual track using a method previously reported  where MSn frames .D) and an anomalous diffusion parameter (α), MSD-t plots were fitted by 4Dtα, for two-dimensional motions.Plotting MSD-time curve was performed using the custom-built MATLAB code. The MSD of particles tracked for more than 20 s were collected and averaged over time to obtain an ensemble MSD graph. For estimation of a diffusion coefficient (


The plant nucleus provides a major hub for environmental signal integration at the chromatin level. Multiple light signaling pathways operate and exchange information by regulating a large repertoire of gene targets that shape plant responses to a changing environment. In addition to the established role of transcription factors in triggering photoregulated changes in gene expression, there are eminent reports on the significance of chromatin regulators and nuclear scaffold dynamics in promoting light‐induced plant responses. Here, we report and discuss recent advances in chromatin‐regulatory mechanisms modulating plant architecture and development in response to light, including the molecular and physiological roles of key modifications such as DNA, RNA and histone methylation, and/or acetylation. The significance of the formation of biomolecular condensates of key light signaling components is discussed and potential applications to agricultural practices overviewed. Shade avoidance response (SAR) is characterized by leaf hyponasty, hypocotyl and leaf elongation, and early flowering initiation to enhance light‐harvesting or temporally overcome competing vegetation by enhancing reproductive success , which begins with the generation of small RNAs and ends with the methylation of cytosines in all sequence contexts CG, CHG and CHH by the DNA methyltransferase DRM2  and 5‐methylcytosine (m5C) have been detected in Arabidopsis messenger RNA (mRNA) and affect mRNA stability, interactions with other molecules, as well as secondary structure  showed no photodependent RNA Pol II (RNPII) activity when using naked DNA as a template, but did so when using reconstituted mammalian chromatin , by recruiting or driving chromatin components at specific loci , by RNPII‐associated Transcription Elongation Factors  that may be particularly dependent on mechanisms facilitating RNPII processivity across nucleosomal physical barriers  mark  and demethylases and recognized by several histone readers. Among the best studied histone post‐translational modifications (PTMs), lysine and arginine residues can be covalently mono‐, di‐, tri‐methylated at different positions along the histone H3 and H4 tails protruding out from the nucleosome core particle  LHCB1.4, LHCB1.5, HCF173 (HIGH CHLOROPHYLL FLUORESCENCE PHENOTYPE) and TZP (TANDEM ZINC‐FINGER PLUS3) genes that correlates with their induction by light , YUCCA9 (YUC9), PRE1 (PACLOBUTRAZOL RESISTANCE 1) and IAA19 (INDOLE‐3‐ACETIC ACID INDUCIBLE 19). In addition, both MRG2 and PIF7 associate to modulate H3K4me3 and H3K36me3 distribution on LREs (such as the G‐box) and TSS regions on the aforementioned shade‐responsive genes  and MRG2 display a significant reduction in hypocotyl elongation upon exposure to shade  1 and GA3OX2 loci, thereby triggering the accumulation of active GA in seeds essential for germination , an ATP‐dependent chromatin remodeling enzyme, was identified through a forward genetic screen as a negative regulator of de‐etiolation  and the plant‐specific HD2 family  promoter as a means of regulating seedling establishment downstream of light and hormone stimuli  in a HY5‐dependent manner by deacetylating ATG5 and ATG8e loci  by modulating nucleosome density  depends on PIF7 association with their promoters. PIF7 can directly interact with the EEN subunit of the INO80‐complex and thereby modulates H2A.Z deposition on key loci. Interestingly, H2A.Z depletion precedes induction of gene expression, suggesting that chromatin remodeling anticipates transcriptional activation , in particular, ACTIN‐RELATED PROTEIN 6 (ARP6) and SWR1 complex subunit 6 (SWC6) that catalyze H2A.Z incorporation into the chromatin  in a PcG (Polycomb group)‐dependent fashion  and PIL1 (PHYTOCHROME INTERACTING FACTOR 3‐LIKE 1) through PRC2‐dependent deposition of the H3K27me3 repressive mark upstream of their TSS  molecule  positively affects R‐loop formation as well as gene expression , removed by ‘erasers’ (RNA demethylases) and recognized by ‘readers’ , METHYLTRANSFERASE B , FKBP12 INTERACTING PROTEIN 37 , VIRILIZER (ortholog of WIRMA) and HAKAI , LATE ELONGATED HYPOCOTYL (LHY) and TOC1, and to increase mRNA levels of key flowering regulatory genes CONSTANS (CO) and FLOWERING LOCUS T  identified approximately 1000 genes with hypomethylated transcripts in fiona1 mutant plants, which predominantly lacks m6A in their 3′ UTR  encodes a R2R3‐MYB transcription factor that promotes the accumulation of UV‐protective flavonoids. Increased P1 expression in leaves of high‐altitude landraces and in response to UV‐B treatments is caused by loss of DNA methylation along its promoter and coding sequences , in which parental ramets exposed to UV‐B produce offspring ramets manifesting an ‘escape strategy’ when foraging in a UV heterogeneous environment, whereas ramets from ‘naïve’ parents do not show any behavioral preference  as well as association with RNA, histones and scaffold proteins facilitate the formation of biomolecular condensates. In future studies, the advancement of bioimaging and genome‐enabled experimental tools such as fluorescence in situ hybridization (FISH), Chromatin Conformation Capture (4C and Hi‐C) and related techniques enabling us to reach a 3D perspective in DNA and protein networks Grob, , and theAll authors  contributed to the conceptualization and writing of the review.


The nucleotide-binding oligomerization and leucine-rich repeat receptor (NLR) protein family consists of important immune sensors that form inflammasomes, a cytosolic multi-protein platform that induces caspase-1 activation and is involved in different inflammatory pathologies. The NLR family pyrin domain containing 6 (NLRP6) is a receptor that can signal by forming inflammasomes, but which can also play an important role without forming inflammasomes. NLRP6 regulates intestinal homeostasis and inflammation, but also is involved in cancer, the nervous system or liver diseases, with both protective and deleterious consequences. In the present article, we review the different roles of NLRP6 in these processes and offer new insights into NLRP6 activation. This review discusses emerging roles for the NLR family pyrin domain containing 6 receptor (NLRP6) in intestinal homeostasis, inflammation, cancer, the nervous system and liver disease. Inflammasomes are important in autoimmune syndromes, chronic inflammatory diseases, and metabolic and degenerative pathologies6. The activation of inflammasomes is related to the cellular sensing of different molecular patterns associated with pathogens (PAMPs), cellular damage or death (DAMPs), microbes (MAMPs) or homeostasis-altering processes (HAMPs)12 (Table 14.Since the first inflammasome was described as a multiprotein cytosolic oligomer for caspase-1 activation and interleukin (IL)-1β processingPodospora anserina HET-E, a telomerase-associated protein TP1 (NACHT) domain, and a C-terminal leucine-rich repeat (LRR) domain. The NLR family includes several members that form inflammasomes and because of the large number of members discovered (23 in humans and more than 30 in mice15) they have been associated with multiple functions of the innate immune system.The NLR proteins contain an N-terminal domain that could be either a pyrin domain (PYD) or a caspase activation and recruitment domain (CARD) leading to the NLRP and NLRC sub-families respectively. NLR members also include a central neuronal apoptosis inhibitory protein NAIP, a major histocompatibility class II transcription activator CIITA, an incompatibility locus protein from 16, covers both inflammasome-dependent and independent functions by activating caspase-1 or caspase-11 or regulating the key transcription factor NF-κB18. NLRP6 has an important role in maintaining intestine homeostasis, and more specifically in regulating the interaction between the host mucosa and the microbiota20. Other physiological roles played by NLRP6 are emerging, such as its role in helping the host to defend itself against pathogens, in tumorigenesis, and in neuroinflammation23, where NLRP6 presents pro- and anti-inflammatory roles. The fact that NLRP6 has differential roles depending on the tissue where it is expressed and activated makes this receptor an exciting and complex new therapeutical target. This review aims to summarize the inflammasome and non-inflammasome-mediated roles of NLRP6 in health and disease.NLRP6, as an NLR family member24 and most of the functional studies of NLRP6 focus on this system and its related diseases. However, NLRP6 is also expressed in other tissues such as the kidney, liver and lung and in cells such as neurons, lymphocytes, or bone marrow-derived cells25. NLRP6 is a gene with multiple alternative transcriptional promoters that determine its distribution and expression across selective tissue and cell types24. In humans, NLRP6 has three alternative transcription starting sites (TSS), the first is located exon 1 (in the 5′ untranslated region), which is selective for intestine expression, the second is located in exon 2 (in the middle of the PYD domain), which is selective for expression in kidney, lung, liver, spleen, and brain, and the third is located in exon 3 (in the region between PYD and NACHT domains)24. All three TSS are present in the kidney, although its predominant transcription starting site is the second (exon 2), meaning that the NLRP6 detected there is truncated. In contrast, in the small intestine, the predominant transcription starting site is the first (exon 1), meaning that in this case, the NLRP6 protein is endogenous, full-length, and highly detectable. Therefore, the translational repression of human NLRP6 occurs in a tissue-specific context  and is the result of alternative promoters24. In mouse, the Nlrp6 gene is expressed in kidney, liver, and intestinal tissue; however, only endogenous NLRP6 protein was detected in the intestine24. This is due to two distinct alternative promoter isoforms of Nlrp6, one expressed in the intestine  and the other expressed in the kidney and liver with an expanded 5′ untranslated region sequence of 1749 bp that results from complex alternative splicing comprising the exon 1 of Nlrp6, two upstream intergenic exons and the exon 1 of the adjacent upstream gene BC024386 (expressed in kidney and liver but not in the colon), thus generating an alternative promoter24. This suggests that mouse Nlrp6 can be regulated by tissue-selective alternate promoters that result in two isoforms, one for the expression in the intestine and the other for the expression in the kidney and liver24. In this regard, it has been shown in a mouse model of nephrotoxic acute kidney injury (AKI) induced by a folic acid overdose that NLRP6 is downregulated in the kidney26.NLRP6 is highly expressed in the digestive systemNlrp6 gene in mouse presents overrepresented peroxisome proliferator-activated receptor γ-retinoid X receptor-α (PPARG-RXRA) binding sites in its promoter region, which are also conserved in human and rat27. In this regard, human epithelial cells from colon tissue treated with rosiglitazone, a PPAR-γ agonist, showed an increase in NLRP6 gene expression, which suggests that modulators of PPAR-γ could be a target to control Nlrp6 transcription27.Mapping of transcription factor binding sites using JASPAR and TRANSFAC databases revealed that the Nlrp6 gene expression in mouse intestine and in fibroblast28, which suggests that NLRP6 plays a role in the host response to viral infection. This role is specifically important in the intestinal tract where Nlrp6 limits the ssRNA virus replication by interacting with the Asp-Glu-Ala-His (DEAH) box helicase 15 (Dhx15)28. The transcriptional expression of mouse Nlrp6 has been found to be negatively regulated by the miR-33-3p in microglial cells treated with hemin in a model of intracerebral hemorrhage29. Another microRNA involved in the downregulation of Nlrp6 is miR-650, which targets the 3′-untranslated regions of Nlrp6 and thus inhibits its expression in ulcerative colitis30. Therefore, the complexity that NLRP6 shows in its transcriptional regulation could reveal different roles for NLRP6 that need to be clarified in other tissues besides the intestine, liver, lungs, or kidney.Viral infections and the production of interferon type I/III upregulate 31 demonstrated that the PYD domain of NLRP6 alone can self-assemble into a filamentous structure that can bind to ASC through homotypic PYD-PYD domain interactions. The interaction between ASC and the PYD of NLRP6 is enhanced when the central domain NACHT of NLRP6 is fused to its PYD domain. Their study suggests that the PYD domain of NLRP6 could seed the inflammasome assembly with the PYD domain in the center and surrounded by the NACHT and LRR domains31, similarly to other described inflammasomes such as NLRP332. These studies imply that NLRP6 undergoes a conformational change to allow the interaction of ASC and therefore suggest that inactive NLRP6 should adopt an auto-inhibited state  is required for LLPS43. After phase separation, the presence of ASC will solidify NLRP6 condensates and will lead to the activation of the inflammasome43  has been found to be associated with NLRP6 activation, and this phenomenon is gaining interest in the study of physiology and disease. Macromolecules such as proteins undergo LLPS when they condense into a dense phase that coexists with a diluted phaseLPS Fig. , which qe43 Fig. . This moNlrp6-deficient mice are more susceptible to chemically induced or spontaneous colitis associated with an increase in Akkermansia muciniphila in the gut, a specific pathobiont sufficient for promoting intestinal inflammation46 , an immunomodulatory c-type lectin whose expression is regulated by NLRP648.NLRP6 is highly expressed in the intestine, where it plays an important role in controlling intestinal homeostasis. It has been shown that a deficiency of NLRP6 affects the microbiota composition and that n46 Fig. . The altCryptosporium or with Candida albicans meant that these mice exhibited a higher parasite burden compared with wild-type mice due to a deficiency in the release of pro-inflammatory IL-18 and IL-1β, which are important cytokines that control the expansion of these parasites50  by shaping the host-microbiome interface and the susceptibility to DDS-induced colitis46. This suggests that intervening in the metabolite-inflammasome-antimicrobial peptide axis in order to treat intestinal diseases could be an exciting area of future search. In this regard, the upregulation of Nlrp6 in infiltrating monocytes after DSS-induced colitis reduces the susceptibility to chemically induced intestinal injury by restoring the intestinal barrier and limiting the induction of bacteria-driven inflammation51. These data suggest that the microbiota composition and function may be related to the therapeutic modulation of NLRP6 function.Recent studies showed that the lack of NLRP6 in mice infected with the intestinal parasite s50 Fig. . NLRP6-d54. NLRP6 in goblet cells has been associated with a crisis in mucus production after the bacterial invasion, where bacterial Toll-like receptor (TLR) ligands stimulate the secretion of Muc2 through NLRP6 activation55 , where host NLRP6 aggravates gastrointestinal symptoms of GVHD and the above-mentioned taurine and the inflammasome action contribute to GVHD mortality65Fig. .66. In line with the importance of NLRP6 in gastric cancer, another recent study shows that a long non-coding RNA named OIP-AS1 interacts with the zeste homolog 2 (EZH2), which is part of an essential polycomb repressive complex 2 whose deregulation is associated with different diseases, especially cancer, and contributes to gastric cancer cell growth and migration due to epigenetic silencing of NLRP6 transcription68  of the 78 kDa glucose-regulated protein (GRP78), which in turn promotes the ubiquitination and degradation of GRP78 and thus reduces its expression and inhibits gastric cancer growthn68 Fig. .70, which is the principal cause of death in patients with Hirschsprung’s disease (HSCR), a congenital disorder characterized by the absence of ganglion cells at the end of the bowel70Fig. . NLRP6 e18. NLPR6 could therefore ameliorate the exacerbated production of pro-inflammatory cytokines in rheumatoid arthritis patients, acting as a docking site to facilitate the interaction of pivotal proteins for NF-κB activation (such as TAB2/3 with tripartite motif 38) and induce their degradation18 73 73 Fig. .S. aureus induces NLRP6 inflammasome activation and loss of neutrophils by pyroptosis and NETosis, thus increasing host mortality74. NLRP6 also limited ROS production and INF-γ after infection72, which suggests that blocking NLRP6 could be a therapeutical approach for augmenting neutrophil-associated bacterial clearance and improving pneumonia.Specifically, LTA from Porphyromonas gingivalis, a pathogen associated with chronic periodontitis, thus inducing pyroptosis and IL-1β and IL-18 release through NLRP6 inflammasome activation75 , a type of cancer that is developed from the mucosal epithelium in the oral cavityC77 Fig. .78. In cell lines derived from malignant glioma, the genetic downregulation of SP1 decreased the migratory and invasive potential of these cells. This SP1 downregulation also reduced the expression of inflammasome components such as ASC, caspase-1, and IL-1β, which were restored after NLRP6 overexpression. This result suggests that SP1 and NLRP6 positively regulate the malignant behaviors of glioma cells and the growth of tumors78 s78 Fig. .79.In other types of cancer, such as skin cutaneous melanoma, the low expression of NLRP6 is associated with poor prognosis85. NLRP6 has an antifibrotic role in the liver because it decreases the levels of the main enzymes responsible for the degradation of the liver matrix, such as MMP2 and MMP981. Furthermore, NLRP6 is downregulated during NASH and alcoholic hepatitis, thus increasing the pro-inflammatory and profibrotic effects of NF-κB, promoting fibrosis, and increasing the severity of the diseases83. In particular, in NASH NLRP6 controls the expression of CD36, which interferes with the accumulation of triglycerides82. In alcoholic hepatitis, NLRP6 is associated with the inhibition of the chemokine C-C motif ligand 20 (CCL20), which is responsible for activating the hepatic stellate cells involved in fibrosis83 , and alcoholic hepatitis (AH)s83 Fig. . Also, NShistosoma mansoni, which is a parasite that causes granulomatous inflammatory reactions87. In contrast to the protective role that NLRP6 plays in NASH, alcoholic hepatitis, and allogeneic transplantation, in this case, it contributes to the formation of hepatic granuloma and aggravates the injury produced by this specific parasite  is becoming more apparent in the context of sterile- and bacterial-induced inflammation, with NLRP6 being involved in inflammasome-independent neuronal recovery from peripheral nerve injury by limiting the activation of extracellular signal-regulated kinase (ERK)21 Fig. . On the e89 Fig. .92, but with opposite effects. In ICH NLRP6 is upregulated in perihematomal brain tissue via TLR4 and ameliorates brain injury, thus reducing brain water content, pro-inflammatory cytokines, NF-κB activity, and neurological deficit scores91. This result is supported by the fact when the miR-331-3p is overexpressed, it negatively regulates the transcription of Nlrp6, which reduces recovery of the neurological functions29. However, in I/R injury, the NLRP6 inflammasome is formed by interaction with the deubiquitinase BRCA1-BRCA2-containing complex subunit 3 (BRCC3) and promotes increased infarct volume and brain water content, together with decreased neurological scores and increased IL-1β92  injury and intracerebral hemorrhage (ICH)β92 Fig. .Nlrp6−/− mice and no Nlrp6 protein is detectable in the kidney24. In kidney injury produced by UUO or NTS administration, NLRP6 seems to play little role in recovery; however, after AKI, its reduced expression and protein levels actually seem to cause or increase AKI26. NLRP6 attenuated nephrotoxic AKI, renal inflammation and infiltration of inflammatory cells (macrophages and neutrophils), and decreased fibrosis, meant that NLRP6 plays a nephroprotective role in the kidney26  and adrenomedullin loci have been related to hypertension95, with NLRP6 being a proposed receptor for AVR96.In vivo assays of the kidney after unilateral ureteric obstruction (UUO) and nephrotoxic serum (NTS) administration suggest that NLRP6 is dispensable after kidney injury, given that no phenotype is observed in y26 Fig. . However 93 Fig. . Kidney 97. NLRP6 was included in the 68 genomic loci reliably associated with variations in MPV and PLT. The NLRP6 gene is placed beside the PSMD13 gene in the human chromosome 11, so these two genes could form a segregation tandem for MPV and PLT, meaning that further experiments are required in order to determine if NLRP6 plays a functional role in platelet formation.In a high-powered genome-wide meta-analysis of more than 66,000 individuals, NLRP6 was associated with variations in mean platelet formation (MPV) and platelet count (PLT)Inflammasomes are highly important as immune sensors due to their role in different pathologies; however, the NLRP6 inflammasome possesses the particular feature of having inflammasome-independent roles and is thus detrimental or beneficial depending on the pathology. In particular, it functions as an inflammasome by exacerbating inflammation and thus contributing to the elimination of intestinal parasites, viruses, and gram-negative bacteria. Also, the activation of NLRP6 inflammasome controls the growth of different types of cancer such as NHSCC, it is implicated in glucose regulation and it promotes the healing of brain injuries after intracerebral hemorrhages. However, the NLRP6 inflammasome does not play a beneficial role in gram-positive infections, GVHD, ulcerative colitis, glioma, ischemia/reperfusion brain injury, or liver infections with parasites. For the inflammasome-independent function, NLRP6 has a protective role in the regulation of NETosis after pneumonia infection, in gastric cancers, in liver diseases, rheumatoid arthritis, certain acute kidney injuries, and intestinal colitis. The recent advances that have shown how NLRP6 is assembled by phase separation will help to tackle the design of drugs to control NLRP6 activation. Drugs affecting the assembly and activation of NLRP6 by directly binding to it and altering its conformation could alleviate or mitigate the different adverse effects when the uncontrolled activation of this inflammasome occurs after gram-positive bacterial infection, GVHD, parasite liver infections, ischemia/reperfusion injury, or glioma. The fact that different metabolites can affect NLRP6 activation opens up the exciting possibility of new methods of metabolite-mediated immune modulation that will improve the treatment of intestinal inflammatory diseases.


The multi-subunit Polycomb Repressive Complex (PRC) 1 and 2 act, either independently or synergistically, to maintain and enforce a repressive state of the target chromatin, thereby regulating the processes of cell lineage specification and organismal development. In recent years, deep sequencing-based and imaging-based technologies, especially those tailored for mapping three-dimensional (3D) chromatin organization and structure, have allowed a better understanding of the PRC complex-mediated long-range chromatin contacts and DNA looping. In this review, we review current advances as for how Polycomb complexes function to modulate and help define the high-order chromatin structure and topology, highlighting the multi-faceted roles of Polycomb proteins in gene and genome regulation. For example, somatic heterozygous gain-of-function mutation of EZH2 at its residue Tyr-646 occurs frequently in human B cell lymphoma patients, which leads to a global increase of H3K27me3 and a more closed chromatin structure  in vitro . An EZH2in vitro . In cellin vitro , a PHC pin vitro . The CBXin vitro . PRC2 antructure . PRC comtructure .Increasing evidence points to critical roles of three-dimensional (3D) chromatin organization and high-order structure during organismal development and cell differentiation, and deregulation of chromatin topology is associated with development of human diseases . In receFirst, segregating chromatin regions can be achieved through DNA loops formed in the interphase cells. Here, the “loop extrusion” model  is the mLoops are further folded into the self-interacting genomic regions, often termed Topologically Associating Domains (TADs), which span hundreds of kilobase pairs (Kbp) of DNA with boundaries usually anchored by CTCF. Chromatin in the same TADs shares similar chromatin state and transcriptional activity and is interactive and generally avoid of interactions to other domains, thereby regulating the transcription of embedded genes in a coordinated fashion .At a larger genome scale (∼1 Mbp or more), chromatin forms two distinctive compartments, referred to as the A (active) and B (inactive) compartment, which display an enhanced frequency of contacts among themselves and correspond to open (A-type) and closed/compact (B-type) chromatin, respectively .Recent studies lend strong support for a notion that PRC complexes have important functions in spatial determination and fine-tuning of high-order chromatin structure. In this review, we discuss about recent advances on these topics.Drosophila melanogaster embryonic cells, Drosophila cells, however, did display binding by Rad21, a subunit of cohesin complex, indicating a possibly distinct loop formation mechanism mediated by cohesin in the fly  Hi-C map in the cultured Kc167  the fly . In addi the fly . Of notein loops .Drosophila embryogenesis and found that PRC1-associated chromatin folding imposes an architectural effect leading to gene repression  are necessary for stabilization of chromosome architecture and maintenance of gene repression in the fly . Using Dpression . This wopression . Also, Ppression . Similarpression . As discIn mammalian cells, Hi-C or Hi-ChIP methods uncovered about ten thousands of DNA loops and 4,101 loops were discovered by Hi-ChIP for H3K27me3 in mouse embryonic stem cells (mESCs) . PRC comLong-range looping interaction between promoter and enhancer represents a common means for modulating transcription. Enhancers are distal gene-regulatory elements, which contain a characteristic mono-methylation of histone H3 lysine 4 (H3K4me1) . In addivia PE-promoter contact in undifferentiated stem cells, which then enables a quick response to differentiation-stimulating signals   . In agre of PRC) . Also, r of PRC) . However of PRC) . Furthervia DNA looping; meanwhile, they can turn into enhancers under different contexts (via (co)activator-associated chromatin looping, these PRC2-bound DREs function as “silencers” and repress target gene transcription through chromatin looping associated with PRC2 and (co)repressors , in animal cells . Similarvia interaction with SUZ12 to repress target genes  was proposed to be crucial for transcriptional control and chromatin structure organization . Ahn et Indeed, studies revealed that cPRC1 can form condensates through LLPS, which is driven by IDRs harbored within cPRC1’s subunits, CBX2 and PHC1, and that PRC1’s condensation and LLPS contribute to chromatin compaction and target gene silencing . Here, LIt becomes increasingly clear that PRC complexes play crucial roles in the determination and/or modulation of 3D chromatin structure, thereby regulating gene expression patterns during organismal development. In future, effort shall be made to further decipher causal roles for the recruitment and binding of PRC1, PRC2, loop anchors and various cofactors in the formation and maintenance of loops. Many of the works covered in this review used either early embryo or ESCs as model system and further research is warranted to investigate the dynamics of PRCs-mediated 3D chromatin organization during development, given that PRC components are dynamically expressed among various differentiated cells and tissue types. Furthermore, a phase separation model for PRC-mediated chromatin compaction  is appea


Gene expression is primarily controlled at the level of transcription, largely due to binding of transcription factors to specific sites on DNA, namely super-enhancers and promoters. Super-enhancers and enhancers can regulate distal target genes via chromatin looping mechanisms. These long-range enhancer–promoter interactions are often mediated by phase-separated condensates. In this review, we summarize recent insights into super-enhancers and phase-separated condensates and their roles in the transcriptional activation of genes involved in development and diseases such as cancer. In addition, we survey a broad array of drugs that target super-enhancers, three-dimensional genome organization, and phase-separated condensates that could be potentially used for the treatment of cancer.3D chromatin organization plays an important role in transcription regulation and gene expression. The 3D genome is highly maintained by several architectural proteins, such as CTCF, Yin Yang 1, and cohesin complex. This structural organization brings regulatory DNA elements in close proximity to their target promoters. In this review, we discuss the 3D chromatin organization of super-enhancers and their relationship to phase-separated condensates. Super-enhancers are large clusters of DNA elements. They can physically contact with their target promoters by chromatin looping during transcription. Multiple transcription factors can bind to enhancer and promoter sequences and recruit a complex array of transcriptional co-activators and RNA polymerase II to effect transcriptional activation. Phase-separated condensates of transcription factors and transcriptional co-activators have been implicated in assembling the transcription machinery at particular enhancers. Cancer cells can hijack super-enhancers to drive oncogenic transcription to promote cell survival and proliferation. These dysregulated transcriptional programs can cause cancer cells to become highly dependent on transcriptional regulators, such as Mediator and BRD4. Moreover, the expression of oncogenes that are driven by super-enhancers is sensitive to transcriptional perturbation and often occurs in phase-separated condensates, supporting therapeutic rationales of targeting SE components, 3D genome organization, or dysregulated condensates in cancer. The regulation of gene expression in eukaryotic cells is a coordinated biological process that relies on the efficient assembly of transcription factors (TFs), transcriptional co-activators such as Mediator complex subunit MED1) and BRD4, and RNA polymerase II (RNA Pol II) at specific genomic sites  [1]. Her and BRD4SEs are clusters of neighboring enhancers spanning over 10 kb with high-fold enhancer activity that drive cell-type specific gene expression . 3D genoSEs are defined by ranking the enriched ChIP-seq signals of master TFs, co-activators BRD4, MED1, p300, or enhancer-specific histone marks, such as H3K27ac and H3K4me1 ,7,8. To These SE profiles can be used to identify master genes that are involved in cell identity and disease condition ,4. Additcis-regulatory elements and target genes [The human genome is organized into higher order structures, and such structures are important for transcriptional regulation . Individet genes . The chret genes ,15. TAD et genes ,17. Mostet genes .Several SE-associated factors, such as the CTCF and cohesin complex mediate chromatin interactions within the SEs . IntegraIL7R locus are restricted within the same CTCF-organized neighborhood [The transcriptional activity of SEs is restricted within insulated neighbourhoods enclosed by CTCFs and cohesin complex such that SEs are specifically tethered to their target genes. Cohesin loss leads to the development of myeloid neoplasms . Higher hborhood . SEs inshborhood .Wap SE (comprised of three constituent enhancers) activated neighboring non-mammary gene Ramp3 separated by three CTCF sites. Although CTCF does not completely block SE activity, deletion of CTCF in mice demonstrated the capacity to muffle gene activation. CRISPR/Cas9-mediated deletion of three CTCF sites did not alter Wap expression, but increased Ramp3 expression (seven-fold) in mammary tissue from parous mice by establishing enhanced chromatin interactions between S3 of Wap SE and the first intron of Ramp3 [The disruption of the insulated chromatin neighborhood by deletion of the CTCF binding site at one of the borders causes dysregulation of intradomain genes and activation of genes outside the neighborhood . Functioof Ramp3 . This inof Ramp3 .MYC oncogene levels are associated with aggressive tumors. One of the ways that this dysregulation is achieved is through the acquisition of large tumor-specific SEs present within 2.8 Mb MYC TAD. In tumor cells, SEs at the MYC locus are looped to a common CTCF site within the MYC promoter  was characterized, in which TAD fusion was observed in the  with SE . TAD fuseraction ,30.Various dCas9 systems, such as dCas9-KRAB , dCas9-DCancer cells can acquire oncogenic SEs either through chromosomal rearrangements, DNA mutations and indels, 3D chromatin structural changes, or viral oncogenes ,37,38. IMYC transcription [Co-activators such as BRDs  and cyclin-dependent kinases  may be targeted to disrupt SEs. Several bromodomain and extra-terminal domain (BET) inhibitors, and CDK inhibitors have been reported to target SEs, as shown in cription . Similarcription , ovariancription , Merkel cription , B-cell cription , and alvcription .CDK7 and CDK9 are important in the initiation and elongation of transcription mediated by the phosphorylation of RNA Pol II. CDK7 inhibitor (THZ1) alters the H3K27ac mark globally. In Chronic Myelogenous Leukemia, THZ1 disrupted the transcription of SE-associated gene XBP1 and eradicated leukaemia stem cells . SeveralAR, FOXA1, and MYC oncogenes, and decreased oncogenic expression in prostate cancer cells [The ATP-dependent chromatin remodelers consist of the SWI/SNF, ISWI, INO80, and CHD families. SWI/SNF complex is a major regulator of distal lineage-specific enhancer activity . Deletioer cells .The INO80 complex occupies SEs and drives oncogenic transcription by regulating Mediator recruitment and nucleosome occupancy . SilenciAnticancer drug Lysine-specific demethylase 1 (LSD1) inhibitor (NCD38) activates GFI1-SE and induces lineage switch from erythroid to myeloid by activating differentiation in leukemic cells ,57. NCD3http://clinicaltrials.gov/). Whereas CDK7 inhibitors such as SY-5609, XL102 and CT7001 are under clinical trials for advanced solid tumors . These inhibitors are in clinical trials either being tested alone or in combination with other drugs.BET inhibitors such as FT-1101, RO6870810 (TEN-010), I-BET762, BMS-986158, OTX-015 (MK-8628), ABBV-075, AZD5153, BI 894999, ODM-207, ZEN-3694, PLX51107, NUV-868, TQB3617, and CPI-0610 are under clinical trials for haematological and solid tumors  in pancreatic cancer [Transcription factor IIH (TFIIH) is a 10-subunit complex  that regulates RNA Pol II transcription. Triptolide inhibits XPB subunit of the TFIIH complex and disrupts SE interactions and down-regulated SE-associated genes , CK2 inhibitor (CX-4945) or PP2A activator (perphenazine) could overcome this resistance [7, CK2 inWNT4, LRP5, BRAF, GNA13, and PDPK1 in breast cancer cells [GNA13 and PDPK1 expression leading to drug resistance in breast cancer [Long-term treatment of JQ1 resulted in the activation of drug-resistant genes in breast cancer cells. BRD4 associates with the repressive complex LSD1/NuRD1 and occupies H3K4me1 defined SEs. BRD4/LSD1/NuRD complex then represses the activation of drug-resistant genes such as er cells . During t cancer . CombineMYC is activated in the absence of BRD4, possibly by activation of the Wnt pathway [Myc expression equivalent to the control cells was observed, as β-catenin occupies the sites where Brd4 is decreased. Inhibition of Wnt signaling resensitizes the cells to BET inhibitors [Myc [Myc expression by recruiting activated Wnt machinery to compensate Brd4 loss. Overall, Wnt signaling acts as a driver and biomarker in acquired BET-resistant leukemia.By contrast, drug-resistant AML cells show yet another mechanism for acquiring drug resistance, in which  pathway ,76. BET  pathway . Despitehibitors . SimilarIn the future, strategies will need to be devised to reduce or delay the development of drug resistance, for example, through combinations of cancer therapies where multiple epigenetic drugs are used to target potential avenues of drug resistance.PDGFRA by contacting the gene with its enhancer outside of the TAD , and cohesin complex ,82. It iThese pieces of data highlight the importance of cohesin complex in genome folding, and thus, inhibitors related to cohesin complex, loading proteins, and release proteins may be potential drugs for affecting 3D genome organization  . Some drYY1 is another well-known chromatin regulator that controls enhancer–promoter loops. Depletion of YY1 can disrupt such interactions . AttemptCTCF is another architectural protein that is absolutely and dose-dependently required for both chromatin looping and TADs formation . No CTCFMYC-associated zinc finger protein (MAZ) has been demonstrated to function as an insulator protein by augmenting the CTCF binding to organize the genome. Depletion of MAZ disrupted the short-range chromatin interactions, as well as decreased the insulation score of TADs, which makes it a promising genome organizer [Since CTCF-associated partners also mediate many of the DNA binding and chromosomal organization responsibilities of CTCF, they may be good candidates to target. For example, the rganizer .Topoisomerase II beta (TOP2B) is another well-known CTCF partner that can bind to CTCF to facilitate supercoiling at the TAD boundaries . ZNF143 Besides individually validated CTCF partners, there are some CTCF mass spectrometry data in different cell lines ,110,111,Polycomb repressive complexes (PRC1 and PRC2) also contribute to the 3D genome organization. PRC2 consists of four core subunits EZH1/2, EED, SUZ12, and RbAp46/48, together with various other components, including AEBP2, PCLs, and JARIDS . Gain-ofPRC1 complex is formed by CBX , PCGF1-6, HPH1-3, and two RING proteins (RING1 and RING2) . Hi-C daSeveral features of SEs in embryonic stem cells and cancer cells include a high density of TFs and co-activators binding, the robustness of driving gene expression, and the ability to simultaneously activate two independent genes ,41. In aEukaryotic cells contain membraneless organelles, such as nucleolus, Cajal bodies, stress granules, nuclear speckles, and P bodies that are formed through a process of liquid-liquid phase separation (LLPS). These membraneless organelles are collectively called biomolecular condensates . Phase sTranscriptional condensate formation at specific genomic loci is a cooperative process, involving a combination of specific structured TF-DNA interactions and weak multivalent interactions between intrinsically disordered regions (IDRs) of TFs, co-activators , and RNA Pol II  139,140,142. AhnEsrrb gene in mouse embryonic stem cells [To link the enhancer DNA sequence and the formation of phase-separated condensates, Shrinivas et al. demonstrated that the DNA sequences encoding TF binding site number, density, and affinity must exceed sharply defined thresholds for transcriptional condensates to form at specific genomic loci that function as enhancers . Sabari em cells .The transcription factor YY1 is overexpressed in prostate , colon , liver , lung 1, and breIt is still unclear how transcriptional condensate formation may contribute to long-range chromatin interactions or higher-order chromatin organization. Shrinivas et al. proposed a model in which transcriptional condensates at SEs are formed by the universal cooperative mechanism of phase separation . In thisHowever, this model does not clarify the sequence of events underlying the formation of long-range chromatin interactions or the formation of transcriptional condensate. It has been proposed that the chromatin loop extrusion mechanism, which is regulated by the insulator protein CTCF and cohesin complex, drives genome 3D organization ,155. LeeThe concentrations and chemical modifications of RNA molecules have been shown to play a regulatory role in the formation and dissolution of condensates. Henninger et al. proposed a non-equilibrium RNA feedback model in which lower concentrations of short RNAs produced during transcriptional initiation enhance condensate formation, whereas increasing concentrations of the longer RNAs produced during elongation result in condensate dissolution . These rThe post-translational modification of proteins involved In the formation of condensate dictates the condensates in which they associate. For example, the C-terminal domain of RNA Pol II is subjected to phosphorylation by CDK7 or CDK9 that alters the partitioning behavior of RNA Pol II into phase-separated condensates that are associated with transcription initiation and elongation at genes driven by SEs . In addiIn contrast to transcriptional condensates, phase separation has also been implicated in the assembly of inactive chromatin compartments. For example, the repressive factor HP1α oligomers induce rapid chromatin compaction into nuclear puncta, suggesting that phase separation can be used to organize constitutive heterochromatin by recruiting known components of heterochromatin, such as nucleosomes, and DNA into HP1 droplets and selectively excluding TFIIB from the condensate . Moreove1,6-hexanediol treatment disrupts condensates by interrupting hydrophobic interactions . This weInterestingly, phase-separated condensates can concentrate small-molecule drugs and influence their pharmacodynamics by enhancing their target engagements . CisplatCells rely on 3D genome organization for transcriptional control. Additionally, normal cells also utilize SEs to regulate transcription and form phase-separated condensates for their normal cellular functions. The critical question is to develop drugs that selectively target oncogenic SEs, specific chromatin interactions or cancer-associated condensates. Thus, an improved understanding of the mechanisms by which SEs and their transcriptional components promote oncogenic transcription and cancer-associated condensate formation could be useful in designing more selective and efficient drugs for cancer treatment.Another critical question is how do we ensure that normal cells can tolerate 3D genome-modulating drugs, SE inhibitors and phase-separated condensate inhibitors? As 3D genome organization and liquid phase condensation are also critical processes in normal cells, we expect that drugs affect these processes might be toxic to normal cells as well as cancer cells. In this way, such drugs will be highly toxic to normal cells, causing a barrier to development in a clinical trial. Some drugs such as curaxins, can specifically kill cancer cells without severe toxicity for normal cells. However, the mechanism of such specificity needs to be addressed. Thus, in-depth research focusing on the differences in 3D genome organization in different types of cancers and normal cells need to be conducted.cis-regulatory elements, such as an enhancer, to activate genes on the same chromosome [trans-regulatory elements, such as extrachromosomal circular DNAs (ecDNAs) can promote intermolecular enhancer-gene interactions and drive oncogene amplification by recruiting RNA Pol II and BRD4 in ecDNA hubs [The field of phase-separated condensates has received much interest in the past few years. Emerging evidence has shown that biomolecular condensates add new insights to the regulation of gene expression. Studies have shown that transcriptional activation in endogenous genomic loci could occur in the form of transactivation hub or phase separation ,170,171.romosome . In addiDNA hubs ,173. WheThe formation of phase-separated condensate is a thermodynamic process, and, so far, most research has given much attention to the formation of condensates. The current view of how condensates regulate transcription activation is often simplistic. How such dynamically condensates achieve reversibility is another direction for future research. It is important to understand that the physicochemical properties determine condensate dynamics in cells in order to design better drugs that target specific condensates. To our knowledge, there is no study comparing the composition and dynamics of different phase-separated condensates in relation to transcription activation or repression.Given the evidence that a gene can be activated by SEs in an additive , synergiConcentrations and chemical modifications in RNA molecules have been shown to play a role in regulating condensate formation and dissociation ,158. HowCondensate formation, dissolution, and function can be modified by the post-translational modifications of constituent components in the transcriptional condensates and are thought to change the selective condensate partitioning behaviour of molecules. Therefore, it is important to gain more insights into the epigenetic modifications of DNA, RNA and proteins that contribute to this selective partitioning behavior of these molecules. For example, third-generation sequencing platforms offered by Oxford Nanopore Technologies allow for direct identification of chemical modifications in DNA and RNA molecules ,183,184,MYC oncogene in ER+ breast cancer cells and tamoxifen disrupts the formation of condensates [cis-elements . The pressing question is to determine the mechanisms by which these mutant molecules contribute to pathogenic condensate processes, and to identify the molecules that are involved therein that might become potential targets for cancer therapeutic intervention [Estrogen enhances formation of MED1 condensates at the densates . ERα poidensates . Togethedensates . Howeverrvention .