Dysregulated gene expression is intrinsic to cell transformation, tumorigenesis and metastasis. Cancer-specific gene-expression profiles stem from gene regulatory networks fueled by genetic and epigenetic defects, and by abnormal signals of the tumor microenvironment. These oncogenic signals ultimately engage the transcriptional machinery on the cis -regulatory elements of a host of effector genes, through recruitment of transcription factors (TFs), co-activators and chromatin regulators. That said, whether gene-expression in cancer cells is the chaotic product of myriad regulations or rather a relatively ordered process orchestrated by few TFs (master regulators) has long remained enigmatic. Recent work on the YAP/TAZ co-activators has been instrumental to break new ground into this outstanding issue, revealing that tumor cells hijack growth programs that are active during development and regeneration through engagement of a small set of interconnected TFs and their nuclear partners. YAP/TAZ are essential mechanosensors of mammalian cells and are found disproportionally active in most human solid tumors. A wealth of experimental evidence has indicated how YAP/TAZ can drive proliferation and other malignant traits in cancer cells . YAP/TAZChIP-seq experiments, thanks to their unbiased nature, have also revealed two unexpected principles of YAP/TAZ transcriptional control. The first one relates to the distribution of YAP/TAZ/TEAD binding sites relative to genes annotated in the human genome. Only a minute fraction of these sites falls on promoters or near to the transcriptional start sites; rather an overwhelming amount  of YAP/TAZ-bound elements correspond to distant enhancer elements –11. The It has been proposed that transcriptional coactivators such as MED1 and BRD4, as well as transcription factors, form phase-separated condensates at superenhancers; this results in the concentration of the transcriptional machinery and robust expression of superenhancer-controlled genes –15. BothYAP/TAZ lack an intrinsic DNA-binding domain, and thus can contact the DNA only indirectly through other transcription factors . A numbede novo appearance of YAP/TAZ-bound peaks represents an unusually common epigenetic blueprint of all CRCs when compared to normal intestinal tissue, independently of their molecular classification. Strikingly, a core of these YAP/TAZ-fueled deregulated enhancers is also active in diverse tumor types, irrespectively of tissue of origin and genetic aberrations [A second principle that has emerged from unbiased ChIP-seq data of a variety of cellular contexts is that, after the TEAD consensus, the second most frequent motif at YAP/TAZ bound peaks corresponded to the consensus for AP-1 TFs , 18, 19.rrations .The above results resonate with those emerging from combined machine-learning and chemicogenomics approaches aimed at the identification of pan-cancer signaling dependencies leading to YAP/TAZ activation . Intriguin vitro and in vivo results validating this model, and of the many ways by which YAP/TAZ activation feedbacks on expression and regulation of AP1 family members. To start, in Drosophila, the YAP/TAZ homologous gene Yorkie contributes to guarantee sufficient expression of ATF3, with loss of Yorkie and AP-1 causing the collapse of the gene-regulatory network sustaining tumor-specific gene signatures initiated by oncogenic Ras [in vivo [The notion that YAP/TAZ and AP1 converge at regulating a core, cancer-specific gene expression program is particularly appealing in light of enic Ras . In mammenic Ras . Conversenic Ras . More di[in vivo .AP-1 is not only a transcriptional partner of YAP/TAZ but also, in a typical self-sustaining positive feedback loop, a transcriptional target of YAP/TAZ. This loop has been documented in several contexts. For example, YAP/TAZ directly promote FOS transcription, in turn contributing to the YAP/TAZ-controlled gene expression program , 22.in vivo caused by overexpression of YAP itself [Inhibition of AP-1 blunts YAP/TAZ-driven tumorigenesis induced by inactivation of the Hippo kinases LAST1/2, and liver overgrowth P itself . MoreoveP itself . These rP itself ) and altP itself , yet thrThe widespread presence of AP-1 sites at YAP/TAZ regulated genes raises questions on to what extent targeting the YAP/TAZ-TEAD interaction may be in fact sufficient to blunt YAP/TAZ-driven tumorigenesis. YAP has been indeed shown to bind directly to AP-1, and recent work by K. Struhl and colleagues proposes that YAP/TAZ may serve as transcriptional co-activators of JUNB to control a set of genes active during cell transformation, but that only in part overlap with those regulated by YAP/TAZ/TEAD . Clearlyde novo recruitment of Pol2 at YAP/TAZ-regulated promoters. Moreover, YAP/TAZ also contact Pol2 through the Mediator complex (MED1), promoting CDK9-mediated phosphorylation of the Pol2 C-terminal tail, as such favoring transcriptional pause-release [Binding of YAP/TAZ to chromatin is just the first of a series of biochemical steps eventually culminating in RNA polymerase II (Pol2) recruitment and activation of YAP/TAZ-driven transcription. To start, YAP/TAZ recruit the general coactivator bromodomain-containing protein 4 (BRD4), in fact dictating the genome-wide association of BRD4 to chromatin, and endowing to YAP/TAZ-bound enhancers the same functional attributes of superenhancers . Thus, Y-release . All in YAP/TAZ has been recently shown to interact also with CDK7, a component of the basal transcriptional machinery . Interesin vivo phenotypes recapitulating those ascribed to YAP/TAZ activation, such as pancreatic acinar to ductal metaplasia, liver regeneration and cardiomyocyte proliferation [Finally, YAP/TAZ nuclear functions are buffered by an inhibitory association with the BAF-SWI/SNF complex, through the ARID1A subunit . Of noteferation . That saferation . This rain vivo and in a variety of organs and tissue types, coupled with the apparent dispensability of YAP/TAZ for the normal homeostasis of those same organs, has fueled translational research aimed to develop drugs specifically targeting YAP/TAZ function [The ability of YAP/TAZ to sustain tumorigenesis function . The disfunction . As mentfunction . Then, tfunction , althougfunction ).in vivo.In conclusion, research on YAP/TAZ transcriptional mechanisms has started to shed some light on essential mediators, mechanisms and genome-wide regulatory elements that are critical for YAP/TAZ biology, laying the groundwork for new routes of pharmacological interventions aimed at controlling YAP/TAZ responses


CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the ATG promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene  The renin–angiotensin–aldosterone system (RAAS) plays an important role in the pathogenesis of cardiovascular diseases ,2,3. AngHypertensive mice that systemically express human renin and human AGT show elevated renal angiotensin II levels . We haveAldosterone is produced in the zona glomerulosa of the adrenal cortex by aldosterone synthase (CYP11B2) and is known to promote cardiac fibrosis and hypertrophy with concurrent elevation of inflammatory and oxidant signaling ,15,16,17DNA methylation is a stable, long-term epigenetic modification that affects gene expression without altering its DNA sequence ,32. In mAGT promoter possesses a number of CpG dinucleotides that are targets for DNA methylation (AGT promoter which is located within a CCAAT enhancer-binding protein (CEBP)-binding site that contains a CpG dinucleotide at positions −218/−217, is hypomethylated in tissues and cells with high AGT expression  but not in those with low expression  [AGT expression. In the human ATG promoter, each of these sites, upstream stimulatory factor (USF)1/estrogen receptor (ESR)1, CEBPB/nuclear receptor (NR)3C1, hepatocyte nuclear factor (HNF)1A, 4A, USF1,2, NR2F1 and ESR1 contains each CpG dinucleotide  proteins, which favor the formation of the transcriptionally inactive forms of chromatin (heterochromatin). Among the MBD proteins, methyl-CpG-binding protein 2 (MECP2), MBD1 and MBD2 repress the transcription of methylated gene promoters. The human hylation . The humR cells) . Thus, tcleotide .AGT promoter and its first exon (−459 to +66) have been analyzed to determine where and how DNA demethylation and remethylation affects the interleukin 6 (IL6) stimulation. DNA demethylation was detected within 12 h of IL6 stimulation and reached a maximum level 24 h following stimulation. The first DNA demethylation event occurred around a CEBP-binding site (positions −225/−217) as well as a transcription start site (TSS); DNA demethylation from CpG9 to CpG12 had started by that time, spreading out and stretching to CpG2 on the 5′-upstream side and to CpG17 on the 3′-downstream side during IL6 stimulation [Overall, 17 out of the 18 CpG dinucleotides within the human mulation .AGT transcription in human hepatocytes. The positive effect of STAT3 on the IL6-induced AGT transcription is mediated by its interaction with its two target DNA sites (positions −278/−269 and positions −171/−162). IL6 also induces the interaction between HNF1A and the AGT promoter at positions −247/−236.27. Additional transcription factors, such as STAT3 and HNF1A, are recruited when chromatin is relaxed and DNA is unmethylated, as MBD proteins have difficulty interacting with DNA without methylated CpG dinucleotides. DNA demethylation on the 5′-upstream side reaches CpG2 (position −434/−433), suggesting that as-yet-unidentified DNA-binding transcription factors are also involved in the IL6-induced AGT transcription. Following the IL6 withdrawal, DNA remethylation concurrently progresses in a reverse manner over the next 24 h [Local DNA demethylation is one of several steps in the serial recruitment of multiple transcription factors. In addition to CEBP, the signal transducer and activation transcription factor 3 (STAT3) and HNF1A are involved in the IL6-induced Excess sodium intake is intimately involved in the pathogenesis of hypertension ,39,40,41AGT gene in both humans and rats [AGT gene. High salt intake increases the AGT mRNA levels in the heart and demethylates the AGT promoter in SSH rats  has been reported with increasing frequency and accounts for 5–10% of the hypertensive population . IncreasAGT promoter with an accompanying upregulation of adipose tissue AGT expression. However, an excess of cortisol in Cushing’s adenoma did not affect the methylation status of AGT. They observed dynamic changes in DNA methylation patterns following IL6 stimulation, the first DNA demethylation event occurred around CpG9 in the AGT promoter. CpG9 exists in a DNA sequence that functions as both a GRE and a CEBP-binding site , Ad4, Ad5 and NBRE-1 (NGFI-B (nerve growth factor-induced clone B) response element). ATF1 or CREB1 bind to Ad1/CRE, leading to increased CYP11B2 transcription . DNA methylation at CpG2 reduced the basal binding activities of NR4A1 (NGF1B) and NR4A2 (NURR1) with Ad5 by 30% and 50%, respectively . PreviouCYP11B2, is mainly controlled by the body sodium status via the RAS. Sodium restriction or Ang II infusion significantly increases CYP11B2 mRNA levels in the rat adrenal gland [CYP11B2 and increased gene expression in the adrenal gland [CYP11B2 and increases the CYP11B2 mRNA levels parallel with the aldosterone synthesis. Plasma renin activity is also increased by a low salt diet. These results clearly indicate the influence of the angiotensin II in the epigenesis of CYP11B2 in the adrenal gland. Nishimoto et al. [In the adrenal cortex, the expression of the aldosterone synthase gene, al gland . We repoal gland . A low so et al.  reportedAldosterone breakthrough is a phenomenon in which plasma aldosterone concentrations increase above pretreated levels after a long-term therapy with angiotensin-converting enzyme (ACE) inhibitors or Ang. II type 1 receptor blocker (ARB) ,69,70,71CYP11B2 was detected in APAs and a negative correlation between the CYP11B2 mRNA levels and the methylation ratio was seen in our clinical samples ). We found that the KCNJ5 mutation was detected in aldosterone-producing microadenoma and APCC in which hypomethylation of CYP11B2 was seen (unpublished data). Sun et al. [CYP11B2 in APCC should be clarified.Hypomethylation of  samples B. Hypomeo in APA . Howard o in APA  reportedi et al.  reportedi et al.  reportedi et al. ,78. Thesi et al.  reportedn et al.  demonstrCYP11B2 in both normal and aldosterone-producing adenoma cells.Medical treatment with a MRA, such as spironolactone, eplerenone or esaxerenone, is recommended to patients with PA due to bilateral adrenal disease or patients with unilateral PA who are unable or unwilling to undergo surgery ,82. SeveHypertrophic cardiomyopathy (HCM) is the most common genetic heart disease, characterized by complex pathophysiology, heterogenous morphology, and variable clinical manifestations. The main goals of pharmacological therapy in HCM include the treatment of the left-ventricular dysfunction and HF and the control of the atrial fibrillation (AF) ,90,91,92CYP11B2 gene expression have been shown to be upregulated in cardiac tissues during hypertrophic cardiomyopathy (HCM), and these are recognized as one of the major modifiers of the HCM phenotype [n = 9) and non-HCM (n = 6) tissues. The CYP11B2 mRNA levels of HCM were 3.9-fold higher than those of non-HCM  plays a central role in the RAAS, which is involved in the pathogenesis of cardiovascular diseases ,107,108.y of SSH . Epigeney of SSH . Inhibity of SSH  reportedy of SSH  reportedy of SSH . FurtherAGT and CYP11B2 is reversibly regulated by an epigenetic modification. Both a high salt diet and an excess aldosterone production influence the methylation status of the AGT and CYP11B2 genes. The relationship between the epigenetic regulation of RAAS by environmental factors or hormone excess and the pathogenesis of cardiovascular diseases should be further clarified.The presence of local RAAS in cardiovascular and adipose tissues and their influence in cardiovascular and metabolic diseases are described. Gene expression of


Histone-modifying and remodeling complexes are considered the main coregulatorsthat affect transcription by changing the chromatin structure. Coordinatedaction by these complexes is essential for the transcriptional activation ofany eukaryotic gene. In this review, we discuss current trends in the study ofhistone modifiers and chromatin remodelers, including the functional impact oftranscriptional proteins/ complexes i.e., “pioneers”; remodelingand modification of non-histone proteins by transcriptional complexes; thesupplementary functions of the non-catalytic subunits of remodelers, and theparticipation of histone modifiers in the “pause” of RNA polymeraseII. The review also includes a scheme illustrating the mechanisms ofrecruitment of the main classes of remodelers and chromatin modifiers tovarious sites in the genome and their functional activities. Fig. 1A).The principal coregulatory complexes involved in the transcription of any geneare chromatin modifiers. They are divided into two large, functionallydifferent groups: complexes that change the position of nucleosomes and thosethat covalently modify histones in chromatin.The general activation process of eukaryotic gene transcription begins with thebinding of an activator protein  to aregulatory element. The activator protein, with the help of protein complexcoregulators, promotes the recruitment of general transcription factors (GTFs)to the gene. Multiprotein coregulatory complexes coordinate the transcriptionprocess; they integrate signals from various DNA-binding activators andchromatin modifications and transmit them to GTFs. AcFig. 1C), 3.CovaFig. 1C).A positFig. 2,which includes references to scientific studies describing the individualproperties of the chromatin modifiers). In more detail, we describe those areasof study pertaining to chromatin modifiers that have advanced significantly inrecent years. In addition, we discuss a number of issues that have not yet beenresolved.This review aims to summarize the available information on the functionalproperties and recruitment mechanisms of chromatin modifier complexes  in the course of evolution, the most important way to regulate genetranscription has been to influence chromatin packaging, determining theavailability of regulatory DNA elements. The protein complexes called chromatinremodelers belong to the transcriptional coregulators that affect the chromatinstate , 59. TheThe molecular mechanisms of pioneer factorsin vitro) [in vivoare capable of affecting chromatin in quite complex ways , which is hardly possible for individualmonomer proteins [DNA-binding transcription factors play the main role in the specificity ofeukaryotic transcriptional regulation. It is the set of transcription factorsassociated with the regulatory element that affects its type of activity . It is agenerally accepted fact that most transcription factors  cannot bind to the regulatory DNA region occupied by nucleosomes. Itis believed that a special class of DNA-binding proteins called pioneer factorsis responsible for the displacement of compacted chromatin from regulatory DNAelements; the FoxA and GATA factors are prominent examples of this class . These pn vitro) . At theproteins .Fig. 3A)[Fig. 3B).Further research is needed to understandhow common this molecular mechanism is in nature.According to current views, it is unlikely that pioneer factors function assingle proteins in living cells. Most likely, their unique ability to act oncompacted chromatin is a consequence of cooperative multiprotein interactions.An example of such joint functioning can be the paired work of a pioneer factorwith a nuclear receptor  . It has Fig. 3A).Most liFig. 3A), 67.TheThe functional activity of chromatin remodeling complexes. Thepossibility of remodeling non-histone proteinsFig. 3C)[While the mechanism that organizes primary access to the regulatory elements ofcompacted chromatin remains unclear, the maintenance of nucleosome-free regionsis undoubtedly the responsibility of chromatin remodeling complexes. Ingeneral, transcriptional remodeling complexes can affect nucleosomes in avariety of ways: to remove them, shift, position, or replace histones withalternative variants. However, all these mechanical functions are based on thesame ability of remodelers to create a DNA loop within the nucleosome andchange its position relative to the nucleosome’s surface. The subFig. 3C). ComplexFig. 3C). The ISWFig. 3C). InaddiFig. 3C).in vitro [in vivothat the SWI/ SNF complex participates in the removal of therepressive effect of Sir3p from its target genes during the activation of theirexpression in the M/G1 phase of the cell cycle[It should be noted that chromatin remodeling complexes can directly influencenot only the position of nucleosomes on DNA, but also the association of otherDNA-binding proteins with chromatin .The abiin vitro .More reFig. 3D).Fig. 4A)[in vivois an extremely complicated methodological problem. The obtainedinformation can almost always be questioned because of the presence of indirectexperimental contributors.The functional role of the SWI/SNF remodeling complex in the withdrawal ofPc-driven repression has been demonstrated as relates to various organisms. The posFig. 4A).The rolThe noncatalytic function of remodeling complexes in the regulation oftranscriptionMany transcriptional chromatin remodeling complexes are characterized by thepresence of a large number of subunits, in addition to the enzymatic subunitresponsible for histone movement .MoreoveFig. 4B).Another remodeler enzyme,the chromodomain-containing ATPase CHD4/ Mi-2, was also shown to be able tointeract with other proteins to form the NuRD complex, which represses genetranscription [This development appears quite logical from an evolutionary point of view.Transcriptional activation and repression are extremely dynamic and complexprocesses. Within these processes, many multicomponent complexes replace eachother at high speed in a limited space . Thisexchange assumes a high probability of contacts between the participants and,accordingly, the possibility of positive or negative mutual regulation.Chromatin remodeling complexes, in the course of their work on a regulatoryelement, bring with them many additional noncatalytic subunits. It is likelythat while the ATPase part of the complex performs its main catalytic activity,the remaining subunits participate in the activation/ repression of thetranscription process . The bescription . This recription . The funcription .Fig. 4C)[Interestingly, the ATPase subunit of the SWI/SNF complex, the BRM protein, wasalso found to exert a repressive effect on transcription, independent of itscatalytic activity , 85. At Fig. 4C), 88, 89.The transcriptional complexes that covalently modify histonesSince the inception of the “histone code” hypothesis, proteinscapable of covalent modification of histones have been the subject of numerousstudies . For a lThe role of covalent histone modifications in the recruitment oftranscriptional complexes to chromatinInitially, the “histone code” hypothesis was actively investigatedin the context of the transcription activation process. Many researchers triedto establish the histone modifications that determine the recruitment of theprotein complexes stimulating transcription. In turn, researchers who studiedprotein complexes worked to determine the protein domains responsible for therecruitment of the complexes to the corresponding “activating”modification. It is worth noting that many of these studies provedunsuccessful. It turned out that such “activating” histonemodifications are often unable to recruit transcription complexes bythemselves. A striking example of such an “activating” modificationwith a complex history of its study is the trimethylation of histone H3 atposition 4. Indeed, there is much evidence of the correlation between thepresence of this modification on the promoter and the active work of thecorresponding gene . HoweverFig. 5A).Apparently, the process of recruitment of protein complexes to the regulatoryelements of DNA is more complicated than we had imagined earlier: it is notrealized through individual protein-protein interactions . The protein complexes that regulate transcription mostoften contain a set of different subunits, many of which carry differentprotein domains . It appearsthat several domains that are part of various subunits are involved in a singleact of recruitment of a transcriptional complex to chromatin. It is the set ofsuch DNA-protein and protein-protein interactions that are realized in aseparate act of recruitment of a complex to a regulatory element that candetermine the type of functional activity of the complex in a given chromatinregion .Thus, the propagation of Pc-dependent repression occurs with theparticipation of the PRC1 and PRC2 complexes, one of which is capable ofrecognizing the H3K27me3 chromatin modification, while the second introducesit. The interrelated molecular work of these complexes organizes thepropagation of Pc-dependent repression around the PRE elements, which areinitiators of Pc-dependent compaction [The role of covalent histone modifications in the spreading of compactedchromatin, which represses transcription, has been investigated much more indetail and unambiguously. The cell uses various systems to create regions ofcompacted chromatin to suppress unwanted gene transcription. Two active systemsof chromatin compaction can be distinguished based on the Pc and HP1 proteins,both containing chromoprotein domains capable of binding specific methylatedresidues of histone H3 , 103. Inmpaction . Apparenmpaction . A positmpaction .In the processes of activation and repression of transcription, the recognitionof histone modifications is often not the primary signal that determines therecruitment of transcriptional regulators. The logical extension of the“histone” code idea was the hypothesis holding that covalentmodifications of histones are necessary for the exchange of transcriptioncomplexes at regulatory sites .This waThe role of covalent histone modifications in the regulation of the RNApolymerase II pauseFig. 5C).Currently, it is believed that this mechanism iswidely used by organisms to regulate the transcription of genes that requirehigh accuracy of induction in space and time  [For a long time, the recruitment of RNA polymerase II to promoters wasconsidered the main mechanism of activation of gene transcription. Later, itbecame obvious that many inactive genes of multicellular organisms containbound RNA polymerase II on their promoters .The tral stage) . ThepreFig. 5D)[For instance, the first description of bivalent nucleosomes was provided in thecontext of studying the “pause” of RNA polymerase II . In mousFig. 5D). ThiscoFig. 5D).For theFig. 5D).It was There are a number of covalent histone modifications that are associated withthe release of RNA polymerase II from a “pause” state and thestimulation of transcription elongation. The acetyl residues of histones arepositive markers of transcription elongation. This role (to overcome thetranscription pause and stimulate elongation) was discovered for the mainacetyltransferase, functioning on enhancers, the CBP protein . CBP wasDrosophila cells)[Fig. 6A).We believe that during the formation of a transcriptional“pause,” not only RNA polymerase II, but also many coregulatorycomplexes that modify chromatin are recruited to the promoters. Thesignal-inducing transcription does not lead to a further increase in the levelof binding of these complexes but stimulates their functional activity, leadingto an increase in the level of chromatin modifications. We plan to test theprevalence of the effect of coactivator “pause” in theDrosophila genome in future studies.Recently, our group has studied the kinetics of recruitment of chromatinmodifiers and the appearance of covalent histone modifications in the firstminutes of transcriptional activation . We haveAre covalent histone modifications actually “sidetargets”?The covalent modifications of histones have long attracted the attention ofresearchers because of the popularity of the “histone code”hypothesis. In particular, the introduction of covalent histone modificationswas described as the main molecular function for a variety of transcriptionalregulators (including multisubunit complexes). Later, it was discovered that anumber of histone modifications make rather modest functional contributions tothe regulation of transcription and that the enzymatic functions of theregulators introducing them have completely different, nonhistone proteintargets, which are of greater importance.Fig. 6B)[A striking example of such a chromatin modifier is the SAGA complex, which iscapable of acetylating lysine residues in the histones H3 and H4. For a longtime, researchers believed that the acetyl groups introduced by this complexare specific labels that are accurately “read” by othertranscriptional regulators using protein “reader” domains. Inparticular, it was assumed that the modifications introduced by the SAGAcomplex are recognized by bromodomains, which are part of the SWI/SNF complex,which acquires the ability to remodel exactly acetylated histones . This hyFig. 6B).Fig. 6C)[Additional protein targets have been described for other chromatin modifiers.Thus, the arginine methyltransferase CARM1, originally described as a specificmodifier of 17-arginine in histone H3, was found to methylate the arginineresidues of many transcriptional regulators, modulating their functions, 126.InFig. 6C), 127.As we can see, a deeper study of transcriptional regulators, initiallycharacterized as chromatin modifiers, leads to the description of theiradditional enzymatic targets. It is likely that further study of theseadditional targets will uncover a higher functional significance in comparisonwith target histones, which for a number of modifiers can only be “sidetargets.” This assumption is supported by the results of some mutationalstudies that aimed at identifying the functional significance of individualhistone modifications. It has been shown that mutations in individual chromatinmodifiers have a stronger effect on the regulation of transcription thanmutations in their target sites in histones, thereby demonstrating the presenceof more significant targets –transcriptional regulators , 129. ItThe growing volume of experimental information on the mechanisms oftranscriptional regulation and the activity of coregulators has not led toanswers to some of the questions that were formulated earlier. Below, we willaddress several problems for which definite solutions have yet to be found,despite the wealth of experimental arsenals available today.Recruiting a transcriptional regulator to chromatin: in a complex with aDNAbinding protein or sequentially?Fig. 7A).DNA-binding proteins determine the specificity of the effect of the coregulatoron gene transcription. They mediate the binding of coregulators to enhancer andpromoter sequences. Until now, the general mechanism of interaction ofcoregulators with DNA has remained unclear. Does the sequential binding of theDNA-binding protein to the regulatory element and the subsequent recruitment ofthe coregulatory complex occur, or is the preformed complex recruited to thegenomic sites? (invivo biotinylation of proteins by their partners) will help answerquestions regarding the mechanisms underlying the interaction betweencoregulators and chromatin.The concept of sequential recruitment looks questionable in the context ofstudies focused on the dynamics of protein binding to chromatin. It was shownthat the association of any proteins with DNA only lasts a few minutes. In thisChanges in the subunit composition of protein complexes duringtranscription: transformation of the same complex or recruitment of a newcomplex?Fig. 7B).Thus, it is known that thetranscriptional coregulator SAGA exhibits acetyltransferase anddeubiquitinylating activities towards histones. Both of these activities arerequired for SAGA to function on the gene promoter, where it promotes theinitiation of transcription[Many coregulatory complexes are involved in the various stages of genetranscription. Often, in the course of the study of such complexes, researchersfocus on examining the distribution and properties of the enzymatic subunits ofthe complex, while the behavior of the other subunits remains unexplored.Nevertheless, for a number of transcriptional complexes, it was shown thattheir composition is not constant and can change depending on the stage of genetranscription[in vivo with processingfactors, while their knockdown led to impaired mRNA transport[The original strategy researchers employed to study the functions of proteinsand protein complexes was to perform an in-depth study of one original functiondescribed for a protein of interest. Subsequently, another direction in thestudy of protein properties became more prominent, in which researchers triedto indentify and describe as many new functions as possible for a singleprotein, even when their molecular processes were sufficiently distant fromeach other. Therefore, a number of metabolic enzymes normally functioning inthe cytoplasm of the cell were observed to shuttle into the cell nucleus understress conditions and control gene transcription, acting as transcriptionalregulators .AnotherFig. 7C).The ORCransport, 140.At some point, the problem of a rethinking of the available data andestablished views on the leading functions of some multifunctional complexesarises. It may well turn out that the initially described functional role formany regulators can only be an indirect result of their leading function, whichwas noticed much later. Given the exponential growth in the amount ofexperimental data, it is likely that we will have to go through such stages ofrethinking of the hierarchy of functions for most of the known proteins. Itseems to us that evolutionary research can be very helpful in this case.Obtaining information about the functional properties of proteins in non-modelorganisms can help trace the history of the emergence of new functions andcreate a hierarchy of their significance.Eukaryotic organisms use coregulatory complexes as one of the ways to controlthe transcription of a certain set of genes. Thus, coregulatory transcriptionalcomplexes may well be promising therapeutic targets for the development ofdrugs aimed at altering the transcription levels of a specific set of genes.Currently, there are a number of such drugs in clinical trials. The followingare considered the most promising transcription coregulator targets for thedevelopment of low-molecular-weight inhibitors: the EZH2 enzymatic subunit ofthe PRC2 complex, the Brd4 transcription elongation coregulator, and variousHDAC histone deacetylases , 143. Th


Intriguingly, gene ontology analysis revealed that promoters of the autophagy pathway-related genes were most significantly occupied by RAD51 in all four cell lines, predicting a non-canonical role of RAD51 in regulating autophagy-related genes.RAD51 is a key enzyme involved in homologous recombination during DNA double-strand break repair. However, recent studies suggest that non-canonical roles of RAD51 may exist. The aim of our study was to assess regulatory roles of RAD51 by reanalyzing RAD51 ChIP-seq data in GM12878, HepG2, K562, and MCF-7 cell lines. We identified 5137, 2611, 7192, and 3498 RAD51-associated cis-regulatory elements in all four cell lines. In addition, genome-wide USF1, USF2, and/or MITF-binding regions significantly coincided with the RAD51-associated cis-regulatory elements in the same cell line. Interestingly, the promoters of genes associated with the autophagy pathway, such as ATG3 and ATG5, were significantly occupied by RAD51 and regulated by RAD51 in HepG2 and MCF-7 cell lines. Taken together, these results unveiled a novel role of RAD51 and provided evidence that RAD51-associated cis-regulatory elements could possibly be involved in regulating autophagy-related genes with E-box binding proteins.RAD51 is a recombinase that plays a pivotal role in homologous recombination. Although the role of RAD51 in homologous recombination has been extensively studied, it is unclear whether RAD51 can be involved in gene regulation as a co-factor. In this study, we found evidence that RAD51 may contribute to the regulation of genes involved in the autophagy pathway with E-box proteins such as USF1, USF2, and/or MITF in GM12878, HepG2, K562, and MCF-7 cell lines. The canonical USF binding motif (CACGTG) was significantly identified at RAD51-bound  RAD51 plays a crucial role in homologous recombination (HR) during DNA double-strand break (DSB) repair ,3,4. RADRAD51 is highly expressed in various cancers including breast, lung, and pancreatic cancers ,13,14. ITwo recent investigations into the molecular mechanism of RAD51 have been extended to the transcriptional level through a genome-wide approach, using chromatin immunoprecipitation followed by sequencing (ChIP-seq). First, RAD51 was recruited to the active chromatin regions, which are preferentially repaired by HR . These scis-regulatory elements coincided with E-box proteins such as USF1, USF2, and/or MITF, especially on the genes associated with autophagy and lysosome. Our findings demonstrated an unrecognized function of RAD51 in transcriptional regulation of genes, which is related to autophagy in cancers.In this study, we investigated the novel role of RAD51 in regulating genes at the transcriptional level, by reanalyzing RAD51 ChIP-seq data in multiple cancer cell lines, such as GM12878, HepG2, K562, and MCF-7. We found that genome-wide RAD51 bindings were more strongly enriched at the active promoters with upstream stimulating factor (USF1 and USF2) motifs. Deeper analysis revealed that RAD51-associated https://www.encodeproject.org/, accessed on 12 March 2021) [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, accessed on 12 March 2021, version 0.6.4) with default parameters. Trimmed reads were aligned to the human genome (hg38 genome assembly) using Bowtie2 (version 2.3.4.1) with default parameters [p-value cutoff of 0.001 was used for the analysis. To further identify reliable peaks, those peaks that did not overlap at least 50% of regions between the biological replicates were discarded, using Bedtools (version 2.26.0) [ChIP-seq data were downloaded from the ENCODE database (ch 2021) . The accrameters . Duplicarameters . Peaks wrameters  with the 2.26.0) .Motifs, which were significantly associated with given binding sites (150 bp), were identified using HOMER (findMotifsGenome.pl script) with the following parameters: -size 150 -mask.http://metascape.org/, accessed on 12 March 2021) [Gene ontology (GO) analysis was conducted using Metascape (ch 2021)  with thehttp://www.interactivenn.net/, accessed on 12 March 2021) [https://igv.org/app/, accessed on 12 March 2021) [A Venn diagram was generated using InteractiVenn (ch 2021) , and snach 2021) . Heatmapch 2021)  based onhttp://www.oncolnc.org/, accessed on 12 March 2021) [RAD51 gene.Kaplan–Meier (KM) survival analysis was performed using Oncolnc (ch 2021) . PatientThe human breast cancer cell line MCF7 and the human liver cancer cell line HepG2 were obtained from the Korean cell line bank (KCLB). MCF7 cells were cultured in RPMI 1640, and HepG2 cells were cultured in minimum essential medium (MEM) . All media were supplemented with 10% fetal bovine serum  and 1% antibiotic–antimycotic (Gibco). All cell lines were routinely cultured in a humidified 5% CO2 incubator at 37 °C. MCF7 and HepG2 cells were seeded onto 12-well plates and incubated with cisplatin  at various concentrations  and RS-1 (medchemexpress) at various concentrations  and B02 (medchemexpress) at various concentrations  for 24 h at 37 °C in an atmosphere containing 5% CO2.ATG3 and ATG5 was normalized to the house-keeping gene TBP. Primers for these genes are indicated in Total RNA was extracted from cells using Ribospin II (GeneAll), and total RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit . Real-time PCR was performed in replicate with TOPreal qPCR 2X PreMIX (SYBR Green with low ROX). Rotor-Gene Q software 2.3.1 was used for capturing and analyzing spectral data. Relative mRNA expression of p-values < 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism 8 .All quantitative data are presented as the mean ± SEM. One-way analysis of variance (ANOVA) was used to identify statistically significant data. https://www.encodeproject.org/, accessed on 12 March 2021) [p-value cutoff of 0.001. To further filter out non-reliable RAD51 binding sites, we discarded the peaks that did not overlap between biological replicates. The analysis revealed that the characteristic of genome-wide RAD51 binding was surprisingly similar to those of typical transcription factors. For example, there were totals of 5137, 2611, 7192, and 3498 RAD51-associated cis-regulatory elements in GM12878, HepG2, K562, and MCF-7 cell lines, respectively  revealed that RAD51 binds to transcriptionally active sites ,29, suggch 2021) . There wch 2021) . RAD51 bectively , and up ectively a. This rectively b, which ectively c. Manualectively d. Accordectively . Collectp-value of 1.0 × 10−1214. Notably, all of these transcription factors are known to bind to a consensus DNA element called an enhancer box (E-box), which harbors the palindromic DNA sequence “CACGTG”, and these E-box motifs were highly specific for RAD51 binding since motifs found at H3K4me3-enriched regions (active histone modification) in the same cell line were much less significant than the E-box motifs in terms of the p-value  regions relative to the center of the RAD51 peaks. Surprisingly, the known binding motifs for the upstream stimulating factors (USF1 and USF2), followed by CLOCK, MITF, bHLHE40, and TFE3, were significantly overrepresented at the RAD51 binding sites in all four cell lines a. For ex p-value . In addi p-value a. Accord p-value , we hypo p-value b. For in p-value . The resOur comprehensive analysis of genome-wide RAD51 binding sites revealed that those in GM12878, HepG2, and K562 cells significantly coincided with E-box binding proteins such as USF1 and USF2 b. The moTo further validate the predicted role of RAD51 in regulating autophagy-related pathways in vitro, we first checked expression levels of the RAD51 gene in various cancer cell lines . As expeSince RAD51 plays a pivotal role in homologous recombination, almost all studies regarding RAD51 have focused primarily on the role of RAD51 in homologous recombination. This is because RAD51 is a recombinase that filaments on double- and single-stranded DNA , in ordeThe paradoxical role of autophagy in cancer cells has been documented. For example, the role of autophagy in cancer initiation and epithelial–mesenchymal transition (EMT) is antitumoral, while its role in the growth of the primary tumor and anoikis resistance is protumoral . Thus, tcis-regulatory elements, the predicted functions of which were related to the autophagy pathway. Our integrative analysis of various transcription factor ChIP-seq data further showed that RAD51 may contribute to gene regulation through USF1 and USF2. Although results from our study provide the first in silico evidence of RAD51 contributing to the autophagy pathway, through RAD51-associated cis-regulatory elements, experimental validations are warranted to verify these findings.We reanalyzed RAD51 ChIP-seq data collected from GM12878, HepG2, K562, and MCF-7 cell lines and identified 5137, 2611, 7192, and 3498 RAD51-associated


We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development  and splicing factors  showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.During positive selection at the transition from CD4 Summary: The pioneer transcription factors Foxa1 and Foxa2 are required for positive selection during T-cell development in the thymus for regulation of RNA splicing. Maturation from DP to SP follows successful rearrangement of the Tcra locus, and requires TCR signalling: positive selection results in appropriate MHC restriction of SP cells, and is followed by negative selection of potentially self-reactive clones and selection of regulatory T cells (Tregs)  . The str (Tregs) . For DP  (Tregs) . Many mo (Tregs) . Current (Tregs) , in whicto CD8SP . The CD4to CD8SP .Many transcription factors contribute to regulation of the CD4/CD8 lineage decision . AdditioHere, we investigate the role of the transcription factors Foxa1 and Foxa2 in T-cell development. The Foxa proteins are a highly conserved subfamily of forkhead box transcription factors, which contain unique wing-helix DNA-binding domains . The FoxFoxa1 and Foxa2 proteins are closely related to each other and are widely co-expressed during embryogenesis and in several tissues postnatally, including lung, liver, intestines, pancreas and thymus . GeneticFoxa2 is a well-recognized target gene of the Shh signalling pathway in the floorplate, and is required for the maintenance of Shh expression, but can also negatively regulate expression of components of the Shh signalling pathway . ExpressLittle is known about the function of Foxa1 and Foxa2 in the immune system. In mouse models of autoimmune inflammation, ectopic Foxa1 expression has been shown to drive the differentiation and suppressive function of a novel subset of Tregs . We receHere, we investigate the function of Foxa1 and Foxa2 in developing αβT cells. We show that Foxa1 and Foxa2 are required at the transition from DP to SP T cell by regulation of RNA splicing. Conditional deletion of both Foxa1 and Foxa2 led to a reduction in positive selection, differentiation and maturation of the SP populations, and a reduction in the peripheral CD4 T-cell pool.Foxa1 and Foxa2 expression by qRT-PCR in FACS-sorted developing thymocytes from the DN3 stage onwards  CD4SP and CD8SP populations . Thus, in the absence of Foxa1 and Foxa2, not only were there fewer CD4SP and CD8SP cells, but their maturation was also affected, such that fewer mature CD62L+CD4SP were produced to egress from the thymus. In contrast, we found no significant difference in the proportion of CD4+CD25+Foxp3+ Tregs, NKT cells or NK cells in the Foxa1/2cKO thymus compared with control , CD69, Qa2 and CD62L. After positive selection, SP thymocytes retain high expression of HSA and CD69, and as they mature HSA and CD69 are downregulated, Qa2 is upregulated, and expression of CD62L indicates that thymocytes are mature and ready to egress from the thymus. The proportion of HSAl thymus A. Furthe control B,C, wher control D,E.Fig.+ T-cell populations mirrored the thymus in the Foxa1/2cKO compared with control, with reductions in the proportion and number of conventional CD4+ T cells overall and of naïve CD44−CD62L+CD4+ T cells in spleen  between Foxa1/2cKO and control datasets (FDR-adjusted P<0.05), of which 109 (62%) were more highly expressed in Foxa1/2cKO than in control (Table S1). Of the 176 DEGs, 85 (∼48%) had previously been identified as Foxa1/2 targets in genome-wide ChipSeq analysis of Foxa1/2-binding sites in dopaminergic neuronal progenitors and 56 of those genes with verified Foxa1/2-binding sites (approximately two-thirds) showed higher expression in the Foxa1/2cKO than in control (Table S1) (Table S1). Among these, DEGs that were more highly expressed in Foxa1/2cKO than in control datasets included genes known to reduce TCR signal strength [Cbl, a ubiquitin ligase that negatively regulates TCR signalling  , 2015. Dable S1)    to compare the overall pattern of gene expression in our datasets with transcriptome data from publicly available datasets prepared from DP thymocytes that were receiving different strengths of TCR signals during selection (SE38909) . The GSESE38909) . We selesduction H. In conlo/negCD69− (pre-selection thymocytes), TCRβintCD69+ , TCRβhiCD69+  and TCRβhiCD69− (more mature population). The proportion of pre-selection thymocytes was higher in the Foxa1/2cKO than control thymus, whereas there was no significant difference in the proportion of the TCRβintCD69+ population, and both TCRβhiCD69+ and TCRβhiCD69− populations were reduced, indicating that Foxa1 and Foxa2 expression in thymocytes promotes initiation of the process of positive selection, and also progression of cells during the differentiation process  was increased in the CD69+DP population in the Foxa1/2cKO thymus compared with control . Among the transcription factors for which expression was downregulated by Foxa1/2 deletion, there were no obvious downstream candidates that might function to promote positive selection, although several showed changes in expression consistent with the developmental phenotype, for example Ptma, an anti-apoptotic gene . Thus, as well as an overall enrichment in immunity-related terms, as expected from the cell type, there was enrichment for terms related to processes required for differentiation to SP, such as lymphocyte migration, as differentiating cells must migrate to the medulla to complete their maturation and repertoire selection. Several terms related to cytokine signalling are also pertinent to the partial arrest at the DP and CD4+CD8lo stage in the Foxa1/2cKO, as cytokine signalling rescues cells for which the MHCI-restricted TCR signal has been interrupted by downregulation of CD8, allowing differentiation to CD8SP, whereas cytokine signalling is inhibited for differentiation to the CD4SP compartment.We then carried out Gene Ontology (GO) term enrichment analysis of all DEGs between Foxa1/2cKO and control CD69Mmp14, a membrane-bound matrix metalloproteinase involved in breakdown of the extracellular matrix  between the conditional knockout and control, which involved 628 different genes, 222 (∼35%) of which had previously been shown to bind to Foxa1/2 in genome-wide ChipSeq analysis of Foxa1/2-binding sites in dopaminergic neuronal progenitors (Table S3) (Table S1). This intersection included several genes involved in the regulation of RNA splicing, with known splicing variants  , 2015. Cable S3) F, 18 of variants G: Ranbp2ormation ; Sf3b1, splicing ; and Smgsplicing .Table S4). Many terms associated with mRNA splicing were also over-represented  and with their known functions in other tissues as epigenetic regulators and pioneer transcription factors   (Table S3). Many of the T-lineage genes that contained significant DEU have been previously described to be alternatively spliced during lymphocyte development or activation, such as the Ikaros family members Fyn, Ptprc (CD45), Stat5a, Stat5b and Cd28  , many of which are important during T-cell development, and are specifically required for the transition from DP to SP cell   encoded transcription factors  B.Fig. 7Here, we show that the pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during T-cell development at the transition from DP to SP thymocyte. Conditional deletion of Foxa1 and Foxa2 from developing DP thymocytes led to reduced positive selection and a partial arrest at the transition from DP to SP thymocyte, with a reduction in SP cell maturation, and a reduced peripheral naïve CD4 T-cell population. Conditional deletion of Foxa1 and Foxa2 also led to significant changes in the expression of genes that regulate RNA splicing in cells undergoing positive selection, and concomitantly to >850 significantly differentially used exons.Socs1, Ptprc, Rasgrp1), and many encode transcriptional regulators of this stage of development . GO term enrichment analysis also highlighted terms associated with positive selection and differentiation to SP. In contrast, few DEGs encoded known relevant transcription factors or regulators of TCR repertoire selection or differentiation from DP to SP, and Themis, a DEG with a verified Foxa1/2-binding site, which was more highly expressed in the conditional knockout, and which can modulate TCR signalling in thymocytes, also showed DEU. The way in which the transcriptional activity of Foxa1/2 relate to their regulation of splicing will require further investigation, as there is increasing evidence that splicing can occur co-transcriptionally as well as post-transcriptionally  and the histone linker H1f0, which may be required for mRNA splice site recognition , 1.5 mM MgClescribed . Primer Foxa1 and Foxa2 WT and floxed gene primers and PCR conditions were as described , DN4 (CD4−CD8−CD25−CD44−), DP (CD4+CD8+), CD4SP (CD4+CD8−CD3+), CD8SP (CD4−CD8+CD3+) and CD69+DP (CD4+CD8+CD69+), thymocyte cell suspensions were sorted using Molecular Flow Cytometer (Cytomation).To obtain DN3 .RNA was extracted using Stratagene StrataPrep Total RNA miniprep kit according to the manufacturer's protocol and cDNA was synthesized using SuperScript III kit (Invitrogen) according to the manufacturer's protocol. RT-PCR was carried out by analysis of cDNA sample in triplicate on an iCycler (Bio-Rad Laboratories) using the iQ SYBR Green Supermix according to the manufacturer's instructions. The housekeeping gene Table S5). Cell suspensions were stained for 30 min on ice in PBS supplemented with 2% fetal calf serum and 0.01% sodium azide. For intracellular Foxp3 and Nr4a1 staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization kit (eBioscience). AnnexinV staining was carried out as described . Therefore, the z-scores returned by DESeq2 were used as input to the CRAN package fdrtool (1.2.16) to re-estimate the null variance and subsequently the P-values . Genes with adjusted P-values <0.05 were considered as DEGs.CD69escribed  and sequescribed . The Bioescribed . P-valueP-values . AdjusteP-values were then calculated by Benjamini–Hochberg false discovery correction (5%).The Bioconductor package DEXSeq (1.36.0) was used to test for DEU . The PytTo identify over-represented gene ontology terms in the set of DEGs and genes with DEU, we used the Bioconductor package GOseq (1.42.0) . For datTo identify verified transcription factors among DEGs and DEU genes, we merged the most recent updated lists from the Riken mouse transcription factor database  and the P-values, calculated by moderated eBayes adjusted for false positives) between DP thymocytes stimulated with a non-selecting control peptide and DP thymocytes stimulated with the positively selecting peptide were used to generate a scale of unstimulated to TCR-signalling-for-positive-selection, and we regressed our datasets onto this axis.Canonical correspondence analysis is a multivariate analysis that allows the comparison of experimental transcriptome data with publicly available datasets from other laboratories . CCA was


Drosophila melanogaster, they were associated with stable sustainment of gene repression and maintenance of cell identity. Acting as multiprotein assemblies with an ability to modify chromatin, through chemical additions to histones and organization of topological domains, they have been involved subsequently in control of developmental transitions and in cell homeostasis. Recent work has unveiled an association of Polycomb components with transcriptionally active loci and the promotion of gene expression, in clear contrast with conventional recognition as repressors. Focusing on mammalian models, I review here advances concerning roles in transcriptional control. Among new findings highlighted is the regulation of their catalytic properties, recruiting to targets, and activities in chromatin organization and compartmentalization. The need for a more integrated approach to the study of the Polycomb system, given its fundamental complexity and its adaptation to cell context, is discussed.The Polycomb system is made of an evolutionary ancient group of proteins, present throughout plants and animals. Known initially from developmental studies with the fly  Specific and coherent gene expression patterns are essential to cell diversity of multicellular organisms. Such a selective use of the encoding potential of genomes is achieved largely through transcriptional regulation. Much of this function relies on transcription factors, proteins that bind DNA with both low and high affinities. Eukaryotic genomes are organised as chromatin, a lineal succession of nucleosomal units resulting from wrapping DNA around histones, thus precluding binding sites from access to transcription factors. This arrangement, further elaborated as high-order and topological domains poses important regulatory opportunities through the modulation of chromatin structure. This is accomplished by the action of chromatin regulators, a vast collection of multiprotein complexes endowed with catalytic and other activities, affecting nucleosomal dynamic or their chemical modification.Hox genes, observed during the genetic analysis of the development of the fly Drosophila melanogaster, constitute the foundation of the discovery of the Polycomb group (PcG) of genes. Both circumstances, their activity in D. melanogaster and its initial association with gene repression has come to impact enormously how the study of the Polycomb system is approached. Its subsequent association with the repression of tumor suppressors and oncogenic transformation in mammals prompted further the interest in the system. It may appear paradoxical, however, that while these early functions link the Polycomb system with transcriptionally silent states, a large number of genomic sites occupied by Polycomb are transcriptionally active, and that in certain cell types even the expression of some of them depends on the presence of Polycomb products.Within the ample collection of chromatin regulators present in eukaryotes, the Polycomb system has achieved a major prominence due to the nature of the genes under its regulatory influence, many of them important developmental regulators. Precisely, the morphological, homeotic alterations associated to the derepression of Drosophila development as antagonists of Polycomb mutations, the TrxG system will not be covered in any detail. Excellent TrxG-centered reviews can be found in [Organised in biochemically heterogeneous, evolutionarily conserved protein complexes, the Polycomb machinery carries out a diversity of functions in transcriptional control and genome stability. Here I review recent advances in its architecture and its recruiting to targets regarding transcriptional functions, with an emphasis in metazoans and mammals in particular. Comprehensive reviews of plant PcGs can be found in ,2. Althofound in ,4,5.O-GlcNAc transferase (OGT), the product of Drosophila Super sex combs, an enzyme that adds N-acetylglucosamine to a multitude of proteins, some of which are PRC subunits [The products of the PcG of genes form multiprotein assemblies termed after their best known transcriptional activity as Polycomb Repressive Complexes (PRCs). From plants to metazoans, PRCs are a collection of biochemical entities containing either of two catalytic modules endowed with histone modifying activity. Not all subunits present in the distinct PRCs correspond to products of  PcG genes. Conversely, some of the PcG gene products are not subunits of Polycomb complexes. An example of the latter is subunits . PRCs arHox genes in flies while it protects genes from PRC1 repression in mammals [Depending on the nucleosomal substrate they modify, two major classes of PRCs are recognised: PRC1, that contains an E3 ubiquitin ligase that monoubiquitylates lysine 119 of histone H2A (H2AK119Ub) , and PRC mammals ,14. More mammals .PRC1 and PRC2 can be thought of as catalytic modules made of a core of subunits shared within either class of complexes, to which a variety of subunits associate, frequently in a mutually exclusive fashion, thus making the heterogenous collection of assemblies that PRCs are. Cell context-dependent combinations of subunits results in sets of related but different PRCs. Recent surveys of PcG subunits, functional domains and evolutive relationship can be found in ,16. HereDrosophila PcG genes or their mammalian homologs, are known as canonical complexes, to distinguish them from a distinct, new set of  PRC1 complexes identified later on. All PRC1 assemblies share a heterodimeric E3 ubiquitin ligase, made of combinations of two types of RING finger-containing paralogs. One of them is made of RING1/RING1A and RNF2/RING1B, homologs of Drosophila Sex combs extra (Sce). The other, by either of the six products of the family of the Polycomb group of genes (PCGFs), whose RING fingers appear conserved in Drosophila Posterior sex combs (Psc), Suppressor of zeste 2 (Su(z)2) or Lethal (3) 73Ah ((l(3)73Ah). The RING finger structure is characterised by a C3HC4 motif that binds two zinc cations in a way that builds a globular, compact domain apt for protein–protein interactions [Drosophila Polycomb (PC). For reasons that are not clear, CBX-containing PRC1 complexes usually contain either PCGF2/MEL18 or PCGF4/BMI1 but no other PCGF members. It is important to note that the PRC1 architecture described above, as that of PRC2 (2.2), is a version distilled from evidence obtained from a rather reduced number of cell types. As research expands into other cell models or developmental times, variant PRC1 complexes may appear that do not conform fully to the above picture. Examples are the presence of CBX proteins associated to subunits of non-canonical complexes [Initial PRC1 preparations, containing the products of characteristic ractions . RING1 aractions ). Both wractions ,20. Giveractions . Bound tractions ,22. Thisractions . CBX subomplexes ,25.Drosophila Polyhomeotic (PH). The relevant BMI1 sequence is conserved in the Ub-like domain of PCGF2–MEL18, the other PCGF family member of canonical PRC1 that also binds PHC2 [The identity of the PCGF member in the heterodimer decisively influences the nature of other subunits that associate with the core complex, in agreement with a modular organisation of PRC1 deduced from reconstitution experiments A. For exnds PHC2 . It is nnds PHC2 ,30,31. Inds PHC2 ,33,34, pLCOR gene, which also encodes C10ORF12, a shorter form of PALI1) or PALI2 (encoded by the LCORL gene) [The catalytic core of PRC2 is rather homogeneous compared to the variety of PRC1 E3 modules resulting from iterations of RING and PCGF pairs. In addition to the lysine methyl transferase (KMT) , the enzymatic module of PRC2 contains subunits EED, SUZ12 and one of the extremely similar paralogs RBBP4, RBBP7. In cells expressing both EZH paralogs, PRC2 complexes contain one or the other ,36. EZHsRL gene)  can be fRL gene) ,38,39. PRL gene) ,38 and oRL gene) ,41. CoexDrosophila embryos [miR-125 and miR-181 families of miRNAs [It is important to notice that the descriptions of PRC1 and PRC2 complexes correspond to the forms found in abundant cell types such as cultured cell lines: human (Human embryo kidney (HEK)) 293 ,13,21,42 embryos ,50. In v embryos , thus ma embryos . When ES embryos ,51. A sif miRNAs ). A simif miRNAs . For PRCf miRNAs ,56,57. Sf miRNAs ,59.Quantitative proteomics analysis during the generation of neural progenitors from ESCs, a widely used model of in vitro cell differentiation, illustrates accurately changes in PRC composition associated to cell identity . Thus, wThe activity of the Polycomb system relies not only on the histone modifications induced by the catalytic modules of PRC1 and PRC2  but also on protein–protein interactions mediated by non-core subunits. These interactions, for example, are decisive for the formation of defined high-order and local chromatin structures. Both functions, catalytic and architectural, can be independent of each other. Additional enzymatic activities found in subsets of PRC complexes, such as CBX4-dependent protein sumoylation , or the Drosophila), and to equivalent lysines in variant histone H2AZ [The content in histone H2AK119Ub per cell is not known in a systematic manner, but work with cells lines indicates that it accounts for, approximately, 10% of total histone H2A . This hione H2AZ ,69.PRC1 recognition of the nucleosomal substrate relies on an interaction (arginine anchor) shared by many other chromatin modifiers . RING1–PDespite the structural characterisation of the complex between PRC1 catalytic modules and the nucleosome, the description of the modification process is still work under progress. For example, in vitro studies indicate that efficient monoubiquitylation requires auto-polyubiquitylation of RING1B . Steady Drosophila proteins where the activity was first identified: Suppressor of variegation 3–9, Enhancer of zeste and Trithorax [Mono-, di- and trimethylated H3K27 are modifications mediated uniquely by PRC2 subunits EZH2 and EZH1. The reactions are catalyzed by SET domains (named after three rithorax ), presenrithorax . Its actThe predominant product of PRC2 activity is the dimethylated form of H3K27 ,80,81. QEZH2 catalytic activity is not only regulated by interrelated interactions among core components but also can be stimulated, indirectly, through non-core subunits. For example, the presence of AEBP2 in reconstituted PRC2 complexes activates K327me3 deposition on nucleosomal templates containing monoubiquitylated H2AK119 . LikewisDrosophila TrxG gene [H3K27me3 spreading, and the associated effects on transcription are maintained in check by chromatin modifiers that antagonise PRC2 catalytic activity. Most relevant is the methylation of histone H3K36, which results in an effective inhibitor of H3K27 trimethylation ,97. ThusrxG gene ,99 or ofrxG gene ,101,102 rxG gene  it is lirxG gene  result fDrosophila and mammals. Regardless of the experimental approach, whether conventional fluorescent in situ hybridisation [Sites occupied by Polycomb proteins, sometimes separated by long genomic distances, can be physically proximate, as seen both in disation ,104, supdisation ,106,107 disation ,110,111,disation ,113. Drosophila Posterior Sex Combs (PSC) [Two types of protein structures, present in subunits of canonical PRC1 assemblies, appear as major determinants of these clustering events . One is bs (PSC) ,116. IDRbs (PSC) ,118).Drosophila, H3K27me3-marked domains form highly compacted structures in which chromatin intermixing occurs at a high rate while, at the same time, remains isolated from other nuclear domains [Drosophila, however, where CTCF loops are not as prevalent as in mammals, looped structures identified within Polycomb-repressed compartments have PC-enriched boundaries [Drosophila [PRC1 SAM-mediated contacts results in hundreds of domains, comprising from small, 20–140 kb genomic segments , to much domains . These r domains ,107. Sim domains ,120. By  domains ,108 they domains ,122. Som domains . In Drosundaries . Whetherosophila ,126 or wDrosophila PSC and mammalian CBX2/M33 [Drosophila Psc or mouse Cbx2/M33 establish their functional relevance in vivo [PRC1 IDRs were identified in a search for protein motifs responsible for the correlation between in vitro compaction of nucleosomal arrays and the inhibition of chromatin remodeling and transcription on defined templates by reconstituted/purified PRC1 complexes ,129. IDRCBX2/M33 ,116. In CBX2/M33 ,131. Phe in vivo ,133. For in vivo ,134 and Meis2 sites occupied by RING1B, at the promoter and downstream the polyA addition site, during mouse midbrain development. These interactions prevent a critical enhancer from reaching the promoter at early stages of development, while the release of RING1B from the promoter, at a later time, permits Meis2 gene activation [The above architectural functions of PRC1 align well with gene repression functions. However, Polycomb-dependent enhancer–promoter and promoter–promoter contacts are also involved in facilitating gene activation. For example, following EED depletion in ESCs, looped structures containing poised enhancers and promoters are severely affected, and although the enhancers do not become activated, the expression of genes  linked to them is defective in their differentiated progeny . The reqtivation .Drosophila, mostly the products of non-PcG genes) and a hierarchical model in which PRC1 complexes were “hooked” onto PRC2-modified nucleosomes, through the recognition of H3K27me3 by CBX subunits. Currently, the localisation and activities of PRCs on chromatin is considered the outcome of a multitude of interactions involving DNA, histone tails, nucleosome and even RNA (The range of residence times (on chromatin) for Polycomb subunits is similar to that of conventional transcription factors . Associaeven RNA .Drosophila pleiohomeotic (Pho) was the only subunit known to encode a bona fide DNA-binding protein [Recruiting via DNA can occur either by direct DNA-mediated interactions, through a PcG subunit or, indirectly, through DNA-bound proteins that do not form part of Polycomb complexes. Direct recruiting to targets has been an elusive mechanism because during some time because  protein . Pho, id protein ,147), pl protein . In mamm protein ,143, its protein ,150.Drosophila. The closest PhoRC-related complex in mammals, PRC1.6, shares some of subunits, including MBT-domain protein L3mbtl2. However, instead of YY1, it uses as DNA-binding proteins MGA–MAX or E2F6-TFDp1 heterodimers [Pho can be isolated as part of a complex—Pho-repressive complex (PhoRC)—in which MBT domain-containing Sfmbt is also present . As PcG rodimers ,156,157.rodimers ,155,156,Despite the important recruiting function of PhoRC, Pho binding by itself is insufficient, and additional PRE-binding proteins are required. Unlike Pho, these DNA-binding proteins Direct recruiting of vertebrate PRC complexes through PcG DNA-binding proteins has come along rather slowly. For example, PRC1 recruitment through MGA–MAX and E2F6–TFDp1 heterodimers, mentioned before, has been verified only recently, despite their presence in PRC1.6 being known for a long time . In otheIn vertebrates, these unmethylated CpGs concentrate on singular genomic regions known as CpG islands (CGIs), in the proximity of a large proportion of promoters ,172. A sDrosophila KDM2B homolog, KDM—a component of dRAF complex [The affinity of PcG DNA-binding motifs for CGI sites is but one of the multi-contact steps involved in recruitment. For example, PRC1 localisation in ESCs after KDM2B/FBXL10 downregulation is affected only partially, as shown by a limited decrease of RING1B occupancy ,179,180. complex —plays anDrosophila mutants carrying a null Pcl allele, the loss of H3K27me3 occurs with little alteration in the levels of H3K27me1,2 [Drosophila PCL binds DNA with no preference for non-modified sequences [The second DNA-binding domain in Polycomb proteins that recognise GC-rich sequences is formed by a region contiguous to the C-terminal end of the second Plant Homeodomain (PHD) domain of PCL subunits, which folds as a winged helix motif ,164. PRCK27me1,2 . The affK27me1,2 . HoweverK27me1,2 , is someK27me1,2 ,172. In equences , perhapsHox genes, for instance) decreases upon inactivation of Dnmt1 [The involvement of JARID2 and AEBP2 in PRC2 targeting is mediated only in part by their DNA-binding activities. JARID2 localizes to GC-enriched genomic sites  and, togof Dnmt1 , the majIt is also worth noting a DNA-binding activity in SUZ12, defined by structures in its N-terminal moiety not folded in a recognisable motif that determines occupancy of CGIs in ESCs, independently of EED or EZH2 .Some PcG subunits, in both PRC1 and PRC2, contain protein motifs for recognition of modified histone tails, including the products of their own enzymatic modules . This prDrosophila PC [Drosophila PC. Some mammalian CBX proteins even show no preference over H3K9me3 tails [The widely accepted notion of H3K27me3-dependent interactions as a major PcG recruiting mechanism originated from work with phila PC . As othephila PC . Mammaliphila PC . The facphila PC . Howevere3 tails . Given te3 tails ,204, thee3 tails , perhapse3 tails . Moreovee3 tails . In summe3 tails .The interaction of PRC2 with H3K27me3 uses an entirely different aromatic cage, defined by residues of the blade-folded WD-repeats of EED ,195. MorXist expression in an ESC model of X-chromosome silencing, occurs at a lower rate when the cells lack RYBP and YAF2 [Monoubiquitylated histone H2A is a bulky modification recognised by the ubiquitin-binding zinc finger of non-canonical PRC1 subunit RYBP ,207 , also present in other chromatin regulators, which binds the most terminal region of histone H3 ,200. ThiDrosophila PCL to recognise methylated histone peptides [Drosophila Trx-G gene Ash1 and murine Nsd1 [Mammalian DNA-binding PCL subunits in PRC2 complexes contain a Tudor motif that binds methylated K36 in histone H3 tail ,198,199.ine Nsd1 ,211, whiine Nsd1 .Altogether, the interactions of Polycomb complexes with modified histone tails may influence residence times, in a recruiting perspective, but they appear to have evolved rather towards the modulation of their catalytic activities. lncRNAs) Xist on the inactive X chromosome (Xi) or HOTAIR on the HOXC cluster and the recruiting of both PRC1 and PRC2 [Another potential pathway for localization of Polycomb activity to defined genomic sites is through the recognition of chromatin-bound RNA. This notion, based initially on correlations between the presence of long non-coding RNAs .ANRIL lncRNA, transcribed from the INK4 locus in orientation antisense to that of mRNAs encoding tumor suppressors (Ink4a/p14 (p16 in mice), Ink4aArf/p19 and Ink4b/p15) has been shown to participate in PRC1 recruiting to this well known Polycomb target [TUG1 or lncRNA MALAT1/NEAT2, resulting in CBX4 localization to Polycomb bodies or to interchromatin granules, respectively [Concerning PRC1 subunits, and in line with previous studies reporting binding of certain chromodomains to RNA , all murb target . Anotherectively . Recent ectively , althougPolycomb localisation on chromatin has been approached mostly by asking how the complexes interact effectively with their components and remain at sites long enough to induce histone modifications or other structural changes. Less studied is the opposite scenario, that of pathways promoting release from chromatin. One obvious scenario is through post-translational modifications, of which phosphorylation-dependent release of PCGF4/BMI1 from chromatin in human HeLa cells is among the first examples  . Switching of silent, PRC1-occupied sites programmed to become active during retinoic-acid triggered differentiation of ESCs is accompanied by RING1B release in a ZRF1-dependent process . Not a fDrosophila homolog BRG). It appears that, in contrast to other remodelers, the major substrate of BAF complexes are not nucleosomes. Instead, they participate in PRC1 and PRC2 eviction from chromatin. Oncogenic mutations in BAF components correlate with defective  or enhanced  ability to evict Polycomb complexes from chromatin [Another eviction mechanism involves the BAF (Brg/Brahma-associated factors) complex, a member of the group of chromatin-remodeling complexes whose DNA helicase belongs to the SNF2 subset of ATP-binding helicases of the DEAD/H family . A measuhromatin ,237. Thuhromatin . Artifichromatin . Such anhromatin . Forced hromatin . Major thromatin ,240.Overall, the impact of components of Polycomb-eviction systems has been studied in a reduced number of situations. For example, neurogenic differentiation of ESCs is particularly affected upon ZRF1 depletion, whereas mesodermal or endodermal derivatives in teratomas derived from these cells are less affected . ZRF1 isIt is likely that cell context complexity has made a systematic characterization of Polycomb targets difficult, and a detailed description of known targets in diverse cell types is beyond the scope of this review. Loci occupied or modified by PcG products are considered candidates for direct transcriptional regulation. Initial genome-wide studies of the DNA bound to PRC1 and PRC2 proteins, isolated by chromatin immunoprecipitation (ChIP) studies, identified sites in embryonic cell lines of flies and mammals corresponding to developmental genes activated during differentiation processes ,245,246.A shared functionality of Polycomb targets in primitive cells, then, is the preservation of their self-renewal and developmental potential. This includes transcription factors with roles in cell commitment, and receptors and their ligands, of important signaling pathways  ,245,246.The study of better resolution and depth ChIP assays have subsequently expanded the range of Polycomb targets, both in flies and mammals, to include loci encoding products with metabolic, cytoskeletal and signaling functions ,76. ThesHow Polycomb transcriptionally regulates these targets can only be described, mechanistically, in rudimentary terms. In part, this is due to a still incomplete understanding of the mechanisms governing transcription  but alsoDrosophila [Recent, unbiased analysis of Polycomb localisation unveils their presence on transcriptionally active promoters and enhancers besides their well known association with repressed/poised promoters. In fact, a large proportion of Polycomb targets identified in mammalian in vitro cell models correspond to transcriptionally active loci ,256,257.osophila ,260,261.Drosophila, where the canonical ones, decorated with H2AK118Ub and H3K27me3, are derepressed when genes encoding either PRC1 or PRC2 subunits are mutated, whereas non-canonic targets, lacking these marks, respond only to mutations in PRC1-encoding genes [On transcriptionally active sites, PcG products are said to perform non-canonical functions ,76, in cng genes ,144. DesPolycomb-dependent negative regulation of gene activity affects both transcriptionally active and inactive loci, corresponding most likely to two very different types of regulatory layouts. For historical reasons, however, the study of mechanisms that maintain repressed states has dominated investigations on Polycomb transcriptional functions. Perhaps under this influence, roles decreasing chromatin accessibility, supported by biochemical activities of PcG products, have appeared as the most important mechanism underlying Polycomb-mediated silencing. Indeed, although very scarce, the few studies that have addressed Polycomb function in global chromatin accessibility in vivo ) show PRC-occupied promoters as less accessible structures than PRC-free promoters . AnotherThe contribution of PRC1 to the nucleosomal configuration derives from the relaxed structure acquired by PcG targets in cells lacking both RING1A and RING1B, particularly at promoters of derepressed genes . As incrUbx, Abd-B) and the homeotic phenotypes in mutant flies whose histone H3 has a K27 to R mutation, demonstrating that PRC2-repression uses this H3K27 modification [Hox genes in similar fashion to that of E(z) mutants [If chromatin accessibility and nucleosome packing are, at least in part, unrelated to Polycomb influence on transcription what about the histone modifications dependent on Polycomb complexes? It is generally accepted that they anticorrelate with transcriptional activity, and that nucleosomes at silent canonical, but not to active PcG targets are marked with H2AK119Ub and H3K27me3 . The corfication . How thefication ,271,272  mutants ,273, theDrosophila  shows upregulation in about a third of these PcG targets [Drosophila cells, changes in the phosphorylated states of RNA polII and decreased recruiting of Spt5  are observed [Polycomb function(s) at transcriptionally active sites have not been studied in detail. PRC1 loss-of-function analysis in  targets ,259. Sim targets ,274, con targets . Followiobserved ,274. TheContrasting with these repressive functions, both at silent and also active sites, a number of observations document a PcG requirement, mostly for PRC1 components, in gene activation  functional diversity in the distinct settings. It is now clear that catalytic and non-catalytic functions can be unlinked, certainly for canonical PRC1 complexes. However, the actual role(s) of Polycomb-dependent histone modifications, even for those so well correlated with gene repression, such as H3K27me3, in any interaction with the transcriptional machinery are unknown. It is likely that progress in unraveling transcriptinal functions, particularly at a subset of active loci, will need simplified cell models and allele-specific analysis, so that the studies are not blurred by contributions of cell/allele diversity. Now that Polycomb is part of the current set of druggable biomolecules, addressing these questions may prove therapeutically beneficial.


The 3D organization of the genome facilitates gene regulation, replication, and repair, making it a key feature of genomic function and one that remains to be properly understood. Over the past two decades, a variety of chromosome conformation capture (3C) methods have delineated genome folding from megabase‐scale compartments and topologically associating domains (TADs) down to kilobase‐scale enhancer‐promoter interactions. Understanding the functional role of each layer of genome organization is a gateway to understanding cell state, development, and disease. Here, we discuss the evolution of 3C‐based technologies for mapping 3D genome organization. We focus on genomics methods and provide a historical account of the development from 3C to Hi‐C. We also discuss ChIP‐based techniques that focus on 3D genome organization mediated by specific proteins, capture‐based methods that focus on particular regions or regulatory elements, 3C‐orthogonal methods that do not rely on restriction digestion and proximity ligation, and methods for mapping the DNA–RNA and RNA–RNA interactomes. We consider the biological discoveries that have come from these methods, examine the mechanistic contributions of CTCF, cohesin, and loop extrusion to genomic folding, and detail the 3D genome field's current understanding of nuclear architecture. Finally, we give special consideration to Micro‐C as an emerging frontier in chromosome conformation capture and discuss recent Micro‐C findings uncovering fine‐scale chromatin organization in unprecedented detail.Gene Expression and Transcriptional Hierarchies > Regulatory MechanismsGene Expression and Transcriptional Hierarchies > Gene Networks and GenomicsThis article is categorized under: Progress in understanding 3D genome structure and function has come from the development of ever more powerful chromosome conformation capture methods. Here we provide an overview of these methods going from 3C to Hi‐C, orthogonal methods, and the recent development of Micro‐C. With all of our cells sharing the same genetic code, regulation of gene expression serves as the root of heterogeneity in cellular identity, response, and role. Given all of the information  that must be encoded in the human genome, it is perhaps no surprise that the diploid human genome is very long, spanning 6 billion base pairs. Stretched end‐to‐end, the DNA in each diploid human somatic cell would measure roughly 2 m; however, a need for space‐efficient storage of DNA results in its compaction by orders‐of‐magnitude to fit inside small nuclei less than 10 μm in diameter , and genes were sequenced for the first time Lis, . By the 22.13C, 4C, and 5C—Chromosome conformation capture technologies have primarily derived from the foundation laid by 3C (Chromosome Conformation Capture) in 2002 . Compartment domains, comprised of self‐associated A or B compartmentalized chromatin, manifest in a checkerboard manner across contact maps and are typically assigned using eigenvector decomposition. TADs, on the other hand, are local domains that manifest as triangles rising off of the contact map diagonal and are usually called based on an insulation score or directionality index  are called CTCF loops. Finally, dots and corner peaks between enhancers and promoters are referred to as enhancer‐promoter (E–P), enhancer‐enhancer (E–E), and promoter‐promoter (P–P) interactions, loops, or links.For the purposes of this review, we use the following terminology. We refer to  de Wit, . MechaniFinally, we note that features such as compartments, TADs, and CTCF loops are generally population‐level terms that are visible in contact maps after averaging across millions of heterogenous cells. Different terminology is sometimes used for domains observed using single‐cell methodologies that may be present in single cells, but sufficiently rare to not be clearly visible in population‐averaged contact maps. For instance, “TAD‐like domains” of contact that manifest in single‐cell data may be a snapshot of the intermediary formation of a domain observed in population‐level data  in 2009 altered this paradigm , and 4C (i4C) forgo cross‐linking and perform digestion and ligation in situ (discussed below); this not only reconstitutes known features of folding, but also improves the signal from more stable chromatin interactions  adopted an approach employed by the nuclear ligation assay by performing cross‐linking, restriction digestion, and ligation within intact nuclei, after which it isolated individual nuclei and proceeded through the rest of the Hi‐C methodology  methodology into chromosome conformation capture protocols. First introduced in 2005, ChIP‐loop  modifies the 3C protocol by enriching ligated fragments for contact with a protein of interest  and achieving finer resolution than Hi‐C . Given similarities in methodology, subsequent advances in whole‐genome ChIP‐based techniques have often drawn from parallel advancements in Hi‐C‐based techniques. For example, HiChIP, which exhibits improved sensitivity to DNA contacts and lowered input material requirements relative to ChIA‐PET, was built by leveraging principles of in situ Hi‐C  in distal elements to expression changes in their cognate genes  Capture‐C as a solution  addresses some of Hi‐C's shortcomings and reveals hubs of interaction within the nucleus , overcomes the limited ability of 3C‐based methods to capture multi‐way simultaneous interactions  and other aspects of organization such as compaction and association with the nuclear periphery , ionizing radiation‐induced spatially correlated cleavage of DNA with sequencing (RICC‐seq) is another noteworthy technique that has revealed nucleosome‐level folding and local structure  , identifying chromatin domains, and distilling cellular heterogeneity. With advancements in super‐resolution microscopy and synthetic oligonucleotide design, techniques such as fluorescence in situ hybridization (FISH), OligoPAINT, three‐dimensional assay for transposase‐accessible chromatin‐photoactivated localization microscopy (3D ATAC‐PALM), stochastic optical reconstruction microscopy (STORM), optical reconstruction of chromatin architecture (ORCA), and live‐cell imaging have uncovered structural insights and multiway interactions not captured by the other methods discussed here   degradation of CTCF established that CTCF loss reduces TAD insulation and that CTCF acts in a dose‐dependent manner, though with some boundaries only being moderately affected  “extrudes” DNA until it stalls at a boundary element   explain, researchers have started unraveling the complexity of the 3D genome across levels of organization and the competing forces at play. At the scale of whole chromosomes (hundreds of megabases), chromosomal territories have been known to segregate chromosomes into preferred locales within the nucleus for over a century   genome architecture by the mid‐2010s  and disuccinimidyl glutarate (protein–protein interactions); this double cross‐linking is unique to the Micro‐C method Figure . Cell me4.14 = 256 bp or 46 = 4,096 bp for 4‐bp or 6‐bp cutters, respectively; however, such an assumption is an oversimplification of the stochasticity of digestion. RE sites are not equally distributed throughout the genome, nor is all DNA readily accessible. The primary level of genomic organization is characterized by nucleosomes, in which 147 bp segments of DNA are tightly wound around histone proteins  digestion. This protocol alteration, though seemingly minor, shifts the resolution of conformation capture to finer scales. REs are endonucleases that cleave DNA at their recognition sequences, which are usually 4 bp, 6 bp, or 8 bp in length. When performing genome‐wide analyses , restriction digestion is therefore predicted to cleave the genome on average every 42+ dependent, MNase can be inactivated by calcium chelators for greater control over the extent of digestion . Micro‐C co‐opts MNase's indiscriminate digestion to achieve nucleosome‐level resolution by taking advantage of local DNA accessibility. With DNA wound around nucleosomal histones largely protected from enzymatic degradation, MNase digestion begins at linker DNA  are more clearly discernable in Micro‐C maps compared to Hi‐C maps Figure . Convers4.2i) and analyzed unique contacts in WT‐CTCF and ΔRBRi‐CTCF mESCs at medium resolution (668 M‐694 M contacts)  transcription‐dependent, enriched for Pol II and TFs; (2) ES‐cell‐specific with Nanog and H3K4me1 enrichment; (3) YY1‐related and found across cell types, with H3K27ac and Mediator enrichment; (4) repressive, enriched for bivalent chromatin related to the Polycomb complex; and (5) CTCF‐ and cohesin‐mediated. These analyses highlight an abundance of boundary factors beyond CTCF, which largely remain to be characterized. Krietenstein et al., clustering dots visible in Micro‐C but not Hi‐C analysis, report similar classification of loops anchors into five groupings. Furthermore, examination of peak enrichment at insulators in human ESCs and fibroblasts reveals boundary localization to nucleosome‐depleted regulatory elements and promoter marks, supporting similar results in yeast and mouse lines  should further improve Micro‐C's signal‐to‐noise ratio and address cell‐to‐cell or region‐specific variability in fine genomic architecture. Furthermore, like most 3C‐based technologies, Micro‐C is reliant upon proximity ligation and is limited to probing primarily pairwise contacts. For instance, with hundreds of known protein interactions with the promoter and enhancer regions demarcating fine‐scale boundaries, it is possible that multi‐way complex interactions at play in E–P and P–P stripes are better captured by non‐3C‐based methods.Beyond chromosome conformation capture technologies, techniques including cryo‐EM, fixed‐cell microscopy, live‐cell imaging, and polymer modeling will be instrumental for disentangling the mysteries of the 3D genome. The elucidation of the roles of CTCF and cohesin, the loop extrusion model, and the different forces at play across scales of organization have been foundational to the greatest breakthroughs in understanding chromosome conformation; however, many missing pieces in each of these stories remain to be found. Although significant biochemical evidence points toward cohesin‐driven loop formation, the structure and molecular mechanism of the extrusion complex has not yet been captured. The biochemical mechanism explaining the convergent rule for CTCF anchoring of cohesin extrusion is still unclear, as are the forces contributing to compartmentalization. And even as the field has shifted away from a static view of TADs and other features of nuclear architecture, active loop formation and dynamics have not yet been observed in vivo. Finally, the extent to which genome organization is instructive for regulating transcription as opposed to a downstream consequence from the act of transcription itself remains an urgent but poorly understood question. Thus, a diverse repertoire of methods will be necessary to tackle looming questions in chromatin organization in coming years.We declare that no competing financial interests exist.Viraat Goel: Conceptualization; investigation; writing‐original draft; writing‐review and editing. Anders Hansen: Conceptualization; funding acquisition; investigation; supervision; writing‐original draft; writing‐review and editing.