Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the composition profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.Intact-organism imaging of  Chromatin is a complex, linear macromolecule comprising DNA and histone proteins which in eukaryotic cells, is localized in the nucleus where it is solubilized in water, salts, and other small molecules . In manyDrosophila larvae nuclei where the chromatin was labeled by H2B-RFP, and the nuclear envelope was labeled by Nesprin/Klar-GFP. These experiments reveal and quantify chromatin-aqueous phase separation and its control by changes in the nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery (whether A or B) within the nucleus, is appropriate when the chromatin self-attraction relative to the chromatin-aqueous phase interaction, as well as the chromatin-lamina interaction are larger than the difference between the AA and BB interactions. The qualitative trends as well as the compositional profiles obtained in our simulations compare well with the observed, intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables, this allows us to identify the generic mechanisms underlying the changes in the phase separation that are observed.In most previous measurements, nuclear-scale phase separation of the chromatin and the aqueous phase has not been considered. However, recent super-resolution microscopy reveals a ‘marshland’ of chromatin and aqueous phase, with a non-uniform distribution of chromatin at the submicron level . Anothereriphery . Here, wDrosophila has only one A-type lamin (lamin C) and one B-type lamin (lamin Dm0) gene  chromatin-lamina, which is attractive and tends to organize the chromatin peripherally (ii) chromatin-chromatin, that if attractive, tends to condense the chromatin and separate it from the aqueous phase. The nuclear envelope contains lamina which is a dense, fibrillar network of proteins that provide anchoring points for various proteins that bind to chromatin on one end and the lamina on the other . These bm0) gene . A-type m0) gene . Those cm0) gene . The ancm0) gene . Experimm0) gene . The rolan cells .Previous models of chromatin-lamina interactions resolved euchromatin and heterochromatin domains in the context of conventional organization of chromatin  where phThe model we study is generic in nature, short-ranged and does not depend on the detailed molecular origin of the chromatin self-attraction. This is an appropriate approach for understanding and predicting the nuclear-scale concentration profile of the chromatin and aqueous regions in vivo, where the identity and function of all the molecular actors are so far unknown. Nevertheless, there is ample reason to believe that such attractions are present and important in determining chromatin organization. For example, the positively charged histone tails attract negative DNA linkers and can thus promote condensation of chromatin . ProteinIn this paper, we systematically examine how the interplay between chromatin-lamina interactions, intra-chromatin interactions, and hydration affect the chromatin organization. The biological observations of different organizational modes are simulated and explained by the polymeric properties of chromatin and the competition of its bulk interactions with the surface interactions with the lamina. Different organizational modes can affect gene expression since different amounts and sections of the chromosome are exposed to the aqueous phase and the proteins and enzymes that it contains. This may be why changes in nuclear volume have been demonstrated to result in changes in gene expression . Our simgeneric physical effects that determine the chromatin concentration profile at the nuclear scale. However, to predict this from computer simulations of chromatin as a self-attracting, confined polymer that is also attracted to the lamina at the nuclear periphery, we must treat a specific polymeric model. We therefore fixed the polymer monomer size, molecular weight, and persistence length, and instead varied the physical properties of the nucleus, including its volume and chromatin-lamina interactions. This was motivated by the intact-organism experiments  potential,rc refers to a cutoff distance beyond which LJ interaction is set to zero. Here In addition to the spring-bead model that accounts for the connectivity of nearest neighbor beads along the polymer chain, we include short-range interactions (both attractive and repulsive) between We model the nucleus to which the chromatin is localized, as spherical shell of confinement radius We varied d nuclei .Drosophila where the distribution of the LAD regions is available online  of e online . That dae online  and usede online .At the microscopic level, there are many possible interactions  which can cause chromatin self-attraction and lead to its separation from the aqueous phase. Since there may be additional bridging proteins and because even the ones we listed are not known in detail, we use a generic model for short-range chromatin-chromatin attractions. As outlined above, we model chromatin self-attraction by an attractive LJ potential  and to model that we use a LJ interaction with However, the most important feature of the chromatin-lamina interaction is the bonding of the LAD and the lamina via specific proteins. This depends on the availability of these proteins and the nature of their binding to the lamina. In the experiments  that indWhen n factor  of the LAdding up all these contributions yields the total potential energy of chromatin in our model nucleus:The total potential energy depends on the positions of the beads that determine their mutual interactions and those of the LAD beads with the lamina; the force on a given bead arises from the gradient of this potential energy.We now summarize the results of the Brownian dynamics simulations and discuss how conventional, peripheral and central organization of chromatin can be achieved by changing hydration  can bind to the lamin beads. This was suggested by the experiments in systems where lamin C was overexpressed and may be due to a reduction in the number of lamin-binding proteins or the obscuring or burial of lamin-binding sites in the overexpressed situation . The parDrosophila larva nuclei had an average value of In the experiments, the volume of live In the results shown above, we see how chromatin organization changes when we change We have shown here that the observed transitions in nuclear-scale chromatin organization in eukaryotic cells can be understood from the physics of a simple polymer model. The comparison of the theoretical and experimental trends indicates that these transitions are due to competition of polymer entropy, self-attraction of the chromatin and the chromatin-lamina attraction. By varying the values of the three generic parameters  The radius of confinement within the nucleus is greater than radius of gyration of chromatin (ψ=1) see .Starting with these parameter values that resulted in peripheral organization, we then varied ≤0.5 see . We alsoψ≤1) see . We plotψ≤1) see . In Appeψ≤1) see  and compψ≤1) see . We haveψ≤1) see .Drosophila nuclei  and heterochromatin  regions do not separate in the radial directions, but are separated in the perpendicular (angular) direction see . Our sim0.5, see . These va nuclei . We notesurfaces .To focus on the essential physics of chromatin self-attraction vs. chromatin-lamina interactions, we have simplified the chromatin self-attractions to be the same for the euchromatin (EC) and heterochromatin (HC) domains. This is appropriate when the chromatin-aqueous phase interaction and the lamin-chromatin interaction are larger than the differences in the interactions of euchromatin and heterochromatin. Our model is the minimal one required to understand how the chromatin separates from the aqueous phase in peripheral and central organization. Although, previous models explicitly accounted for different interactions of euchromatin and heterochromatin domains, they did not consider the various types of nuclear-scale chromatin/aqueous phase/laminar organization and typically focused only on conventional chromatin organization. In addition, in the Appendix 7, we have discussed simulation results that show coexistence (micro phase separation) of more condensed and less condensed regions of chromatin due to different self-attractions of the LAD and non-LAD regions. This is analogous to the observed differences in concentration of chromatin in heterochromatin and euchromatin. Those results also show that peripheral organization cannot be achieved if difference in interaction of the LAD and non-LAD regions is too large. This justifies the original model presented in the main text in which differences in the self-attraction of the LAD and non-LAD regions are neglected, as a first approximation. But at the next order of approximation where we include differences in the self-attraction of different chromatin regions, the simulations show micro phase separation.ks and The stretching energy of the chromatin chain is calculated as:kb is bending stiffness which is taken as lp is persistence length, σ is diameter of the bead, The chromosome has a persistence length of 2 beads (1.2 kb) which is based on a previous estimate  of a perThe system is simulated calculating the Brownian dynamics of the chromatin beads in which the aqueous solvent is treated implicitly by the average frictional force it exerts on the moving beads and by the deviations from this average, represented by a stochastic force. For the use in computer simulations, the discrete time  equation systems . The LAM systems .rb of the lamina to create/break bonds with lamin beads, via the spring model, allowing for both bond creation and breaking dynamics. After equilibration, we changed the chromatin-chromatin interactions to allow for self-attraction and let the system re-equilibrate, again using the LAMMPS package  that are mixed along the chain as indicated by the sequence data . The cen package . The parExperimental documentation techniques, sample sizes etc. are described in our experimental paper . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The authors study theoretically chromatin distribution in the nucleus. This is a very well-written study that identifies the physical variables underlying observed nuclear phase separation. It is of broad interest for understanding chromatin organization.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Helmut Schiessel (Reviewer #2).Thank you for submitting your article "Mesoscale phase separation of chromatin in the nucleus" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife.As the editors have judged that your manuscript is of interest, but as described below there are some doubts on whether your work goes sufficiently beyond your Ref.[12] of the manuscript to merit publication in Drosophila nuclei. This is a very well-written study that presents. The most important finding being that of the effect of the solvent quality on phase separation. However, the fact that the configuration of a spherically confined polymer can steered in the manner shown by directly manipulating the relative self-interaction versus the affinity of (parts of) the chain to the boundary is not really surprising. Also it is not clear, whether the difference between eu- and heterochromatin can indeed be neglected in this context.You study theoretically the effect of nuclear volume and the interaction of chromatin with the lamina on the distribution of chromatin within the nucleus using a simple polymer model. In your model, you also consider self-attraction of the chromatin. You find that depending on the relative strength of these effects, the chromatin is either homogeneously distributed within the nucleus or is localized peripherally. An intermediate state with the chromatin "partially wetting" the nuclear lamina is also observed. These states of chromatin distribution were also observed in eLife, please, make sure to comment in particular on why you think that the results presented here and that go beyond Ref.[12] merit publication in eLife rather than a more physics oriented journal like Soft Matter.If you decide to submit a revised version to Essential revisions:1. Why do you need a second potential in addition to the LJ potential after bond formation between an LAD bead and a lamina bead?2. Are the effects of fixation well described by changing the chromatin volume fraction alone?breakP}. One would expect to find in the exponent the difference between the elastic energy at σ and at br.3. The paragraph on page 8 about bond formation is a little difficult to follow. Could you clarify the probability for bond breaking C. elegans ) seems to suggest that the specific affinity of heterochromatin to the lamina is important to understand the global structure of the nucleus. The authors definitely appear aware of this issue, by explicitly stating that their model holds whenever the generic interactions between chromosome and lamins are more important than the e.g. eu/hetero cross-interactions. However, in the discussion they appear to want to have it both ways in the end, by appealing (without further references) to a correlation between the occurrence of LAD domains and the eu/hetero nature of regions of the chromosome. Could you clarify this issue?4. Earlier work in  Drosophila nuclei. This is a very well-written study that presents. The most important finding being that of the effect of the solvent quality on phase separation.You study theoretically the effect of nuclear volume and the interaction of chromatin with the lamina on the distribution of chromatin within the nucleus using a simple polymer model. In your model, you also consider self-attraction of the chromatin. You find that depending on the relative strength of these effects, the chromatin is either homogeneously distributed within the nucleus or is localized peripherally. An intermediate state with the chromatin "partially wetting" the nuclear lamina is also observed. These states of chromatin distribution were also observed in We thank the reviewers for their positive approach regarding our article; we are also grateful to the reviewers for their insightful comments and questions that have helped us to improve the manuscript. Below we address the reviewers’ concerns in detail. Based on the referee comments, we have done new simulations and have revised the manuscript to address all the concerns. The changes in the manuscript have been marked in red in the revised manuscript.However, the fact that the configuration of a spherically confined polymer can steered in the manner shown by directly manipulating the relative self-interaction versus the affinity of (parts of) the chain to the boundary is not really surprising.eLife and not to a physics journal, proper. The observations, model simulations and explanations change the biological paradigm and show that the competition of the surface and bulk interactions, can indeed be fine-tuned to lead to the transitions between peripheral, conventional and central (wetting droplet) organization. Our paper provides the conceptual and modeling basis for these transitions which are indeed observed in the experiments as we discuss in the paper.We believe that our paper is important for biologists because it presents a new paradigm that the chromatin can be (depending on hydration) phase separated in the nucleus. This is the first time that, to our knowledge, chromatin phase separation is discussed based on experimental data. That is why we submitted the paper to Moreover, we present a guide for further experiments that can map the global state diagram showing the transitions between conventional, peripheral and central (wetting droplet) organization of chromatin. An additional interesting observation, simulation result and explanation involves the wetting droplet  as well as the non-equilibrium droplet with arm-like extensions that are kinetically “stuck” to the lamin layer ; these are not commonly used in biology or biophysics. In our simulations in Appendix 5, we show that these different situations are kinetically determined.eLife. In light of the referee comment, we have further explained the novelty and importance of the paper on page 3 of the Introduction where we added the following text: “In this paper we systematically examine how the interplay between chromatinlamina interactions, intra-chromatin interactions, and hydration affect the chromatin organization. […] Our simulation results also provide suggestions to experimental groups on how to vary the minimal number of parameters that will experimentally result in different modes of chromatin organization in vivo.”Because chromatin organization can have profound effects on gene expression (see just below for the text we inserted in the paper), it is important to explain these effects and quantify them for biologists and biophysics even though some of the qualitative properties may not surprise experts in polymer physics, per se. That is precisely the reason we are submitting to Also it is not clear, whether the difference between eu- and heterochromatin can indeed be neglected in this context.We justify our use of the same self-attractions of eu and hetero chromatin, as a first approximation only, from the experiments themselves. The experimental figure of the peripheral organization shown in Figure 6(c), indicates that both heterochromatin and euchromatin are found in the peripheral part of the nucleus. They both separate from the aqueous phase in the center. This suggests that the chromatin attractions driving this phase separation are to a first approximation, similar for heterochromatin and euchromatin. Of course to the next approximation, the self-attraction of heterochromatin and of euchromatin differ and that is why the experiments imaged alternating regions of heterochromatin and euchromatin along the angular direction in the nuclear periphery ). Our simulations show that this can indeed be the case when the LAD interactions with the lamin are larger than the smaller differences between the self-attraction of euchromatin vs. heterochromatin ). We explained this in the paper on page 10 of the Discussion part.Based on the referee comments, we have added new simulation results to the paper that do take into account different self-attraction for the LAD compared to the self-attraction of the non LAD regions. Note that we are aware that there are other differences between heterochromatin and euchromatin such as histone modifications. These new simulations are not meant to replicate hetero and euchromatin in detail, but rather to show that micro phase separation within the chromatin (as opposed to chromatin/aqueous phase separation) can occur even for different self-attractions of the LAD and non-LAD regions. This is analogous to eu and heterochromatin whose detailed study is outside the scope of our paper. We have done this in the limiting case that the attractions of the LAD regions  are larger than the non LAD regions . In Appendix 7, motivated by previous models, we considered the non-LAD regions as polymers in good solvent and the LAD regions as polymers in poor solvent [3]. These new simulation results show phase separation of the LAD and nonLAD regions for the relatively high volume fraction regime where the overall chromatin is conventionally packed and not phase separated from the aqueous phase (see the new Appendix 7 and Appendix 7–figure 1 in revised manuscript). Our new results also demonstrate that peripheral organization cannot be achieved if the difference in interactions of the self-attractions of the LAD and non-LAD is large. This justifies our original model as presented in the main text in which differences in the self-attraction of the LAD and non-LAD are neglected to a first approximation. The primary effect is then phase separation of chromatin from the aqueous phase as observed. Our new simulations demonstrate that the largest interactions are the chromatin-chromatin self-attractions and that the differences in those attractions due to other modifications (such as LAD and non-LAD) are secondary.We have referred to these new results of Appendix 7, in the main text on page 5, where we added the following text:“In addition, in the Appendix 7, we also consider the case in which interactions between two LAD beads are more strongly attractive compared with the interactions of two nonLAD beads or a LAD non-LAD pair. […] But at the next order of approximation where we include differences in the self-attraction of different chromatin regions, the simulations show micro phase separation.”If you decide to submit a revised version to eLife, please, make sure to comment in particular on why you think that the results presented here and that go beyond Ref.[12] merit publication in eLife rather than a more physics oriented journal like Soft Matter.eLife.We believe that paper is important for biologists because it presents a new paradigm that the chromatin can (depending on hydration) phase separate from the aqueous phase in the nucleus. To our knowledge, this the first time that chromatin phase separation is analyzed using simulations and discussed physically in comparison with experimental data. This evidence changes the biological paradigm and shows that the competition between the surface (lamin) and bulk chromatin interaction can indeed be fine tuned in both simulations and experiments to result in transitions between peripheral, conventional and central organization. Those transitions are indeed observed in the experiments as we discuss. Moreover, we present a guide to further experiments that can map the global state diagram of conventional, peripheral and central (wetting droplet) organization of chromatin. Because the organization can have profound effects on gene expression, it is important to explain these effects and quantify them for biologists and biophysicists even though the qualitative polymer properties may not surprise experts in polymer physics. That is precisely the reason we are submitting to Based on the referee remarks we have further highlighted the biological implications of our results in a manner that relates to experiment. We have now included the results of additional calculations that relate our predictions of phase separation to the Hi-C contact maps of chromatin, which are of great interest to biologists. In particular, to relate the new paradigms of chromatin organization to the contact maps, we calculated those contact maps from our simulation results and compared the maps of peripheral, conventional and central organization (see Appendix 6 and Appendix 6–figure 1 in revised manuscript). Our new results show that chromatin in peripheral organization is condensed (in the sense of Hi-C maps) in a manner that is comparable to conventional organization but is more condensed (in the Hi-C sense) than central organization (Appendix 6–figure 1(a) and (b)). We have also shown that the lamin attraction prevents peripherally organized chromosomes from mixing and help chromosomes remain in well-define regions  (see Appendix 6–figure 1(c)). We have also shown that similar to conventional organization, peripheral chromatin is also organized in a fractal globule fashion (see Appendix 6–figure 1(d)).We have summarized the new results on the Hi-C contact map (presented in detail in the new Appendix 6) and how they impact the various chromatin concentration profiles in the main paper of the paper on page 10, where we added the following text:“In Appendix 6 and Appendix 6–figure 1 we calculated contact map from our simulation results (whose snapshots are shown in Appendix 1–figure 1(a)) and compared the contact maps of peripheral, conventional and central organization. […] We have also shown that like conventional, peripheral chromatin is also organized in a fractal manner (see Appendix 6–figure 1(d)).”Essential revisions:1. Why do you need a second potential in addition to the LJ potential after bond formation between an LAD bead and a lamina bead?We used harmonic potential in addition to the LJ potential for following reasons:BT, rck= 2.5σ) was not strong enough to have localize LAD beads in the periphery. Even for cases where the LJ simulation parameters are larger (but still reasonable) and should favor peripheral organization, this potential was not enough to localize in periphery. That is accomplished by a more specific bonding model which introduces a strong energy penalty for changing the bond length. The main difference with the LJ interaction (which causes “polymer adsorption” but not bonding) is that once a bond is formed, there can be a strong energy penalty for changing the bond length (see below for the discussion of bond breaking).i. The short-range LJ attraction . This corresponds to the experiments on overexpression of Lam A/C (see Appendix 1–figure 1). These qualitative changes in chromatin organization as a function of the fraction of bonds that can be formed, cannot be studied using the LJ interaction which results in polymer adsorption to the lamina but not bonding, which can be limited due to the bonding restrictions.ii. The LAD regions are initially uniformly distributed within the nucleus. The LJ attraction allow the LAD regions to kinetically become localized at the periphery where the bond formation further stabilizes their connection to the lamin. Biologically, the anchor protein lamin B receptor (LBR) binds lamin B and acts as a tether between chromatin and the nuclear lamina. To mimic LBR protein binding, we used a bonding potential between lamin and some fraction of the LAD beads. We restricted only a fraction, iii. In our model, the bond between lamin and LAD is dynamic and can break (see below for a discussion of the breaking probability). Also, a single LAD bead can form a maximum of one bond with a given lamin bead. So, to simulate the transition from peripheral to central, we need only change the breaking bond properties of the LAD and lamin (see Appendix 5 and Appendix 5–figure 1 in revised manuscript).In light of the referee comment, we have further explained the importance of bonding potential in the paper on page 5 of the Model where we added the following text:“These qualitative changes in chromatin organization as a function of the fraction of bonds that can be formed, cannot be studied using the LJ interaction which results in polymer adsorption to the lamina and not bonding, which can be restricted due to a finite number of binding sites or available binding proteins.”2. Are the effects of fixation well described by changing the chromatin volume fraction alone?Drosophila larva nuclei as having an average value of 1183µm3, while fixed nuclei had an average volume of 381µm3 [9].It has been reported that tissue fixation can cause a reduction in both the cellular and nuclear volumes due to dehydration of the cell [4–6]. For example, in Hi-C experiments, cells are fixed by formaldehyde. It has been reported the fixation of cells with formaldehyde leads to cell shrinkage and the cell volume of fixed cells can be significantly smaller than the cell volume of living cells . Also, our experiments measured the volume of live break. One would expect to find in the exponent the difference between the elastic energy at σ and at rb.3. The paragraph on page 8 about bond formation is a little difficult to follow. Could you clarify the probability for bond breaking PbreakPthat does not require a detailed formulation of the interaction potential responsible for bonding, other than its curvature near the minimum, as we now explain here and in the revised text:We apologize for the confusion. The bond breaking kinetics are parameterized in a general manner by i. In Langevin simulations, per se, only forces are relevant.r (which is LAD-lamin distance) is less than or equal to br≡ 2.5σ. Changes in the LAD lamin distance results in a spring-like force that restores the bond to its optimal length of r = σ with a spring constant of 10BT/σk2.ii. In the bond model the relevant force is due to a spring (bond) that is formed whenr = σ enters only indirectly since the bond breaking probability  is used by the LAMMPS software. iii. The bond stabilization energy i.e. energy when iv. Two parameters enter to describe bond breaking in LAMMPS.breaka. P: Probability that over many time steps the bond will stochastically break.bb. r: Bond breaking only occurs if r (the LAD-lamin distance) obeys br > r. That means, when r is too small, bond breaking does not occur and the bond distance fluctuates due to the competition of thee thermal forces and the spring.v. These two features allow us to describe bond formation and bond breaking withoutincluding a detailed attractive potential that describes how the bond is formed with all intermediate configurations.breakP= eUbond/kBT−where we set bondU= 10BTk. This represents a fairly long-lived bond. We took br= 2.5σ. For bonds whose formation/breaking is given by detailed balance, bondUis the energy gained upon formation of the bond (relative to the unbonded state). In Appendix 5 where we show the wetting droplet, we have compared a value of br= 1.1225σ where the unbonding is fast so that the equilibrium wetting droplet (Appendix 5–figure 1(a)) is obtained. For br= 2.5σ, the unbonding is slow and the system remains “stuck” in the non-equilibrium state with many arms as show in Appendix 5–figure 1(a).vi. We have therefore taken breakPin the paper on page 5 of the Model section where we added the following text: “The difference between the LJ interactions of the lamin and LAD and the bonding energy is seen when one considers the detachment of the lamin and LAD. […]  ”In light of the referee comment, we have further explained the probability for bond breaking C. elegans ) seems to suggest that the specific affinity of heterochromatin to the lamina is important to understand the global structure of the nucleus. The authors definitely appear aware of this issue, by explicitly stating that their model holds whenever the generic interactions between chromosome and lamins are more important than the e.g. eu/hetero cross-interactions. However, in the discussion they appear to want to have it both ways in the end, by appealing (without further references) to a correlation between the occurrence of LAD domains and the eu/hetero nature of regions of the chromosome. Could you clarify this issue?4. Earlier work in First, we thank to referee for the reference [11]; we have now added this to the revised manuscript. In the revised manuscript (and in our comments to the referees and the paper revisions noted above), we have now clarified that the simulations relate to LAD and non-LAD regions of chromatin. These is no modeling of hetero and euchromatin as relates to histone modifications. However, in the new Appendix 5 we now present a study of a system where the attractions of the LAD and non-LAD beads are different.This results in micro phase separation of those regions and is analogous to micro phase separation associated with eu and heterochromatin.In the peripheral organization, the experiment image in Figure 6(c) shows that both heterochromatin and euchromatin are found in the peripheral part of the nucleus. They both separate from the aqueous phase in the center. This suggests that the chromatin attractions driving this phase separation are to a first approximation, similar for heterochromatin and euchromatin as we assumed in the main text. Of course in the next order of approximation, the self-attraction of heterochromatin and of euchromatin differ and that is why the experiments imaged alternating regions of heterochromatin and euchromatin along the angular direction in the nuclear periphery ). In Discussion part, based on our simulation results ), we suggest that this can be the case when LJ attraction between LAD-lamin and nonLAD-lamin are same but LAD-lamin also have bonding attraction.References:1. Jain N, Iyer KV, Kumar A, Shivashankar G (2013) Cell geometric constraints induce modular gene-expression patterns via redistribution of hdac3 regulated by actomyosin contractility. Proceedings of the National Academy of Sciences 110:11349–11354.2. Thomas CH, Collier JH, Sfeir CS, Healy KE (2002) Engineering gene expression and protein synthesis by modulation of nuclear shape. Proceedings of the National Academy of Sciences 99:1972–1977.3. Chiang M, et al. (2019) Polymer Modeling Predicts Chromosome Reorganization in Senescence. Cell Reports 28:3212–3223.e6.4. Xie K, Yang Y, Jiang H (2018) Controlling cellular volume via mechanical and physical properties of substrate. Biophysical journal 114:675–687.5. 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Genes and development 24:766–782.11. Meister P, Towbin BD, Pike BL, Ponti A, Gasser SM (2010) The spatial dynamics of tissue-specific promoters during


CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for  Although the majority of CpGs in mammalian genomic DNA is methylated2, about 70% of human gene promoters are associated with unmethylated DNA sequences called CpG islands (CGIs)4. CGIs, which are rich in C and G nucleotides and defined by a high density of CpG dinucleotides, are often refractory to methylation and characterized with a chromatin state permissive for transcription. Recent studies indicate that deregulation of the tissue-specific methylation state of different classes of CGI promoters could contribute to the initiation/progression of cancer6. The establishment and maintenance of CGI-specific chromatin conditions are mediated by structurally and functionally distinct zinc-finger (ZF)-CXXC family proteins. The ZF-CXXC proteins preferentially interact with unmethylated CpG dinucleotides through a highly conserved ZF-CXXC domain characterized by two consecutive cysteine-rich motifs (CXXCXXC) tetrahedrally coordinated with Zn2+ ions forming zinc-finger structures. Upon binding to DNA, the ZF-CXXC proteins establish a chromatin architecture directly through chromatin-modifying enzymatic activities and/or indirectly through the recruitment of chromatin-modifiers9.DNA methylation is one of the mechanisms of gene silencing and primarily occurs in CpG dinucleotides of the genome. Methylation of cytosine residues results in the recruitment of methyl-CpG-binding proteins (MBPs) that act as transcription repressors10. The CXXC5 gene located on chromosome 5q31.2 is ubiquitously expressed, albeit at varying levels, in human tissues10. Evidence indicates that morphogenic retinoic acid11, multifunctional cytokine family member transforming growth factor-β12, bone morphogenetic protein 414, the Wnt family of secreted glycolipoprotein Wnt3a17 or estrogen20 alters the CXXC5 expression as the primary response gene, whose protein product subsequently leads to changes in cell type-specific secondary gene expressions26. These changes are manifested as the modulation of cellular metabolism, proliferation, differentiation, or death in developmental processes and tissue maintenance31. Consistent with the functional importance of CXXC5 in physiology, de-regulated expressions of CXXC5 have been reported to correlate with the development of various pathologies including acute myeloid leukemia (AML), gastric, prostate, and breast cancer38.CXXC5, also known as RINF and WID, is a member of the ZF-CXXC family40. Nevertheless, promoters as diverse and complex architectural DNA segments primarily located adjacent to the transcriptional start sites (TSSs) of genes constitute the key platform for the assembly of pre-initiation complexes to mediate transcription40. Delineation of promoter features of the CXXC5 gene is essential for understanding the mechanisms of the CXXC5 gene regulation in a signal pathway- and cell type-dependent manner that could underlie its role in physiology and pathophysiology.Despite the involvement of CXXC5 in diverse cellular events mediated by distinct signaling pathways, the mechanism responsible for the expression of the CXXC5 gene remains largely unknown. Spatio-temporal control of gene expression is achieved at the transcriptional level. This requires the integrated effects of sequence-specific trans-factors, general transcription regulators, and cis-acting DNA regulatory elements including promoters, promoter-proximal elements, distance-independent elements, locus control regions, and insulator within a highly dynamic chromatin environmentCXXC5 transcripts, transcript variant 2, which is composed of Exon3, 10, and 11 of CXXC5 and has the highest expression level among transcript variants, represents the main CXXC5 transcript in cell models. We also identified a DNA segment in and at the 5′ sequences of the first exon of transcript variant 2 (Exon3) as the core promoter region. Based on DNA sequence composition and motifs, chromatin configuration of as well as the presence of multiple TSSs together with an active RNA polymerase II at the core promoter, we suggest that a CGI promoter drives the expression of CXXC5. A promoter pull-down approach revealed the potential association of various transcription factors (TFs)/co-regulatory proteins, as well as of proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. We found here that of the transcription factors, ELF1 and MAZ contribute to the CXXC5 expression. Moreover, the DNA sequence within the first exon of transcript variant 2 was found to form a G-quadruplex (G4) structure in vitro that could modulate the CXXC5 expression in cellula.We found here that of the 14 annotated 43. Alternative splicing of a single gene can generate a repertoire of protein isoforms with distinct features. Alternative promoters of a gene could have different tissue specificity, developmental activity, expression levels, and they may produce protein isoforms with distinct amino-termini43. The CXXC5 gene located on the long arm of chromosome 5, in the 5q31.2 region, is approximately 35 kb in length according to the human genome assembly GRCh38. The gene contains 11 exons and 10 introns. Transcript annotations by databases including Ensembl (https://www.ensembl.org/index.html), NCBI (https://www.ncbi.nlm.nih.gov) indicate that the CXXC5 gene can generate 14 transcript variants in different tissues , 924 bp of which is in Exons10, and 45 bp including a stop codon is in Exon11. All 14 transcript variants of CXXC5 encode the same protein with a calculated molecular mass (MM) of 33 kDa.Genes can have multiple transcript variants encoding the same protein or different variants of a protein, and/or non-coding transcript variants as a result of alternative promoter usage and/or alternative splicing47, we predicted that the identification of TSS(s) of the main transcript variant(s) as the quantitatively most abundant one(s) could be used to define the promoter region(s) of CXXC5. The expression of CXXC5 as a retinoid-inducible nuclear factor critical for retinoid-induced cellular differentiation was first reported in cell models, including HL60 cells, derived from leukemia48. Similarly, we showed using breast adenocarcinoma-derived cell lines, including MCF7 cells, that CXXC5 is an E2-ER responsive gene19 and CXXC5 as an unmethylated CpG binder contributes to E2-mediated gene expressions critical for cellular proliferation29. We, therefore, explored the presence and the extent of transcript variant expressions of CXXC5 in MCF7 cells and HL60 cells as cell models. To identify transcript variants of CXXC5 expressed in MCF7 and HL60 cells, cDNA libraries generated from total RNA were subjected to multiple nested rounds of PCR using progressively nested primers specific to each variant together with a primer specific to Exon10, which is common to all CXXC5 transcript variants, followed by cloning and sequencing of the PCR amplicons  indicate that although transcript variants of CXXC5 are expressed at varying levels in distinct tissues, transcript variant 2 and transcript variant 3 in breast tissue and transcript variant 2 in blood show the highest expression levels  nt. We used a biotinylated probe complementary to the joint boundaries of Exon10 and Exon11 (343 nt in length) which are present in all transcript variants, and a biotinylated probe targeting Exon3 sequences (390 nt). We also used a GAPDH probe (589 nt) targeting Exon5-8 to detect the GAPDH transcript of 1525 nt in length as the control . Results revealed that both probes specific to CXXC5 detect primarily a CXXC5 transcript with an electrophoretic migration of approximately 2500 nt in length similar to that of the annotated transcript variant 2, 2601 nt, while, as expected, a single GAPDH transcript of about 1500 nt was detected  analyses using ribosomal-RNA depleted RNA samples from MCF7 or HL60 cells. The length of the annotated human CXXC5 resides in a transcript variant 2 region that encompasses a transcription start site (TSS). For the identification of TSS(s), we used the 5’ Rapid Amplification of cDNA Ends (5’RACE) approach, designed for the amplification of nucleic acid sequences from a messenger RNA (mRNA) template between a defined internal site and unknown sequences at the 5′-end of the mRNA through the use of an adaptor RNA probe49. Although prone to biases introduced by various factors including RNA secondary structures, G–C nucleotide content, adaptor ligation efficiency50, 5′RACE has been successfully used for the identification of 5′-ends of numerous RNA transcripts51. We also used TFF1, a well-studied estrogen-responsive gene52, as a control for 5′RACE studies. 3′RACE was also used for the identification of 3′-transcript sequences of both CXXC5 and TFF1 transcripts.Because of these findings, we predicted that the promoter of CXXC5 or TFF1 transcript  promoter effectively repressed the Luciferase enzyme activity , (2) a greatly enriched CpG dinucleotide repeats (https://www.biologicscorp.com/tools/GCContent/), (3) an asymmetric GC distribution, GC skew, which is used as a measure of DNA strand asymmetry in the GC nucleotide distribution (http://genskew.csb.univie.ac.at/GenSkewServlet) as a property of CpG islands54, and (4) the presence of a CpG island (CGI) . These analyses suggest that the transcription start region, including Segment A, of the transcript variant 2 is located in a CpG island, a conclusion consistent with the CGI annotation track of the CXXC5 locus in the human genome (https://genome.ucsc.edu/).Based on the conclusion that transcript variant 2 is the main 55. Acting as stable and heritable epigenetic marks, methylated CpGs are present in 80% of CpGs in the genome and involve both genic and intergenic regions2. Although the majority of CpGs are methylated, about 70% of human gene promoters are associated with unmethylated CGIs4. CGIs are short (200–2000 bp) DNA segments that display high G-C content with enriched CpG dinucleotide repeats56. CGI promoters, often define promoters of housekeeping, developmental and tissue‐specific genes, show a transcriptionally permissive state, within which transcription initiation can occur at several closely spaced locations56. To examine the methylation state of Exon3 and the surrounding region including the putative CXXC5 promoter segment, we explored a targeted methylation profile of the region as well as Exon10 as a control for the methylated gene body of the CXXC5 locus using bisulfite-sequencing. Genomic DNA of MCF7 cells was subjected to bisulfite reaction to convert unmethylated cytosine residues to uracil followed by bisulfite PCR. PCR amplicons generated with bisulfite primers were cloned and sequenced. Sequences were then aligned to the genomic sequence of the corresponding CXXC5 regions using QUMA57 (http://quma.cdb.riken.jp/). Results from MCF7  for chromatin digestion  or a fragment of Exon10 (220 bp) as control DNA followed by incubation with streptavidin-conjugated magnetic beads. Proteins bound to beads/DNA were then subjected to MS. Subtractive analysis of MS results obtained with proteins bound to beads, the control DNA, and Segment A revealed 94 proteins that specifically associate with the core CXXC5 promoter  and DAVID63 (https://david.ncifcrf.gov/) databases suggest that Segment A associated-proteins are mainly grouped in the regulation of gene expression, which can further be sub-grouped into proteins as TFs and transcription co-regulatory proteins as well as proteins involved in histone/chromatin, DNA, and RNA processing  database, a resource of human and mouse cis-regulatory information derived from ChIP-seq, DNase-seq, and ATAC-seq chromatin profiling assays to map the genome-wide locations of transcription factor binding sites64. Due to the availability of information on TFs in Cistrome, the possible association of 16 TFs  with Exon3 and surrounding sequences was analyzed with datasets generated by the use of MCF7 cells and/or of other cell lines for which datasets were available. Results revealed that while ATF7, CREB1, MGA, MYNN, NFIA, NFIB, ZBTB2, or ZBTB7B does not appear to interact with the Exon3 region, the association of ELF1, TFAP4, or TFAP2C with the region in cells seems to be dependent on tissue-of-origin. On the other hand, AFF1, MAZ, PRDM10, RB1, or ZBTB7A could be involved in the regulation of CXXC5 expression in MCF7, and also in other, cells by interacting with the Exon3 region   with which ELF1 is shown to interact67 as control. Similarly, primer sets for the promoter of MYC  were used to assess the interaction of, as shown previously, MAZ69 or RB170 as control. We also used Exon2 of MB (Myoglobin) as control. In addition, ChIP using an antibody specific to CREB1 was conducted to ensure that CREB1 does not interact with the Exon3 region as the findings of the Cistrome database suggested.MAZ binds to DNA sequences with high G nucleotide contentMB  with an estimated MM of 37 kDa  database73, which is a resource for curated, non-redundant TF-binding profiles stored as position frequency matrices (PFMs) for TFs. We identified potential binding sites for MAZ and ELF1 proteins in Segment A  assays, as we described previouslylls Fig. . When thlls Fig. a or MAZ lls Fig. b was incΔELF1-RE) or MAZ (SegAΔMAZ-RE) motif compared to the native sequence in MCF7 cells whether or not cells transfected with the expression vector bearing the HA-ELF1 or HA-MAZ cDNA. These results indicate that Segment A contains sequences for the binding of ELF1 and MAZ critical for the promoter activity.To assess the effects of ELF1 or MAZ on the expression of reporter enzyme driven by Segment A, we transiently transfected MCF7 cells with the expression vector bearing the HA-ELF1 or HA-MAZ cDNA for 24 h Fig. c ResultsCXXC5 expression in a chromatin context, we transiently transfected MCF7 cells with the expression vector bearing none (EV), the HA-ELF1, or HA-MAZ cDNA for 48 h. HA-ELF1  is used to identify potential G-quadruplexes from the primary sequence76.G-rich sequences can self-associate into stacks of G-quartets to form complex structural motifs known as G-quadruplexes (G4s) which arise from Hoogsteen hydrogen bonding of four guanines arranged within a planar quartet (G‐quartet) linked by loop nucleotides77 (http://bioinformatics.ibp.cz/) and G4CatchAll78  revealed the possible presence of a G4 on the positive strand , 34 nt long, in the presence of 70 mM KCl led to a substantial fluorescence increase which was determined to be as F − F0 = 720 ± 15  present in the promoter region of the VEGF  gene, which was previously characterized to form a G4 structure81. These together with low fluorescence intensities of ThT at 488 nm with various mutant sequences of SegC-G4 designed to disrupt the G4 formation (SegC-Mut1-3), SegC complementary (SegC-Comp), or dT32 suggest the possible adaptation of a G4 structure by SegC-G4 sequence.Analysis of the sequence of Segment C strands with G4 prediction tools including G4Hunter83. The presence of a parallel G4 is characteristically associated with a positive band around 260 nm and a negative band around 240 nm. On the other hand, the formation of an antiparallel G4 reveals a positive band around 295 and 240 nm and a negative band near 260 nm. The hybrid type G4 structure is associated with a positive band at 290 nm together with a shoulder band at 260 nm and a negative band at around 240 nm84. In the CD spectrum of SegC-G4, a negative peak around 240 nm, a positive peak around 260 nm and another positive peak around 290 nm were observable  did not show the characteristic peak intensities of G4s. Compared to SegC-Mut1 and SegC-Mut2, the high intensity of the CD spectrum at 220 nm of SegC-Mut3 likely results from the A-rich content of this sequence86.To verify that the SegC-G4 sequence indeed forms a G4 structure, we also used the Circular Dichroism (CD) approach, which is commonly utilized to determine the G4 topology of G-rich sequencesble Fig. d. The pr87. CD thermal denaturation experiments were conducted to further examine the G-quadruplex structure of SegC-G4. CD spectra were recorded as a function of temperature (between 15 °C and 95 °C)  but not Segment D (CMV-Pr-D) was indeed effectively alleviated with the deletion of the G4 sequence in Segment C (CΔG4)  Fig. f.74. While sequence motifs for TATA-box and downstream promoter element (DPE) positioned at distinct locations relative to TSS tend to characterize non-CpG promoters, CGI promoters generally lack these elements74. We identified here transcript variant 2 with the highest expression level among transcript variants in MCF7 and HL60 cells as the main transcript of CXXC5. We also defined a DNA segment within and at the 5’ surrounding sequences of Exon3 as the core promoter region required for CXXC5 expression. Based on DNA sequence composition and motifs, chromatin configuration of, and the presence of multiple TSSs together with an active PolII at the CXXC5 promoter, we suggest that a CGI promoter drives the expression of CXXC5.The majority of human gene promoters of housekeeping, developmental and tissue‐specific genes are located within unmethylated CGIs that display a chromatin state permissive for transcription which is initiated at multiple closely spaced TSSs by ‘broad or dispersed’ promoters in contrast to ‘focused or sharp’ non-CpG promoters of cell-type-specific genes within which a single TSS initiates transcription89 and inherently unstable nucleosome architecture90, the chromatin accessibility of CGIs is critical for the binding of various transcription factors including the ZF-CXXC family proteins and the subsequent recruitment of DNA/histone modifiers and RNA polymerase machinery for transcription74. Our studies coupled with bioinformatics analyses suggest that AFF1, ELF1, MAZ, PRDM10, TFAP2C, TFAP4, and ZBTB7A transcription factors may be involved in the regulation of the CXXC5 expression; of these, ELF1, MAZ, TFAP2C, TFAP4, and ZBTB7A appear to be capable of interacting with sequences enriched with C and/or G nucleotides within the nucleosome-free CXXC5 promoter. We verified here that ELF1 and MAZ are critical components of the CXXC5 expression by directly interacting with cognate sequence motifs present in the CXXC5 promoter.Transcription is the result of the integrated effects of multiple inputs mediated by TFs whose activities are dynamically modulated in response to internal and external signaling cascades. Studies indicate that due to a high CpG densityCXXC5 expression is likely multifactorial involving many transcription factors with activator or repressor functions responding to distinct signaling pathways. ELF1 (E74 Like ETS Transcription Factor 1), a ubiquitously expressed gene product, is a member of the ELF subfamily of the ETS transcription factor family which plays diverse roles in regulating many essential processes including embryonic development, cell cycle control, cell proliferation, apoptosis, cell migration, hematopoiesis, and angiogenesis92. ELF-1 interacts with a permutation of a consensus core sequence, AGGAA, , which are often found within the same gene, are very similar: GGGAGGG and GGGCGG, respectively66. Studies further showed that SP1 binds, and competes with MAZ for binding, to the same GC-rich DNA-binding sites101.As CXXC5 expression is unclear, alterations in histone modifications, upon binding to DNA, of target gene promoters appear to be critical for gene expressions102. MAZ, for example, represses transcription by recruiting histone deacetylases including HDAC1, HDAC2, and HDAC365. Moreover, the interaction of FAC1 , a truncated isoform of the chromatin remodeler BPTF (bromodomain and PHD domain transcription factor), with MAZ is shown to alter the transcriptional activity of the protein103. It is therefore likely that upon association with DNA, MAZ and/or ELF1 directly or through co-regulatory proteins interact with histone modifiers, as we find here with the pull-down assay of the association of histone acetyltransferase KAT2A as well as histone demethylases JMJD1C, KDM1A, and KDM2A with Segment A, and establishes a chromatin state and structure permissive/restrictive for the transcriptional regulation of CXXC5.Although how ELF1 or MAZ modulates CXXC5 gene, the surrounding regions may contribute to gene expression as they contain potential binding motifs for various transcription factors (data not shown) as well as structural features, exemplified here with the presence of a G4 conformation in Segment C. As non-canonical nucleic acid secondary structures formed within G-rich sequences in both DNA and RNA, G4s are widely found in promoter regions, immunoglobulin class switch regions, ribosomal DNA, mitochondrial DNA, replication initiation regions as well as in the extended repeat sequences in various pathologies105. G4s play fundamental roles in transcription, replication, genome stability, and epigenetic regulation as well as post-transcriptional events including RNA transport, localization, and translation105. G4 structures could act as modifiers of various TFs, as exemplified with p53106, at target promoter sites in the regulation of gene expression. The abilities of various proteins including helicases, chromatin/histone modifiers, and transcription factors to interact with G4 may be critical for the dynamic regulation of gene expressions. For example, the binding of nucleolin to the nuclease hypersensitive element III1 (NHE III1) of MYC induces the formation of a G4 structure and reduces the MYC transcription107; the binding of NME , on the other hand, unfolds the G4 structure and promotes the transcription of MYC108. MAZ is also shown to bind to secondary DNA structures including G4s, which appears to be critical for transcriptional events of target genes111. In addition to Segment A, we also observe adjacent binding motifs for MAZ (GGGGAGGGGGAGGAGGG) in Segment C  or ThermoFisher . Chemicals were obtained from Sigma-Aldrich (Germany) or ThermoFisher. Pageruler Prestained Protein Ladder  or Pageruler Plus Prestained Protein Ladder  was used as the molecular mass (MM) marker.l-Glutamine  and 1% Penicillin/Streptomycin  as described previously114. HL60 cells derived from acute promyelocytic leukemia were grown in phenol red-free, low glucose (1 g/L) containing DMEM supplemented with 10% fetal bovine serum, 1% L-Glutamine , and 1% Penicillin/Streptomycin. MCF7 cells were transiently transfected with Turbofect transfection reagent  for 48 h if not otherwise specified. Protein concentrations in extracts were assessed with a Bradford protein assay kit .MCF7 cells were grown in phenol red-free, high glucose (4.5 g/L) containing Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , 1% CXXC5, we generated Luciferase reporter vectors bearing a DNA fragment containing the putative promoter elements of the CXXC5 gene. We used the pGL3-Basic Luciferase Reporter vector that bears the Firefly Luciferase cDNA as the reporter enzyme . For the engineering of the reporter vector bearing the putative CXXC5 promoter containing genomic region, a DNA fragment of 1975 bp of the CXXC5 locus  generated by PCR using the genomic DNA of MCF7 cells as a template was inserted into the pGL3-Basic vector with appropriate restriction enzymes. To increase the resolution of the putative CXXC5 promoter region, we carried out deletions from both the 5’ and 3’ ends of the region by PCR and inserted them into the pGL3-Basic vector with appropriate restriction enzymes. All constructs were sequenced for PCR fidelity. TFF1  is a well-studied estrogen-responsive gene52. The human TFF1 gene confers E2 responsiveness through the binding of ER to a non-consensus ERE117. The pGL3-TFF1 reporter construct is responsive to E2 in transiently transfected cells synthesizing estrogen receptor(s) exemplified with MCF7 cells119. We used the pGL3-TFF1 reporter vector bearing the estrogen-responsive TFF1 gene promoter as control under the steady-state cellular growth condition in that the growth medium contains unprocessed fetal bovine serum (FBS) as opposed to charcoal–dextran treated FBS to remove endogenous steroid hormones, including estrogens. In transfections, transfection efficiency was monitored with a reporter vector bearing CMV promoter that drives the expression of the Renilla Luciferase cDNA . For luciferase studies, cells, 4 × 104 cells/well, were seeded in 48-well plates for 48 h. Cells were then transiently transfected with a 125 ng reporter vector together with 0.5 ng pCMV-RL using Turbofect. Luciferase assays were performed with a Dual-Luciferase Assay Kit  as described previously19.To assess the promoter activity of the genomic region of −ΔCT method120 and normalized using the RPLP0 expression levels. Relative expression levels of CXXC5, and ELF1- or MAZ-regulated genes were assessed using 2−ΔΔCT method120 and normalized using the RPLP0 expression levels. Results were adjusted to the expression level of transcript variant 1, which was arbitrarily chosen, as one. In all RT-qPCR experiments, MIQE Guidelines were followed121.Isolated total RNA from MCF7 or HL60 cells was used for the cDNA synthesis  and transcript variant identification was carried out by PCRs with transcript variant-specific primer sets  to enrich the mRNA concentration in the total RNA population from MCF7 cells. We performed rRNA removal according to the manufacturer’s instructions. The method is based on the selective depletion of the rRNAs by the hybridization of rRNA to Locked Nucleic Acid (LNA) probes conjugated to magnetic beads. LNA referred to as inaccessible RNA is a modified RNA nucleotide that significantly increases the hybridization properties to DNA or RNA. After the hybridization, LNA probe bound rRNAs were captured with the help of a magnetic stand and the supernatant containing largely mRNAs depleted of rRNA was recovered. Phenol:chloroform:isoamyl alcohol and ethanol precipitation was then used for the purification of mRNAs.CXXC5 transcripts as well as of TFF1 as control, we used the FirstChoice RLM-RACE Kit  as directed by the manufacturer. For 5’RACE, in brief, purified mRNA (500 ng) was subjected to calf intestinal alkaline phosphatase (CIP) at 37 °C for one hour. Following the termination of the CIP reaction, RNA was extracted with phenol:chloroform:isoamyl alcohol, and ethanol precipitated. Resuspended RNA in nuclease-free water was then treated with tobacco acid pyrophosphatase at 37 °C for one hour and subjected to the ligation using a 5'RACE adapter and T4 RNA ligase. Ligated RNA products were subsequently reverse transcribed with M-MLV reverse transcriptase (M-MLV-RT) using random decamers at 42 °C for one hour. An aliquot of reactions was then used for outer 5′ RLM-RACE PCR with a 5′RACE CXXC5- or TFF1-specific 5′RACE outer primer  according to the manufacturer’s instructions. In brief, 10 µg RiboMinus-treated RNA samples as well as 3 µl of RNA ladder  were mixed with 3 volumes of formaldehyde loading dye and incubated at 65 °C for 15 min. Samples were then loaded to a denaturing agarose-LE gel and electrophoresed for 2 h and were transferred onto a positively charged PVDF membrane . Transferred mRNAs were then cross-linked to the membrane with a UV transilluminator (312 nm wavelength) for 10 min. Membranes were placed into 15 ml sterile falcon tubes and pre-hybridization was initiated using 6 ml of ULTRAhyb buffer pre-heated to 42 °C in a vertical rotator for 40 min. Biotin-tagged probes were diluted tenfold with 10 mM EDTA containing TE buffer to a final volume of 100 µl and denatured at 90 °C for 10 min. The mixture was immediately added onto the membranes in falcon tubes and placed in the oven for hybridization overnight. Membranes were then washed twice with a low stringency buffer at RT for 5 min, followed by washing twice with a high stringency buffer for 15 min at 42 °C. Membranes were subsequently subjected to the Chemiluminescence Nucleic Acid Detection Module  which enables the detection of biotin-tagged nucleic acids with the utilization of HRP-conjugated streptavidin, according to the manufacturer’s instructions. Membranes were visualized with the Chemidoc MP system .PCR amplicons for targeted identification of Exon3, Exon boundaries of Exon10 and Exon11 of CXXC5 locus, we subjected 500 ng of isolated gDNA from MCF7 cells to bisulfite reaction for the conversion of unmethylated cytosine residues to uracil using the EZ-DNA Methylation Lightning Kit . The bisulfite converted DNA was subsequently used as the template for PCR with bisulfite primers . It should be noted that bisulfite primers with no or at most one CpG position conserved were readily designable for sequences of − 930 to + 103th nucleotide of Exon3 of the CXXC5 gene. However, due to the very high number of CpG positions at the center of Exon3 until the end of Exon4, designing bisulfite primers were precluded. We instead designed methyl-specific primers, which were based on methylated and unmethylated DNA sequences generated after bisulfite conversion. Methyl-specific PCRs were carried out with primer sets containing three or more CpG sites and these regions were PCR amplified as 3 overlapping segments. PCR was carried out with 2.5 units LongAmp Taq Polymerase  in 50 µl total reaction containing 0.5 µM forward and reverse primers. Template DNA was added to the reaction at 90 °C to prevent nonspecific primer annealing and the first two cycles of the reaction were carried out only in the presence of reverse primer complementary to the sense strand to avoid the formation of primer dimers. The subsequent 10 cycles were performed 5 °C above the annealing temperature followed by 30 cycles of PCR123. In all PCRs with bisulfite-converted DNA templates, we also used a bisulfite-converted “Universal Methylated Human DNA Standard”  as control. PCR amplicons with expected sizes were excised from agarose gels, purified, and cloned into the pGEM-T vector  by TA-cloning and sequenced . Sequences were analyzed using the QUMA57 tool.For methylation analyses of CpG rich regions of the CXXC5 gene, MCF7 cells, grown in six-well plates for 48 h were fixed with 2% formaldehyde in 1xPBS for 15 min at RT with gentle shaking. To quench the formaldehyde, 0.125 M Glycine  solution was added and cells were incubated for 10 min. Cells were then washed with 1xPBS twice and 0.4% Triton X-100 was added onto the cells for permeabilization. Cells were subsequently washed twice with 1 × Micrococcal Nuclease (MNase) buffer . 500 or 1000 Gel Units (GU) of MNase  diluted in MNase buffer was added and cells were incubated for 30 min at 37 °C for chromatin digestion. Digestion reaction was stopped by the addition of 10 mM EGTA . Cells were then collected in a lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris–HCl pH 8.0, 0.5 mM PMSF, Protease Inhibitor (PI) by scraping. To reverse crosslink, 200 mM NaCl as the final concentration was added onto cell lysates and incubated at 95 °C for 5 min. To digest RNA, 0.2 mg/ml RNAse A  was added onto the lysate and incubated at 37 °C for 30 min. Lastly, to digest cellular proteins, 0.25 mg/ml Proteinase K  was added into the lysate and incubated overnight at 65 °C. DNA was subsequently purified by phenol:chloroform:isoamyl alcohol  and ethanol precipitation. To analyze the digestion pattern, purified DNA was loaded on an agarose gel and bands corresponding to the tri-nucleosomal and mono-nucleosomal DNAs were excised and gel purified using Zymoclean Gel DNA Recovery Kit (D4001). 50 ng of purified DNA or uncut genomic DNA was used as a template in PCR reactions to assess nucleosome occupancy. For the initial scanning, five overlapping regions  and actively sonicated for 20 min. Cell debris was pelleted and the supernatant was collected. After pre-clearing of nuclear extracts, the supernatant was incubated with a species-specific (Mouse or Rabbit) IgG , PolII antibody  for the precipitation of hypo- and hyper-phosphorylated PolII; or Ser5-PolII  for the precipitation of Ser5 phosphorylated PolII overnight. Nuclear extracts were also incubated with an antibody specific to CREB1 , ELF1 , MAZ , or RB1  overnight.ChIP assays were carried out as described previouslyChIP assays for total Histone 3 and H3K4me3 were performed following MNase digestion  and sonication for 5 min followed by the incubation with a species-specific IgG, histone H3-1B1B2 mouse mAb , or Histone 3 trimethylation at lysine 4, Tri-Methyl-Histone H3 (Lys4)-C42D8 Rabbit mAb .CXXC5 promoter, the promoter of OAS1 (2′-5′-Oligoadenylate Synthetase 1), MYC , or GAPDH , or the Exon2 of MB (Myoglobin) as a negative control.Samples were then subjected to immunoprecipitation with Protein G-coupled magnetic beads (New England BioLabs) for anti-mouse antibodies or Protein A/G coupled magnetic beads (New England BioLabs) for anti-rabbit antibodies. After washes, de-crosslinking, and protein digestion, DNA was recovered with phenol:chloroform: isoamyl alcohol followed by ethanol precipitation. Samples (1 µl of 60 µl elution) were subjected to qPCR using ChIP primers (Supplementary Information Table g for 5 min at 4 °C. Pellet was washed with ice-cold PBS twice and packed cell volume (PCV) was determined. Cells were resuspended in 1xPCV of Buffer A  and rested on ice to allow cells to swell. Cells were then lysed by passaging 25 times through a 25-gauge needle. Lysed cells were centrifuged to pellet crude nuclei at 12,000×g for 20 s at 4 °C. The crude nuclear pellet was washed twice with 1xPCV of IB . After centrifugation, the pellet was resuspended in 2/3 PCV of Buffer B  and rested on ice for 30 min with occasional agitation. Samples were then centrifuged for 5 min at 4 °C. Supernatant was then diluted isovolumetrically to decrease the salt concentration to 125 mM with Buffer D .MCF7 cells grown in T75 flasks were trypsinized and collected by centrifugation at 1000×CXXC5 promoter  and inserted it into a vector with appropriate restriction enzyme cut sites. Similarly, a 220 bp in length DNA fragment within Exon10 of the CXXC5 gene as control DNA was cloned into the vector. 5′ end-biotinylated forward and reverse primers specific to the vector were then used for the amplification of Segment A and the control Exon10 DNA sequences by PCR. Biotinylated double-stranded PCR amplicons were recovered from agarose gels with Zymoclean Gel DNA Recovery Kit (Zymo Research).Based on luciferase reporter results, we amplified a 220 bp in length DNA fragment by PCR from the genomic DNA of MCF7 cells that includes the Streptavidin magnetic beads  were blocked using 2% BSA in PBS for 2 h at 4 °C followed by washes with 1xPBS twice. The blocked SMBs were resuspended in 200 µl PBS containing 0.5 mM Phenylmethylsulfonyl Floride (PMSF), 0.5 mM DTT, and 1xPI, which were then mixed with one ml of nuclear extracts for pre-clearing for 1 h at 4 °C in 300 µl 1xPBS. Subsequently, the pre-cleared nuclear extract was divided into three equal (about 400 µl) aliquots. One aliquot of the extract was then mixed with 10 µg biotinylated double-stranded Segment A, control DNA or beads alone in the presence of 10 µg of Poly[d(I-C)] to form the protein-DNA complexes overnight at 4 °C on a rotator. The SMB–DNA–protein mixtures were subsequently washed with 1xPBS three times for 5 min each and resuspended in 200 µl 1xPBS for Mass Spectrometry (MS) analyses.4HCO3, followed by reduction with 100 mM DTT in 50 mM NH4HCO3 at 56 °C for 45 min, and alkylation with 100 mM iodoacetamide at RT in the dark for 30 min. MS Grade Trypsin Protease (Pierce) was added onto the beads for overnight digestion at 37 °C (enzyme to protein ratio of 1:100). The resulting peptides were purified using C18 StageTips (ThermoFisher). Peptides were analyzed by online C18 nanoflow reversed-phase HPLC  linked to a Q-Exactive Orbitrap mass spectrometer (ThermoFisher). The data sets were searched against the human SWISS-PROT database version 2014_08. Proteome Discoverer  was used to identify proteins. The final protein lists were analyzed using the STRING v11125 and DAVID126 databases.MS analyses of two biological replicates were carried out at the Koç University Proteomic Facility . The SMB–DNA–protein mixtures were washed with 50 mM NH72 using all the available ChIP-Seq datasets at the Cistrome64 database. This tool obtains: (1) a set of binding locations on a sample of Chip-Seq reads using MACS2 peak locations, (2) the reference sequence of the genomic locus to analyze, and (3) the binding motifs for a specific Transcription Factor from the JASPAR73 database as inputs. The program conducts and approximates string search on binding locations of the reference sequence using the consensus binding motif as the query sequence. The program generates both the forward and reverse strand hits which are ranked to a logarithmic sequence similarity score on binding locations.To find TF binding motifs, we developed a motif search tool19. 5′ end biotin-labeled oligomers bearing ELF1 or MAZ binding motif sequence were purchased from Integrated DNA Technologies  and annealed. Double-stranded DNA fragments were incubated in the presence or absence of (45 μg) nuclear extracts for 15 min. Reactions were further incubated without or with the ELF1- or MAZ-specific antibody for another 15 min. Samples were subjected to electrophoresis on 5% non-denaturing polyacrylamide gel. Gel contents were subsequently electrophoretically transferred to a nylon membrane and processed for EMSA using the LightShift Chemiluminescent EMSA kit (Thermo-Fisher). In brief, the membrane was UV cross-linked and blocked for non-specific binding using a blocking buffer. The membrane was then probed with Streptavidin–Horseradish Peroxidase Conjugate in the blocking buffer for image development. Images were then captured using ChemiDoc Imaging System (Bio-Rad).EMSA was conducted as described previously29. Briefly, cells were grown in six-well tissue culture plates in medium supplemented with 10% FBS for 48 h and transfected with a siRNA pool targeting ELF1 or MAZ and an expression vector bearing the HA-tagged ELF1 or the MAZ cDNA for 48 h. Cells were collected and protein isolation was performed using the NE-PER protein extraction kit (Thermo-Fisher). Protein concentration was determined using Bradford Proteins Assay (Bio-Rad). Nuclear extracts were subjected to denaturing SDS-PAGE, transferred to a membrane and proteins were probed with an antibody specific to ELF1 or MAZ followed by a secondary antibody conjugated with the horseradish peroxidase (Advansta). The membranes were then re-probed with the HA antibody  and subsequently with an HDAC1 antibody  as a loading control. Images were developed using the ECL-Substrate (Advansta) and captured with ChemiDoc Imaging System (Bio-Rad).IB was carried out as described previouslyCXXC5 Exon3 with a predicted G-quadruplex forming sequence (SegC-G4), its complementary strand (SegC-Comp), and mutant sequences  were purchased from Oligomer Biotechnology Inc. . The Pu22-G4 forming sequence of the VEGF promoter80 and dT32 were acquired from IDT. Concentrations of oligonucleotide stock solutions were determined by UV–Vis spectroscopy, using the molar extinction coefficient values obtained by IDT OligoAnalyser Tool. All nucleic acid samples were prepared in 25 mM K-phosphate buffer at pH 7.0 in the presence of 70 mM KCl in Millipore water, where the DNA concentration was 3.0 µM per strand. The nucleic acid samples were heated at 93–95 °C for 5 min and cooled down to room temperature overnight in a water bath to assure the formation of the proper secondary structures. Thioflavin T molecule (ThT) was purchased from Sigma Aldrich . ThT stock solution was prepared in Millipore water, and the concentration of the stock solution was determined by UV–vis spectroscopy using the molar extinction coefficient value of 36,000 M−1 cm−1 at 412 nm79. Igor Pro Software  was used for data analysis.Oligonucleotides for segment C of the For CD spectroscopy, a Circular Dichroism JASCO J-815 spectropolarimeter  equipped with a Peltier-type temperature control system was used. Spectra of all samples for comparison were recorded at 5 °C using 10 mm quartz cells . The CD thermal denaturation experiment for SegC-G4 was performed by varying the temperature from 15 to 95 °C (and reverse) with a 5 °C/min increment and a 1-min waiting period for each temperature point. The Tm value for SegC-G4 was determined by the differentiation of the Normalized Ellipticity (mdeg) at 262 nm vs Temperature (°C) curve.Cary 8454 spectrophotometer  equipped with a Peltier-type temperature control system was used for the recording of the UV–Vis absorption spectrum of the samples. UV–Vis thermal denaturation experiments were performed by changing the temperature between 15 and 95 °C (and reverse) with 2 °C/min increments.Fluorescence spectroscopy was performed by Cary Eclipse Fluorescence Spectrometer . All oligonucleotide samples were prepared before experiments with the same annealing procedure described above. Parameters for the fluorescence experiments were: Emission spectra collected between 430 and 700 nm, an excitation wavelength of 412 nm, 5.0 nm excitation and emission slit widths, operation at 800 V and 600 nm/min scan rate.0, where F0 is the fluorescence of ThT alone and F is the fluorescence of ThT after the addition of the oligonucleotides at 488 nm.For the fluorescence experiments, 0.5 µM ThT and 2.0 µM nucleic acid concentrations were selected as the optimal amounts. The results were demonstrated by plotting a bar graph of F − FExperiments were repeated at least two independent times. Results, where and when appropriate, were presented as the mean ± standard error (S.E.) of three biological replicates. Statistical analyses were performed using a two-tailed unpaired t-test with a confidence interval, minimum, of 95%.Supplementary Information.


The insights into how genome topology couples with epigenetic states to govern the function and identity of the corneal epithelium are poorly understood. Here, we generate a high-resolution Hi-C interaction map of human limbal stem/progenitor cells (LSCs) and show that chromatin multi-hierarchical organisation is coupled to gene expression. By integrating Hi-C, epigenome and transcriptome data, we characterize the comprehensive 3D epigenomic landscapes of LSCs. We find that super-silencers mediate gene repression associated with corneal development, differentiation and disease via chromatin looping and/or proximity. Super-enhancer (SE) interaction analysis identified a set of SE interactive hubs that contribute to LSC-specific gene activation. These active and inactive element-anchored loop networks occur within the cohesin-occupied CTCF-CTCF loops. We further reveal a coordinated regulatory network of core transcription factors based on SE-promoter interactions. Our results provide detailed insights into the genome organization principle for epigenetic regulation of gene expression in stratified epithelia. Genome topology provides a structural basis for epigenome-mediated transcriptional regulation in eukaryotes. Here the authors characterized the 3D genome of stratified squamous epithelia. They generated a Hi-C map of human limbal stem/progenitor cells (LSCs) and integrated these data with epigenomics, transcription factor binding maps, and transcriptome data. Adult stem cells undergo self-renewal and differentiation throughout life, which is governed by lineage-restricted transcription factor (TF) networks and epigenetic landscapes4. The master regulator p63 is essential for self-renewal of stratified epithelial stem cells and initiation of the stratification program6. The non-keratinized fate of the corneal epithelium is important for ocular surface homeostasis and visual clarity. The corneal epithelium often switches into keratinized epidermal-like epithelium under pathological conditions, such as infection, injury, keratohelcosis, alkaline burn, and squamous metaplasia9. Our previous study depicted the active and repressive histone modification profiles of LSCs and revealed the epigenetic regulatory mechanism of RUNX1, PAX6, and SMAD3 (RPS) in maintaining the non-keratinizing fate of LSCs9. However, due to the lack of high-resolution maps of chromatin 3D organization, it remains unclear how genome topology couples with TF-mediated epigenetic structure to determine LSC function and identity.Understanding the relationship between three-dimensional (3D) epigenomic architecture and function is critical for unlocking the underlying regulatory circuits of gene expression. Stratified squamous corneal epithelium maintains integrity and homeostasis through the limbal stem/progenitor cells (LSCs) residing in the basal layer of the limbus10. The genome is organized into active compartment A and inactive compartment B10. The large-scale A/B compartments are further segregated into megabase-sized topologically associating domains (TADs) and DNA loops that typically occur within TADs11. TAD boundaries are demarcated by the CTCF/cohesin complex11. While TADs remain largely stable across distinct cell types and species12, the epigenetic states and cohesin-associated interaction loops within TADs show cellular specifications13. The active promoters are flanked by H3K27ac and H3K4me3, whereas enhancers are enriched for H3K27ac, EP300, and/or H3K4me114. In contrast, inactive elements, including repressive and heterochromatic regions, are marked by H3K27me3 and H3K9me2, respectively16. These active and inactive regulatory regions bound by chromatin regulators influence gene expression via extensive intra-TAD DNA looping in a cell-type-specific manner19. Accordingly, the comprehensive 3D epigenomic landscape provides a powerful platform for the spatial regulation of gene transcription.The chromosome conformation capture (Hi-C) approach has characterized the multi-hierarchical 3D genome structure that is organized by the architectural proteins CTCF (an insulator protein) and cohesin in mammalsHere, using Hi-C technology, we characterize the multi-hierarchical genome organization of human LSCs, including chromosomal compartments, TADs, and high-resolution chromatin loops. The combination of chromatin organization, epigenome, TF occupancy, and transcriptome creates comprehensive 3D epigenomic landscapes that contribute to gene activation and repression Fig. . The inar > 0.82). We totally collected ~279 million valid pairwise contacts and generated a genome-wide chromatin interaction profile at a 10-kb resolution by pooling the replicate data  for CTCF and the heterochromatic mark H3K9me2 in LSCs. ChromHMM segmentation annotation based on a combination of these epigenome data identified multiple chromatin states, including active and primed enhancers, active promoters, repressive regions, heterochromatin, and insulators  and repressive (H3K27me3) genomic regulatory elementsors Fig. . Althougors Fig. . As expeors Fig. . Compartors Fig. . The majors Fig. . These rors Fig. . CTCF anors Fig. . In contKRT15 and KRT19 were co-regulated by an enhancer network , 7002 enhancer-enhancer (E–E), and 2689 promoter-promoter (P–P) loops Fig. . The enhSFR1 promoter was enriched for H3K27ac and H3K4me3 signals, but its expression was negligible. This promoter interacted with the active promoter of the highly expressed collagen gene COL17A1, a structural component of hemidesmosomes, with two anchors of this P–P loop co-bound by CTCF and SMC1 . We also identified a set of E–P loop-associated TFs, such as the TEAD family, TFAP2C, ETS family, MAFK, MAFB, KLF6, and KLF10  of the p63 motif in E–P anchors and the master role in stratified epithelia, in this study, we also portrayed the genome-wide binding map of p63 by ChIP-Seq in LSCs. Most p63 peaks were located at introns and intergenic regions, while ~14% were located at the promoters  were connected to distal SEs that were co-occupied by p63 and RPS  loopsors Fig. . The obsvel Fig. , which cThis study was conducted in accordance with the criteria set by the Declaration of Helsinki and was approved by the Ethics Committee of Zhongshan Ophthalmic Center of Sun Yat-sen University.9. Briefly, human limbus biopsies from donors were cut into small pieces and digested with 0.2% collagenase IV (Gibco) and then 0.25% trypsin–EDTA (Gibco). After low-speed centrifugation, precipitates were seeded on the Matrigel-coated polystyrene plates and cultured with LSC medium. The components of LSC medium refer to the previous publication9. All human limbus tissues were obtained from four male donors aged from 20 to 46 years with the approval of the Ethics Committee of Zhongshan Ophthalmic Center of Sun Yat-sen University. These donors do not have any ocular surface diseases.LSCs were isolated and cultured as previously described7 cells were used for Hi-C. Following cellular cross-linking with 1% formaldehyde and nuclei extraction in hypotonic solution, the genomic DNA was digested with restriction enzyme MboI (NEB) at 37 °C overnight and labeled with biotinylated nucleotides when the cohesive ends were filled. Then, blunt-end proximity ligation was performed with T4 DNA ligase (NEB) at 16 °C for 4 h. After de-crosslinking with proteinase K (Thermo) at 65 °C overnight, ligated DNA was purified by QIAamp DNA Mini Kit (Qiagen) and sheared to ~400 bp. Next, Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen) was used to pull down the biotin-marked ligation junctions. The Hi-C library was generated using NEBNext Ultra™ II DNA Library Prep Kit (NEB) according to the manufacturer’s instructions and then sequenced with paired-end 150 reads using Illumina HiSeq X10.1 × 10GAPDH locus.Cells were fixed in 1% formaldehyde solution for 15 min and lysed with Hi-C lysis buffer . Permeable cells were treated with 0.5% SDS at 62 °C for 8 min and 1% Triton X-100 at 37 °C for 15 min. Next, chromatins were digested with MboI restriction enzyme (NEB) overnight at 37 °C and DNA Polymerase I (NEB) was added to incubate for 1 h. The digested DNA fragments were ligated with T4 DNA Ligase (NEB) for 4 h. Decrosslinking was performed with Proteinase K at 55 °C for 1 h. The ligated DNA fragments were purified with ethanol and sodium acetate. qRT-PCR was used to quantify 3C enrichment signals by normalizing them to the 58 (version 1f8815d0cc9e). QuASAR-Rep analysis (3DChromatinReplicateQC v 0.0.1)59 showed a high correlation between the two biological replicates, and we pooled the valid pairs of the two replicates together for further analysis. PCA of the normalized Hi-C matrices at 100-kb resolution was used for the identification of chromatin A/B compartments according to the published description10. The PC1 value of each bin defined the A (positive score) and B compartments (negative score). We determined the location and number of TADs and identified the locations of TAD borders using an insulation score algorithm60 based on normalized contact matrices at 40-kb resolution. The intra- and inter-chromosome interactions at 10-kb resolution were determined by Ay’s Fit-Hi-C software20 (v1.0.1). The interactions with P-value < 0.01, FDR < 0.01 and contact count > 2 were considered to be significant.The clean Hi-C reads were iteratively mapped to the human genome by the ICE software package9. Briefly, 1 × 107 cells were fixed with 1% formaldehyde for 10 min. Cross-linked cells were lysed and sonicated  using a Covaris M220 to generate 300–500 bp DNA fragments. The Salt ion concentration of the cell lysate was then diluted to 300 mM for immunoprecipitation. Chromatin extract was incubated with primary antibodies overnight at 4 °C and then Dynabeads A/G beads (Invitrogen) were added to incubate for another 1 h. The beads with immunocomplexes were washed thrice in high-salt buffer, twice in low-salt buffer with lithium chloride, and once in TE buffer. After elution and de-crosslinking, DNA fragments were purified using the MinElute PCR Purification Kit (Qiagen). ChIP-Seq DNA libraries were constructed using KAPA Hyper Prep Kit  and sequenced using the NovaSeq instrument. Primary antibodies used are as follows: p63  CTCF , SMC1 , EP300  and H3K9me2 .ChIP-Seq assays were performed in duplicate as previously described9. Briefly, reads were trimmed using Trimmomatic tool61 and BWA software62 were used to align reads to human hg19 reference genome downloaded from Ensemble database. Picard Markduplicates tool was used to remove duplicated reads and only uniquely mapped reads were retained for downstream analysis. Peak callings for CTCF, SMC1, p63, and EP300 were achieved by MACS263 with the parameters: -f BAMPE -B —SPMR -q 0.001 —call-summits —fix-bimodal —seed 11521 —extsize 200. For peak callings of H3K9me2 and H3K27me3, the following parameters were used: -f BAMPE -B —SPMR —fix-bimodal —extsize 500 —broad —broad-cutoff 0.01 —seed 11521 -c input file. HOMER mergePeaks tool was used to generate a list of merged peaks for two biological replicates. Bigwig files were generated by deepTools bamCoverage and visualized by Integrative Genomics Viewer. Chromatin states were annotated by ChromHMM (version 1.22)64. Motif analysis was performed using HOMER’s findMotifsGenome.pl program65. Intervene66 tool was used to intersect the SuReR-associated genes across distinct cell types.ChIP-Seq data analysis was performed according to previous workflow29. Clusterprofiler67 R package was used for enrichment analysis of GO biological processes with p-value cutoff = 0.01. The Metascape online tool68 was used for KEGG enrichment analysis.Super-silencers were identified and annotated to genes using the ROSE algorithmhttp://www.ehbio.com/Cloud_Platform/front/#/) was used to perform the ANOVA analysis and generate the violin plots, bubble plots of GO analysis, bar plots, and boxplots.The online imageGP tool (Further information on research design is available in the Supplementary informationReporting Summary