Distinct Chromatin Research Articles

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.

Distinct chromatin modifications at the Il33 locus in mast cells contribute to sex dimorphic susceptibility to EAE, an autoimmune CNS demyelinating disease

Abstract Many autoimmune diseases, including multiple sclerosis (MS) exhibit a striking female bias, but the underlying mechanisms remain poorly understood. Using the SJL mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which recapitulates this sex dimorphism, we previously demonstrated that preferential IL-33 expression contributes to male protection. IL-33 expression by mast cells in PLP139–151-immunized males activates type 2 innate lymphoid cells, which in turn drives a non-pathogenic Th2 response to myelin peptide. Testosterone directly activates Il33 in male but not female mast cells, but this male-specific response is also maintained with other modes of activation, including FcɛR1-crosslinking, suggesting there are constraints on the chromatin landscape that limit Il33 expression in females. Here we use ChIP and ATAC-seq to show that the dynamics of chromatin modifications at the Il33 locus is sex-dimorphic. At steady state, ChIP assays reveal activating H3K4me3 and H3K9ac histone modifications at the promoter and conserved non-coding sequences within the Il33 gene are most prevalent in male-derived cells, while H3K27me3 repressive marks dominate in females. This pattern is similar in IgE-DNP-activated mast cells. Increased basal Il33 chromatin accessibility assessed by ATAC-seq is evident in males. Together, these results indicate that while testosterone can directly stimulate IL-33 production, it also exerts effects on Il33 during development conferring a higher potential for expression in males.

Regulation of UV damage repair in quiescent yeast cells

Non-growing quiescent cells face special challenges when repairing lesions produced by exogenous DNA damaging agents. These challenges include the global repression of transcription and translation and a compacted chromatin structure. We investigated how quiescent yeast cells regulated the repair of DNA lesions produced by UV irradiation. We found that UV lesions were excised and repaired in quiescent cells before their re-entry into S phase, and that lesion repair was correlated with high levels of Rad7, a recognition factor in the global genome repair sub-pathway of nucleotide excision repair (GGR-NER). UV exposure led to an increased frequency of mutations that included C->T transitions and T > A transversions. Mutagenesis was dependent on the error-prone translesion synthesis (TLS) DNA polymerase, Pol zeta, which was the only DNA polymerase present in detectable levels in quiescent cells. Across the genome of quiescent cells, UV-induced mutations showed an association with exons that contained H3K36 or H3K79 trimethylation but not with those bound by RNA polymerase II. Together, the data suggest that the distinct physiological state and chromatin structure of quiescent cells contribute to its regulation of UV damage repair.

Natural and trained innate immunity against Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains a major global health emergency. It is estimated that one third of global population are affected, predominantly with latent granuloma form of the disease. Mtb co-evolved with humans, for its obligatory intra-macrophage phagosome habitat and slow replication, balanced against unique mycobacterial innate immunity, which appears to be highly complex. TB is transmitted via cough aerosol Mtb inhalation. Bovine TB attenuated Bacillus Calmette Guerin (BCG) live vaccine has been in practice for protection of young children from severe disseminated Mtb infection, but not sufficiently for their lungs, as obtained by trials in TB endemic community. To augment BCG vaccine-driven innate and adaptive immunity for neonates and better protection against adult pulmonary TB, a number of BCG pre-vaccination based, subset vaccine candidates have been tested via animal preclinical, followed by safe clinical trials. BCG also enhances innate macrophage trained immunity and memory, through primordial intracellular Toll-like receptors (TLRs) 7 and 9, which recognise distinct mycobacterial molecular pattern signature. This signature is transmitted by TLR signalling via nuclear factor-κB, for activating innate immune transcription and expression of gene profiling in a mycobacterial signature-specific manner. These are epigenetically imprinted in reprogramming of distinct chromatin areas for innate immune memory, to be recalled following lung reinfection. Unique TB innate immunity and its trained memory are considered independent from adaptive immune B and T cells. On the other hand, adaptive immunity is crucial in Mtb containment in granulomatous latency, supported by innate immune cell infiltration. In nearly 5-10 % of susceptible people, latent TB may be activated due to immune evasion by Mtb from intracellular phagosome within macrophage, perpetrating TB. However, BCG and new recombinant BCG vaccines have the capacity, as indicated in pre- and clinical trials, to overcome such Mtb evasion. Various strategies include pro-inflammatory-bactericidal type 1 polarisation (M1) phenotype of the infected macrophage, involving thrombospondin-TLR pathway. Saprophytic M. smegmatis-based recombinant vaccines are also promising candidates against TB. BCG vaccination of neonates/infants in TB endemic countries also reduced their pneumonia caused by various microbes independent of TB immunity. Here, we discuss host immune response against Mtb, its immune evasion strategies, and the important role innate immunity plays in the development of protection against TB.

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection

BackgroundCTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem.ResultsUsing CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin “loop extrusion” model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice.ConclusionsThese findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.

Peroxidase‐Like Nanozymes Induce a Novel Form of Cell Death and Inhibit Tumor Growth In Vivo

AbstractNanomaterials with intrinsic enzyme‐like properties, termed nanozymes, have attracted significant interest, although limited information is available on their biological characteristics in cells or in vivo. Here, it is shown that nanomaterials with peroxidase‐like activity trigger a novel form of cell death through an ATP‐citrate lyase (ACLY)‐dependent rat sarcoma viral oncogene (RAS) signaling mechanism. The peroxidase nanozyme‐induced cell lethality, which is termed as nanoptosis, is morphologically and biochemically distinct from the currently well‐defined apoptosis, necrosis, autophagy, pyroptosis, and ferroptosis. It is revealed that nanoptosis is typically characterized by the massive accumulation of cellular vesicles, swelling mitochondria, and distinct chromatin condensation and margination. Using RNA sequencing and protein quantitative mass spectrometry, an ACLY‐dependent RAS signaling pathway is identified that mainly mediates this nanozyme lethality process, and it is observed that RAS‐knockout cells are highly resistant to nanoptosis. Finally, it is demonstrated that this newly discovered nanozyme lethality can be used as an effective therapeutic strategy for inhibiting tumor growth in vivo.

ChromID identifies the protein interactome at chromatin marks.

Chromatin modifications regulate genome function by recruiting protein factors to the genome. However, the protein composition at distinct chromatin modifications remains to be fully characterized. Here, we use natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 a H3K27 residues. We first demonstrate their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localisation, genomic distribution and histone modification–binding preference. By fusing eCRs to the biotin ligase BASU, we establish ChromID, a method for identifying the chromatin-dependent protein interactome based on proximity biotinylation, and apply it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncover the protein composition at bivalent promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.

Rearrangement of macronucleus chromosomes correspond to TAD-like structures of micronucleus chromosomes in Tetrahymena thermophila.

The somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in T. thermophila is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of T. thermophila using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the T. thermophila and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.

Alterations in chromatin at antigen receptor loci define lineage progression during B lymphopoiesis

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype.

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.

Biased visibility in Hi-C datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide

BackgroundProximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature.ResultsThrough analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development.ConclusionsOur observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.

Chromatin maturation of the HIV-1 provirus in primary resting CD4+T cells.

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.

PHF1 fusions cause distinct gene expression and chromatin accessibility profiles in ossifying fibromyxoid tumors and mesenchymal cells.

Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor frequently displaying gene fusions, most of which affect the PHF1 gene. PHF1 encodes plant homeodomain finger protein 1, which is involved in various processes regulating gene transcription, including those orchestrated by the polycomb repressor complex 2. Here, a series of 37 OFMTs, including 18 typical, 9 atypical, and 10 malignant variants, was analyzed with regard to transcriptomic features, gene fusion and copy number status, and/or single-nucleotide variants. The effects on gene expression and chromatin accessibility of three detected fusions (EP400-PHF1, MEAF6-PHF1, and PHF1-TFE3) were further evaluated in fibroblasts. Genomic imbalances showed a progression-related pattern, with more extensive copy number changes among atypical/malignant lesions than among typical OFMTs; loss of the RB1 gene was restricted to atypical/malignant OFMTs, occurring in one-third of the cases. RNA sequencing identified fusion transcripts in >80% of the cases analyzed, including a novel CSMD1-MEAF6. The gene-expression profile of OFMT was distinct from that of other soft tissue tumors, with extensive transcriptional upregulation of genes in OFMT. These findings were largely recapitulated in gene fusion-expressing fibroblast lines, suggesting that genes involved in, e.g., Wnt signaling and/or being regulated through trimethylation of lysine 27 in histone 3 (H3K27me3) are pivotal for OFMT development. The genes showing differentially higher expression in fusion-expressing cells paralleled increased chromatin accessibility, as revealed by ATAC sequencing. Thus, the present study suggests that OFMT develops through gene fusions that have extensive epigenetic consequences.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Role of Histone Deacetylase (HDAC) Inhibitors for the Treatment of Cutaneous T-Cell Lymphoma (CTCL)

HDACs regulate variety of cell functions. Several structurally distinct classes of HDAC inhibitor have been developed. Cutaneous T-cell lymphoma(CTCL) is a clinically and immunologically defined neoplasm which encompasses epidermotropic (mycosis fungoides, Sezary syndrome) and non-epidermotrepic variants. CTCL lymphoma comprises a heterogenous group of lymph proliferative disorders, characterised by clonal expansions of mature post thymic T-cells that infiltrate the skin. The USFDA have been approved many HDAC inhibitors such as Vorinostat, Romidepsin, Belinostat, Panobinostat in which the Vorinostat and Romidepsin has been approved for treatment of CTCL. The response rate shown in Histone deacetylase inhibitor in CTCL for Romidepsin was 34%-35%. Whereas the result of overall response rate of Vorinostat is 24%-30% in refractory advanced patients with CTCL. The impact on cell survival signalling varied with molecular phenotype this study suggests that cellular response to HDACs can be viewed as two distinct effect chromatin effect and a cell death effect. The divergent apoptotic responses observed reflect the variable clinical outcome of HDACi treatment. Romidepsin is potent inhibitors offers a promising new treatment for a disease with few existing therapies. We also found Romidepsin to be a more effective than Vorinostat. Our further objective was to further study the clinical benefits of Romidepsin in patients that had the best response of Stable Disease (SD).

Mechanical properties of nucleoprotein complexes determined by nanoindentation spectroscopy

ABSTRACT The interplay between transcription factors, chromatin remodelers, 3-D organization, and mechanical properties of the chromatin fiber controls genome function in eukaryotes. Besides the canonical histones which fold the bulk of the chromatin into nucleosomes, histone variants create distinctive chromatin domains that are thought to regulate transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether histone variants translate distinctive biochemical or biophysical properties to their associated chromatin structures, and whether these properties impact chromatin dynamics as the genome undergoes a multitude of transactions, is an important question in biology. Here, we describe single-molecule nanoindentation tools that we developed specifically to determine the mechanical properties of histone variant nucleosomes and their complexes. These methods join an array of cutting-edge new methods that further our quantitative understanding of the response of chromatin to intrinsic and extrinsic forces which act upon it during biological transactions in the nucleus.

Low-Phosphate Chromatin Dynamics Predict a Cell Wall Remodeling Network in Rice Shoots.

Phosphorus (P) is an essential plant macronutrient vital to fundamental metabolic processes. Plant-available P is low in most soils, making it a frequent limiter of growth. Declining P reserves for fertilizer production exacerbates this agricultural challenge. Plants modulate complex responses to fluctuating P levels via global transcriptional regulatory networks. Although chromatin structure plays a substantial role in controlling gene expression, the chromatin dynamics involved in regulating P homeostasis have not been determined. Here we define distinct chromatin states across the rice (Oryza sativa) genome by integrating multiple chromatin marks, including the H2A.Z histone variant, H3K4me3 modification, and nucleosome positioning. In response to P starvation, 40% of all protein-coding genes exhibit a transition from one chromatin state to another at their transcription start site. Several of these transitions are enriched in subsets of genes differentially expressed under P deficiency. The most prominent subset supports the presence of a coordinated signaling network that targets cell wall structure and is regulated in part via a decrease of H3K4me3 at transcription start sites. The P starvation-induced chromatin dynamics and correlated genes identified here will aid in enhancing P use efficiency in crop plants, benefitting global agriculture.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity.

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2-phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II-mediated transcription in cancer cells.

Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices.

During embryogenesis, chromatin accessibility profiles control lineage-specific gene expression by modulating transcription, thus impacting multipotent progenitor states and subsequent fate choices. Subsets of cardiac and pharyngeal/head muscles share a common origin in the cardiopharyngeal mesoderm, but the chromatin landscapes that govern multipotent progenitors competence and early fate choices remain largely elusive. Here, we leveraged the simplicity of the chordate model Ciona to profile chromatin accessibility through stereotyped transitions from naive Mesp+ mesoderm to distinct fate-restricted heart and pharyngeal muscle precursors. An FGF-Foxf pathway acts in multipotent progenitors to establish cardiopharyngeal-specific patterns of accessibility, which govern later heart vs. pharyngeal muscle-specific expression profiles, demonstrating extensive spatiotemporal decoupling between early cardiopharyngeal enhancer accessibility and late cell-type-specific activity. We found that multiple cis-regulatory elements, with distinct chromatin accessibility profiles and motif compositions, are required to activate Ebf and Tbx1/10, two key determinants of cardiopharyngeal fate choices. We propose that these 'combined enhancers' foster spatially and temporally accurate fate choices, by increasing the repertoire of regulatory inputs that control gene expression, through either accessibility and/or activity.

The prevalence, evolution and chromatin signatures of plant regulatory elements.

Chromatin accessibility and modification is a hallmark of regulatory DNA, the study of which led to the discovery of cis-regulatory elements (CREs). Here, we characterize chromatin accessibility, histone modifications and sequence conservation in 13 plant species. We identified thousands of putative CREs and revealed that distal CREs are prevalent in plants, especially in species with large and complex genomes. The majority of distal CREs have been moved away from their target genes by transposable-element (TE) proliferation, but a substantial number of distal CREs also seem to be created by TEs. Finally, plant distal CREs are associated with three major types of chromatin signatures that are distinct from metazoans. Taken together, these results suggest that CREs are prevalent in plants, highly dynamic during evolution and function through distinct chromatin pathways to regulate gene expression.