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Mammalian chromosomes form a hierarchical structure within the cell nucleus, from chromatin loops, megabase (Mb)-sized topologically associating domains (TADs) to larger-scale A/B compartments. The molecular basis of the structures of loops and TADs has been actively studied. However, the A and B compartments, which correspond to early-replicating euchromatin and late-replicating heterochromatin, respectively, are still relatively unexplored. In this review, we focus on the A/B compartments, discuss their close relationship to DNA replication timing (RT), and introduce recent findings on the features of subcompartments revealed by detailed classification of the A/B compartments. In doing so, we speculate on the structure, potential function, and developmental dynamics of A/B compartments and subcompartments in mammalian cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The nucleus of eukaryotic cells is constantly subjected to different kinds of mechanical stimuli, which can impact the organization of chromatin and, subsequently, the expression of genetic information. Experiments from different groups showed that nuclear deformation can lead to transient or permanent condensation or decondensation of chromatin and the mechanical activation of genes, thus altering the transcription of proteins. Changes in chromatin organization, in turn, change the mechanical properties of the nucleus, possibly leading to an auxetic behavior. Here, we model the mechanics of the nucleus as a chemically active polymer gel in which the chromatin can exist in two states: a self-attractive state representing the heterochromatin and a repulsive state representing euchromatin. The model predicts reversible or irreversible changes in chromatin condensation levels upon external deformations of the nucleus. We find an auxetic response for a broad range of parameters under small and large deformations. These results agree with experimental observations and highlight the key role of chromatin organization in the mechanical response of the nucleus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Abnormal cell nuclear shapes are hallmarks of diseases, including progeria, muscular dystrophy, and many cancers. Experiments have shown that disruption of heterochromatin and increases in euchromatin lead to nuclear deformations, such as blebs and ruptures. However, the physical mechanisms through which chromatin governs nuclear shape are poorly understood. To investigate how heterochromatin and euchromatin might govern nuclear morphology, we studied chromatin microphase separation in a composite coarse-grained polymer and elastic shell simulation model. By varying chromatin density, heterochromatin composition, and heterochromatin-lamina interactions, we show how the chromatin phase organization may perturb nuclear shape. Increasing chromatin density stabilizes the lamina against large fluctuations. However, increasing heterochromatin levels or heterochromatin-lamina interactions enhances nuclear shape fluctuations by a "wetting"-like interaction. In contrast, fluctuations are insensitive to heterochromatin's internal structure. Our simulations suggest that peripheral heterochromatin accumulation could perturb nuclear morphology, while nuclear shape stabilization likely occurs through mechanisms other than chromatin microphase organization.

Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs) with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The highly and slightly condensed forms of chromatin, heterochromatin and euchromatin, respectively, segregate in the cell nucleus. Heterochromatin is more abundant in the nucleus periphery. Here we study the mechanism of heterochromatin segregation by modeling interphase chromosomes as diblock ring copolymers confined in a rigid spherical shell using molecular dynamics simulations. In our model, heterochromatin and euchromatin are distinguished by their bending stiffnesses only, while an interaction potential between the spherical shell and chromatin is used to model lamin-associated proteins. Our simulations indicate that in the absence of attractive interactions between the nuclear shell and the chromatin, most heterochromatin segregates towards the nuclear interior due to the depletion of less flexible heterochromatin segments from the nuclear periphery. This inverted chromatin distribution,which is opposite to the conventional case with heterochromatin dominating at the periphery, is in accord with experimental observations in rod cells. This "inversion" is also found to be independent of the heterochromatin concentration and chromosome number. The chromatin distribution at the periphery found in vivo can be recovered by further increasing the bending stiffness of heterochromatin segments or by turning on attractive interactions between the nuclear shell and heterochromatin. Our results indicate that the bending stiffness of chromatin could be a contributor to chromosome organization along with differential effects of HP1α-driven phase segregation and of loop extruders and interactions with the nuclear envelope and topological constraints.

Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks., Competing Interests: Competing interests statement:The authors declare no competing interest.

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. Examples of the former are BRCA2 and POLL, and of the latter the FANC complex and ATM. Moreover, in a diversity of human cancer types, loss of several of these proteins alters the distribution of pathway-specific mutations between heterochromatin and euchromatin. Together, these results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner., (© 2024. The Author(s).)

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3 K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1 , suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to the loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways., Competing Interests: CH, ZZ, LI, SF, SJ No competing interests declared, (© 2023, Harris, Zhong et al.)

H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function., (© 2024 Keenan et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive., Results: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes., Conclusions: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity., (© 2024. The Author(s).)

Importance: The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging., Competing Interests: The authors declare no conflict of interest.

The heterochromatin position effect is manifested in the inactivation of euchromatin genes transferred to heterochromatin. In chromosomal rearrangements, genes located near the new eu-heterochromatin boundary in the rearrangement (cis-inactivation) and, in rare cases, genes of a region of the normal chromosome homologous to the region of the eu-heterochromatin boundary of the chromosome with the rearrangement (trans-inactivation) are subject to inactivation. The In(2)A4 inversion is able to trans-inactivate the UAS-eGFP reporter gene located on the normal chromosome. We knockdown a number of chromatin proteins using temperature-controlled RNA interference and investigated the effect of knockdown on trans-inactivation of the reporter. We found suppression of trans-inactivation by knockdowns of Su(var)2-HP2, a protein that binds to the key heterochromatin protein HP1a, SAYP, a subunit of the chromatin remodelling complex, and Eggless histone methyltransferase (SETDB1), which introduces a H3K9me3 histone mark, recognized by the HP1a protein. The method of studying the effects of gene knockdown on heterochromatin position effects presented in this work is of independent methodological interest., (© 2023. Pleiades Publishing, Ltd.)

Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced genes that are important for pathogenicity. This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kramer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chromosomes are arranged in distinct territories within the nucleus of animal cells. Recent experiments have shown that these territories overlap at their edges, suggesting partial mixing during interphase. Experiments that knock-down of condensin II proteins during interphase indicate increased chromosome mixing, which demonstrates control of the mixing. In this study, we use a generic polymer simulation to quantify the dynamics of chromosome mixing over time. We introduce the chromosome mixing index, which quantifies the mixing of distinct chromosomes in the nucleus. We find that the chromosome mixing index in a small confinement volume (as a model of the nucleus), increases as a power-law of the time, with the scaling exponent varying non-monotonically with self-interaction and volume fraction. By comparing the chromosome mixing index with both monomer subdiffusion due to (non-topological) intermingling of chromosomes as well as even slower reptation, we show that for relatively large volume fractions, the scaling exponent of the chromosome mixing index is related to Rouse dynamics for relatively weak chromosome attractions and to reptation for strong attractions. In addition, we extend our model to more realistically account for the situation of the Drosophila chromosome by including the heterogeneity of the polymers and their lengths to account for microphase separation of euchromatin and heterochromatin and their interactions with the nuclear lamina. We find that the interaction with the lamina further impedes chromosome mixing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bajpai, Safran. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density "euchromatin" and high-density "heterochromatin", with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies.

The survival of malaria parasites in the changing human blood environment largely depends on their ability to alter gene expression by epigenetic mechanisms. The active state of Plasmodium falciparum clonally variant genes (CVGs) is associated with euchromatin characterized by the histone mark H3K9ac, whereas the silenced state is characterized by H3K9me3-based heterochromatin. Expression switches are linked to euchromatin-heterochromatin transitions, but these transitions have not been characterized for the majority of CVGs. To define the heterochromatin distribution patterns associated with the alternative transcriptional states of CVGs, we compared H3K9me3 occupancy at a genome-wide level among several parasite subclones of the same genetic background that differed in the transcriptional state of many CVGs. We found that de novo heterochromatin formation or the complete disruption of a heterochromatin domain is a relatively rare event, and for the majority of CVGs, expression switches can be explained by the expansion or retraction of heterochromatin domains. We identified different modalities of heterochromatin changes linked to transcriptional differences, but despite this complexity, heterochromatin distribution patterns generally enable the prediction of the transcriptional state of specific CVGs. We also found that in some subclones, several var genes were simultaneously in an active state. Furthermore, the heterochromatin levels in the putative regulatory region of the gdv1 antisense noncoding RNA, a regulator of sexual commitment, varied between parasite lines with different sexual conversion rates. IMPORTANCE The malaria parasite P. falciparum is responsible for more than half a million deaths every year. P. falciparum clonally variant genes (CVGs) mediate fundamental host-parasite interactions and play a key role in parasite adaptation to fluctuations in the conditions of the human host. The expression of CVGs is regulated at the epigenetic level by changes in the distribution of a type of chromatin called heterochromatin. Here, we describe at a genome-wide level the changes in the heterochromatin distribution associated with the different transcriptional states of CVGs. Our results also reveal a likely role for heterochromatin at a particular locus in determining the parasite investment in transmission to mosquitoes. Additionally, this data set will enable the prediction of the transcriptional state of CVGs from epigenomic data, which is important for the study of parasite adaptation to the conditions of the host in natural malaria infections.

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4 ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins., Competing Interests: EA, AE, MN, EV, CH, RD, AS, DF No competing interests declared, LS Lu Sun is employed by Epicypher, Inc, a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study, MK Michael C Keogh is employed by Epicypher, Inc, a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study, (© 2022, Andreyeva, Emelyanov et al.)

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae . We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3 . Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus.

Background: Interactions of chromatin with the nuclear lamina via lamina-associated domains (LADs) confer structural stability to the genome. The dynamics of positioning of LADs during differentiation, and how LADs impinge on developmental gene expression, remains, however, elusive., Results: We examined changes in the association of lamin B1 with the genome in the first 72 h of differentiation of adipose stem cells into adipocytes. We demonstrate a repositioning of entire stand-alone LADs and of LAD edges as a prominent nuclear structural feature of early adipogenesis. Whereas adipogenic genes are released from LADs, LADs sequester downregulated or repressed genes irrelevant for the adipose lineage. However, LAD repositioning only partly concurs with gene expression changes. Differentially expressed genes in LADs, including LADs conserved throughout differentiation, reside in local euchromatic and lamin-depleted sub-domains. In these sub-domains, pre-differentiation histone modification profiles correlate with the LAD versus inter-LAD outcome of these genes during adipogenic commitment. Lastly, we link differentially expressed genes in LADs to short-range enhancers which overall co-partition with these genes in LADs versus inter-LADs during differentiation., Conclusions: We conclude that LADs are predictable structural features of adipose nuclear architecture that restrain non-adipogenic genes in a repressive environment., (© 2022. The Author(s).)

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level., (© 2021. The Author(s).)

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Deleterious genetic variants in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Although it is a high-confidence ASD risk gene, the neurodevelopmental functions of POGZ remain unclear. Here we reveal the genomic binding of POGZ in the developing forebrain at euchromatic loci and gene regulatory elements (REs). We profile chromatin accessibility and gene expression in Pogz -/- mice and show that POGZ promotes the active chromatin state and transcription of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with ADNP, another high-confidence ASD risk gene, and provide evidence that POGZ regulates other neurodevelopmental disorder risk genes as well. Our results reveal a neurodevelopmental function of an ASD risk gene and identify molecular targets that may elucidate its function in ASD., Competing Interests: Declaration of interests J.L.R. is cofounder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles., (© 2021. The Author(s).)

Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in regulating gene expression DNA replication. Furthermore it also protects the DNA from being damaged. Various proteins are involved in making a specific complex within a chromatin and the knowledge about these interacting partners is helpful to enhance our understanding about the pathophysiology of various chromatin associated diseases. Moreover, it could also help us to identify new drug targets and design more effective remedies. Due to the existence of chromatin in different forms under various physiological conditions it is hard to develop a single strategy to study chromatin associated proteins under all conditions. In our current review, we tried to provide an overview and comparative analysis of the strategies currently adopted to capture the DNA bounded protein complexes and their mass spectrometric identification and quantification. Precise information about the protein partners and their function in the DNA-protein complexes is crucial to design new and more effective therapeutic molecules against chromatin associated diseases.

Nuclear chromosomes transcribe far more RNA than required to encode protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin, and most nuclear RNA from the nucleus but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome "scaffold." RNA-seq analysis reveals that most RNA in the nuclear scaffold is repeat-rich, non-coding, and derived predominantly from introns of nascent transcripts. Insoluble, repeat-rich (C 0 T-1) RNA co-distributes with known scaffold proteins including scaffold attachment factor A (SAF-A), and distribution of these components inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei. We further show that RNA is required for SAF-A to interact with chromatin and for enrichment of structurally embedded "scaffold attachment regions" prevalent in euchromatin. Collectively, the results indicate that long nascent transcripts contribute a dynamic structural role that promotes the open architecture of active chromosome territories., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains. Without Pob3, Spt16 recruitment is partially reduced, exhibiting a silencing defect and impaired H2A/H2B organization. Second, heterochromatin protein 1 (HP1)/Swi6 mediates Spt16 recruitment onto the heterochromatin by physical interaction of the Swi6 chromo-shadow domain (CSD) and Spt16 peptidase-like domains. Several CSD mutants are tested for Spt16 binding activity, and the charged loop connecting β1 and β2 is critical for Spt16 binding and heterochromatic silencing. Loss of these pathways causes a severe defect in H3K9 methylation and HP1/Swi6 localization in the pericentromeric region, exhibiting transcriptional silencing defects and disordered heterochromatin. Our findings suggest that FACT and HP1/Swi6 work intimately to regulate heterochromatin organization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chromatin consists of a complex of DNA and histone proteins as its core components and plays an important role in both packaging DNA and regulating DNA metabolic pathways such as DNA replication, transcription, recombination, and chromosome segregation. Proper functioning of chromatin further involves a network of interactions among molecular complexes that modify chromatin structure and organization to affect the accessibility of DNA to transcription factors leading to the activation or repression of the transcription of target DNA loci. Based on its structure and compaction state, chromatin is categorized into euchromatin, heterochromatin, and centromeric chromatin. In this review, we discuss distinct chromatin factors and molecular complexes that constitute euchromatin-open chromatin structure associated with active transcription; heterochromatin-less accessible chromatin associated with silencing; centromeric chromatin-the site of spindle binding in chromosome segregation.

DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments., Competing Interests: Declaration of interests B.v.S. is a member of the Advisory Board of Molecular Cell., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin., Competing Interests: The authors have declared that no competing interests exist.

Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ's cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1-24 h in the presence of 0.05 mM-1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA sequences containing consecutive guanines organized in 4-interspaced tandem repeats can form stable single-stranded secondary structures, called G-quadruplexes (G4). Herein, we report that the Polycomb group protein BMI1 is enriched at heterochromatin regions containing putative G4 DNA sequences, and that G4 structures accumulate in cells with reduced BMI1 expression and/or relaxed chromatin, including sporadic Alzheimer's disease (AD) neurons. In AD neurons, G4 structures preferentially accumulate in lamina-associated domains, and this is rescued by re-establishing chromatin compaction. ChIP-seq analyses reveal that G4 peaks correspond to evolutionary conserved Long Interspersed Element-1 (L1) sequences predicted to be transcriptionally active. Hence, G4 structures co-localize with RNAPII, and inhibition of transcription can reverse the G4 phenotype without affecting chromatin's state, thus uncoupling both components. Intragenic G4 structures affecting splicing events are furthermore associated with reduced neuronal gene expression in AD. Active L1 sequences are thus at the origin of most G4 structures observed in human neurons.

A characteristic spatial distribution of the main chromatin fractions is observed in most mammalian cell nuclei, with euchromatin localized in the interior and heterochromatin at the nuclear periphery. It has been shown that interactions of heterochromatin with the nuclear lamina are necessary to establish this conventional architecture. Adipocytes are specific cells in which a reduction in lamin A/C expression is observed. We hypothesize that the loss of lamin A/C during adipogenic differentiation of mesenchymal stem cells (MSCs) may be associated with the reorganization of the main classes of chromatin in the nucleus. Thus, in this study, we examine the abundance and nuclear distribution of selected heterochromatin (H3K9me3, H3K27me3 and H4K20me3) and euchromatin (H4K8ac, H3K4me3 and H3K9ac) histone marks during in vitro adipogenesis, using the pig as a model organism. We found that not only did the expression of lamin A/C decrease in our differentiation system, but so did the expression of lamin B receptor (LBR). The level of two heterochromatin marks, H3K27me3 and H4K20me3, increased during differentiation, while no changes were observed for H3K9me3. The levels of two euchromatin histone marks, H4K8ac and H3K9ac, were significantly higher in adipocytes than in undifferentiated cells, while the level of H3K4me3 did not change significantly. The spatial distribution of all the examined histone marks altered during in vitro adipogenesis. H3K27me3 and H4K20me3 moved towards the nuclear periphery and H3K9me3 localized preferentially in the intermediate part of adipocyte nuclei. The euchromatin marks H3K9ac and H3K4me3 preferentially occupied the peripheral part of the adipocyte nuclei, while H4K8ac was more evenly distributed in the nuclei of undifferentiated and differentiated cells. Analysis of the nuclear distribution of repetitive sequences has shown their clustering and relocalization toward nuclear periphery during differentiation. Our study shows that dynamic changes in the abundance and nuclear distribution of active and repressive histone marks take place during adipocyte differentiation. Nuclear reorganization of heterochromatin histone marks may allow the maintenance of the nuclear morphology of the adipocytes, in which reduced expression of lamin A/C and LBR is observed.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Heterochromatin protein 1a (HP1a) is a well-known component of pericentromeric and telomeric heterochromatin in Drosophila. However, its role and the mechanisms of its binding in the chromosome arms (ChAs) remain largely unclear. Here, we identified HP1a-interacting domains in the somatic cells of Drosophila ovaries using a DamID-seq approach and compared them with insertion sites of transposable elements (TEs) revealed by genome sequencing. Although HP1a domains cover only 13% of ChAs, they non-randomly associate with 42% of TE insertions. Furthermore, HP1a on average propagates at 2-kb distances from the TE insertions. These data confirm the role of TEs in formation of HP1a islands in ChAs. However, only 18% of HP1a domains have adjacent TEs, indicating the existence of other mechanisms of HP1a domain formation besides spreading from TEs. In particular, many TE-independent HP1a domains correspond to the regions attached to the nuclear pore complexes (NPCs) or contain active gene promoters. However, HP1a occupancy on the promoters does not significantly influence expression of corresponding genes. At the same time, the steady-state transcript level of many genes located outside of HP1a domains was altered upon HP1a knockdown in the somatic cells of ovaries, thus pointing to the strong indirect effect of HP1a depletion. Collectively, our results support an existence of at least three different mechanisms of HP1a domain emergence in ChAs: spreading from TE insertions, transient interactions with the chromatin located near NPCs, and targeting to the promoters of moderately expressed genes.

Background: Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear., Results: We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin., Conclusion: These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

Histone acetylation, balanced by histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, affects dynamic transitions of chromatin structure to regulate transcriptional accessibility. However, little is known about the interplay between HAT and HDAC complexes in Fusarium graminearum, a causal agent of Fusarium Head Blight (FHB) that uniquely contains chromosomal regions enriched for house-keeping or infection-related genes. In this study, we identified the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and found that it specifically interacts with the FgEsa1 HAT of the NuA4 complex. Deletion of FNG1 led to severe growth defects and blocked conidiation, sexual reproduction, DON production, and plant infection. The fng1 mutant was normal in H3 acetylation but significantly reduced in H4 acetylation. A total of 34 spontaneous suppressors of fng1 with faster growth rate were isolated. Most of them were still defective in sexual reproduction and plant infection. Thirty two of them had mutations in orthologs of yeast RPD3, SIN3, and SDS3, three key components of the yeast Rpd3L HDAC complex. Four mutations in these three genes were verified to suppress the defects of fng1 mutant in growth and H4 acetylation. The rest two suppressor strains had a frameshift or nonsense mutation in a glutamine-rich hypothetical protein that may be a novel component of the FgRpd3 HDAC complex in filamentous fungi. FgRpd3, like Fng1, localized in euchromatin. Deletion of FgRPD3 resulted in severe growth defects and elevated H4 acetylation. In contract, the Fgsds3 deletion mutant had only a minor reduction in growth rate but FgSIN3 appeared to be an essential gene. RNA-seq analysis revealed that 48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3, respectively. Taken together, our data showed that Fng1 is important for H4 acetylation as a component of the NuA4 complex and functionally related to the FgRpd3 HDAC complex for transcriptional regulation of genes important for growth, conidiation, sexual reproduction, and plant infection in F. graminearum., Competing Interests: The authors have declared that no competing interests exist.

In recent years, high-throughput techniques have revealed considerable structural organization of the human genome with diverse regions of the chromatin interacting with each other in the form of loops. Some of these loops are quite complex and may encompass regions comprised of many interacting chain segments around a central locus. Popular techniques for extracting this information are chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and high-throughput chromosome conformation capture (Hi-C). Here, we introduce a physics-based method to predict the three-dimensional structure of chromatin from population-averaged ChIA-PET data. The approach uses experimentally-validated data from human B-lymphoblastoid cells to generate 2D meta-structures of chromatin using a dynamic programming algorithm that explores the chromatin free energy landscape. By generating both optimal and suboptimal meta-structures we can calculate both the free energy and additionally the relative thermodynamic probability. A 3D structure prediction program with applied restraints then can be used to generate the tertiary structures. The main advantage of this approach for population-averaged experimental data is that it provides a way to distinguish between the principal and the spurious contacts. This study also finds that euchromatin appear to have rather precisely regulated 2D meta-structures compared to heterochromatin. The program source-code is available at https://github.com/plewczynski/looper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Understanding the packaging of DNA into chromatin has become a crucial aspect in the study of gene regulatory mechanisms. Heterochromatin establishment and maintenance dynamics have emerged as some of the main features involved in genome stability, cellular development, and diseases. The most extensively studied heterochromatin protein is HP1a. This protein has two main domains, namely the chromoshadow and the chromodomain, separated by a hinge region. Over the years, several works have taken on the task of identifying HP1a partners using different strategies. In this review, we focus on describing these interactions and the possible complexes and subcomplexes associated with this critical protein. Characterization of these complexes will help us to clearly understand the implications of the interactions of HP1a in heterochromatin maintenance, heterochromatin dynamics, and heterochromatin's direct relationship to gene regulation and chromatin organization.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

The recent discovery of N 6 -methyladenine (N 6 -mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism 1 . However, the biological role of N 6 -mA and the molecular pathways that exert its function remain unclear. Here we show that N 6 -mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N 6 -mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD) 2-4 . Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures 3,5,6 . We show that the presence of N 6 -mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N 6 -mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N 6 -mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N 6 -mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N 6 -mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication., Competing Interests: The authors declare no competing interest.

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Malaria caused by Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation., Methods: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' UTR of the PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq., Results: The inducible knockdown of PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes., Conclusions: Our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum.

DNA viruses can hijack and manipulate the host chromatin state to facilitate their infection. Multiple lines of evidences reveal that DNA virus infection results in the host chromatin relocation, yet there is little known about the effects of viral infection on the architecture of host chromatin. Here, a combination of epigenomic, transcriptomic and biochemical assays were conducted to investigate the temporal dynamics of chromatin accessibility in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The high-quality ATAC-seq data indicated that progressive chromatin remodeling took place following BmNPV infection. Viral infection resulted in a more open chromatin architecture, along with the marginalization of host genome and nucleosome disassembly. Moreover, our results revealed that chromatin accessibility in uninfected cells was regulated by euchromatic modifications, whereas the viral-induced highly accessible chromatin regions were originally associated with facultative heterochromatic modification. Overall, our findings illustrate for the first time the organization and accessibility of host chromatin in BmNPV-infected cells, which lay the foundation for future studies on epigenomic regulation mediated by DNA viruses., Competing Interests: The authors have declared that no competing interests exist.