Chromatin Disruption Research Articles

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Neuron-specific chromatin disruption at CpG islands and aging-related regions in Kabuki syndrome mice.

Many Mendelian developmental disorders caused by coding variants in epigenetic regulators have now been discovered. Epigenetic regulators are broadly expressed, and each of these disorders typically shows phenotypic manifestations from many different organ systems. An open question is whether the chromatin disruption-the root of the pathogenesis-is similar in the different disease-relevant cell types. This is possible in principle, because all these cell types are subject to effects from the same causative gene, which has the same kind of function (e.g., methylates histones) and is disrupted by the same germline variant. We focus on mouse models for Kabuki syndrome types 1 and 2 and find that the chromatin accessibility changes in neurons are mostly distinct from changes in B or T cells. This is not because the neuronal accessibility changes occur at regulatory elements that are only active in neurons. Neurons, but not B or T cells, show preferential chromatin disruption at CpG islands and at regulatory elements linked to aging. A sensitive analysis reveals that regulatory elements disrupted in B/T cells do show chromatin accessibility changes in neurons, but these are very subtle and of uncertain functional significance. Finally, we are able to identify a small set of regulatory elements disrupted in all three cell types. Our findings reveal the cellular-context-specific effect of variants in epigenetic regulators and suggest that blood-derived episignatures, although useful diagnostically, may not be well suited for understanding the mechanistic basis of neurodevelopment in Mendelian disorders of the epigenetic machinery.

Abstract B016: Targeting the epigenome through combined Facilitates Chromatin Transcription (FACT) and Bromodomain and Extra-Terminal Domain (BET) inhibition in Diffuse Midline Glioma (DMG)

Abstract Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable tumor that primarily arises in the brainstem of young children. The H3K27M driver histone mutation and resulting permissive chromatin indicates epigenetic targeting as a key therapeutic strategy against this aggressive cancer. One such epigenetic target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in phase I/II clinical trial for pediatric cancer patients (NCT04870944). This study aims to decipher the mechanism of CBL0137 to find effective combination therapies for the next round of clinical implementation. To this end, we applied a multi-omics approach by integrating epigenetic profiling with transcriptomic data sets (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the therapeutic mechanism of CBL0137 in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy. We found FACT to be enriched at the promoters of developmental genes, coinciding with regions of open chromatin and binding motifs of the core DMG regulatory transcription factors TCF12 and OLIG2. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4, suggesting cooperation between FACT and BRD4 in DMG. Given that BRD4 is an established therapeutic target in DMG and other cancers, we investigated the combination of the FACT-inhibitor CBL0137 with the BET-inhibitor JQ1. Combination treatment was cytotoxic against DMG in vitro, with enhanced cytotoxicity observed against H3K27M-mutant cells. We demonstrated a significant survival extension in three independent orthotopic PDX mouse models treated with CBL0137 and JQ1 in combination. Mechanistically, CBL0137 and JQ1 decreased chromatin accessibility and transcription of genes involved in RNA regulatory processes, such as RNA methylation and splicing. Consistently, combination treatment led to increased intron retention, highlighting a possible link between chromatin disruption and the splicing machinery in DMG. Future work will investigate whether treatment-induced intron retention acts as a source of neoantigens, with the potential to enhance DMG immunogenicity. In sum, this study has identified a promising new epigenetic combination therapy – FACT + BET inhibition – while uncovering intriguing new insights into DMG epigenetics and pathobiology. Citation Format: Holly Holliday, Aaminah Khan, Nisitha Jayatilleke, Chelsea Mayoh, Samuel E. Ross, Yolanda Colino Sanguino, Anjana Gopalakrishnan, Anahid Ehteda, Benjamin Rayner, Maria Tsoli, David S. Ziegler. Targeting the epigenome through combined Facilitates Chromatin Transcription (FACT) and Bromodomain and Extra-Terminal Domain (BET) inhibition in Diffuse Midline Glioma (DMG) [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B016.

A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae reveals novel insights into factors that stabilize chromatin structure on newly replicated DNA.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

Ability of a selfish B chromosome to evade genome elimination in the jewel wasp, Nasonia vitripennis

B chromosomes are non-essential, extra chromosomes that can exhibit transmission-enhancing behaviors, including meiotic drive, mitotic drive, and induction of genome elimination, in plants and animals. A fundamental but poorly understood question is what characteristics allow B chromosomes to exhibit these extraordinary behaviors. The jewel wasp, Nasonia vitripennis, harbors a heterochromatic, paternally transmitted B chromosome known as paternal sex ratio (PSR), which causes complete elimination of the sperm-contributed half of the genome during the first mitotic division of fertilized embryos. This genome elimination event may result from specific, previously observed alterations of the paternal chromatin. Due to the haplo-diploid reproduction of the wasp, genome elimination by PSR causes female-destined embryos to develop as haploid males that transmit PSR. PSR does not undergo self-elimination despite its presence with the paternal chromatin until the elimination event. Here we performed fluorescence microscopic analyses aimed at understanding this unexplained property. Our results show that PSR, like the rest of the genome, participates in the histone-to-protamine transition, arguing that PSR does not avoid this transition to escape self-elimination. In addition, PSR partially escapes the chromatin-altering activity of the intracellular bacterium, Wolbachia, demonstrating that this ability to evade chromatin alteration is not limited to PSR’s own activity. Finally, we observed that the rDNA locus and other unidentified heterochromatic regions of the wasp’s genome also seem to evade chromatin disruption by PSR, suggesting that PSR’s genome-eliminating activity does not affect heterochromatin. Thus, PSR may target an aspect of euchromatin to cause genome elimination.

Structure and Dynamics of Compact Dinucleosomes: Analysis by Electron Microscopy and spFRET.

Formation of compact dinucleosomes (CODIs) occurs after collision between adjacent nucleosomes at active regulatory DNA regions. Although CODIs are likely dynamic structures, their structural heterogeneity and dynamics were not systematically addressed. Here, single-particle Förster resonance energy transfer (spFRET) and electron microscopy were employed to study the structure and dynamics of CODIs. spFRET microscopy in solution and in gel revealed considerable uncoiling of nucleosomal DNA from the histone octamer in a fraction of CODIs, suggesting that at least one of the nucleosomes is destabilized in the presence of the adjacent closely positioned nucleosome. Accordingly, electron microscopy analysis suggests that up to 30 bp of nucleosomal DNA are involved in transient uncoiling/recoiling on the octamer. The more open and dynamic nucleosome structure in CODIs cannot be stabilized by histone chaperone Spt6. The data suggest that proper internucleosomal spacing is an important determinant of chromatin stability and support the possibility that CODIs could be intermediates of chromatin disruption.

Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters.

Anthracyclines are a class of widely prescribed anticancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we used Cleavage Under Targets and Tagmentation (CUT&Tag) to profile RNA polymerase II during anthracycline treatment in Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation affect chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of noncanonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the cancer-killing activity of aclarubicin is driven by the disruption of nucleosomes and RNA polymerase II.

Pyroptosis-related noncoding RNAs and cancer involvement

Abstract Cell pyroptosis, an inflammatory and programmed cell necrosis, is also an important cause of multiple organ damage and immunosuppression. Pyroptosis, a new kind of programmed cell death, leads to cleavage of the pyroptotic protein Gasdermin family and ultimately to membrane pore formation, cell shrinkage, plasma membrane cleavage, chromatin disruption and release of pro-inflammatory components. Various studies found that pyroptosis had a key role in cancer and inflammatory diseases. Gasdermin family is key protein in pyrotopsis. Non-coding RNAs have a potential role in cell pyrotosis which regulate immunity and cell death. This article reviews cell pyroptosis, GSDM family and the regulatory role of non-coding RNAs.

DIPG-45. ELONGIN B REGULATES H3K27M CHROMATIN INCORPORATION TO PROMOTE MALIGNANCY IN DIFFUSE MIDLINE GLIOMA

Abstract Chromatin dysregulation is both a cause and consequence of aberrant RNA Polymerase II (Pol2) transcription in cancer. Some of the most striking examples of chromatin disruption in cancer include somatic mutations in genes encoding histone H3 (H3.3/H3.1K27M) observed in ~80% of diffuse midline glioma (DMG). H3K27M mutant histones act in a dominant manner to disrupt chromatin regulation and Pol2-dependent transcription in DMG. How H3K27M impinges on the Pol2 transcriptional machinery and whether aberrant Pol2 transcription can reciprocally induce chromatin dysregulation in DMG remain open questions. We conducted epigenome-focused CRISPR dropout screens and identified multiple regulators of Pol2 elongation, including Elongin B and C (ELOB/ELOC) as DMG genetic dependencies. Follow-up studies confirm that knockout (KO) of ELOB inhibits DMG proliferation in neurosphere culture and abrogates DMG tumorigenesis in orthotopic xenografts. Additional chromatin profiling studies reveal that ELOB binding sites are enriched in H3K27M mutant histones, suggesting that ELOB works together with H3K27M to promote malignant gene expression in DMG. Indeed, reciprocal knockout studies suggest that while ELOB chromatin binding patterns were not altered by H3K27M KO, ELOB KO significantly reduced H3K27M chromatin incorporation at thousands of genomic loci. Further RNAseq and PROseq analyses reveal that ELOB KO alters Pol2 transcription resulting in the up-regulation of genes associated with neuronal and muscle differentiation and the repression of glial stem cell genes. Together, these findings suggest that H3K27M chromatin binding patterns and aberrant gene regulation in DMG are, in part, dictated by ELOB and the Pol2 elongation machinery. Finally, analyses of patient datasets indicate that ELOB complex genes are overexpressed in high grade glioma and DMG and correlate with decreased patient survival. Collectively, these studies suggest that a functional interaction between ELOB and H3K27M mutant histones induces DMG-specific aberrations in chromatin and gene regulation to drive malignancy.

Chromatin and Cancer: Implications of Disrupted Chromatin Organization in Tumorigenesis and Its Diversification.

A hallmark of cancers is uncontrolled cell proliferation, frequently associated with an underlying imbalance in gene expression. This transcriptional dysregulation observed in cancers is multifaceted and involves chromosomal rearrangements, chimeric transcription factors, or altered epigenetic marks. Traditionally, chromatin dysregulation in cancers has been considered a downstream effect of driver mutations. However, here we present a broader perspective on the alteration of chromatin organization in the establishment, diversification, and therapeutic resistance of cancers. We hypothesize that the chromatin organization controls the accessibility of the transcriptional machinery to regulate gene expression in cancerous cells and preserves the structural integrity of the nucleus by regulating nuclear volume. Disruption of this large-scale chromatin in proliferating cancerous cells in conventional chemotherapies induces DNA damage and provides a positive feedback loop for chromatin rearrangements and tumor diversification. Consequently, the surviving cells from these chemotherapies become tolerant to higher doses of the therapeutic reagents, which are significantly toxic to normal cells. Furthermore, the disorganization of chromatin induced by these therapies accentuates nuclear fragility, thereby increasing the invasive potential of these tumors. Therefore, we believe that understanding the changes in chromatin organization in cancerous cells is expected to deliver more effective pharmacological interventions with minimal effects on non-cancerous cells.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

Super-Resolution Imaging Reveals T Lymphocyte Activation-Induced Chromatin Decondensation and Disruption of Nuclear Envelope Facilitate the Release of Nuclear DNA

Super-Resolution Imaging Reveals T Lymphocyte Activation-Induced Chromatin Decondensation and Disruption of Nuclear Envelope Facilitate the Release of Nuclear DNA

Morphological events at the time of oocyte maturation and sperm–egg interactions during fertilisation in a dasyurid marsupial

In marsupials there have been several studies on oocyte maturation and sperm–egg interactions at the time of fertilisation, but controversy exists as to when and how some of the processes occur. Here we present a summary of relevant data from a species of dasyurid marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). The findings show that in oocytes of developing ovarian follicles cortical granules are produced with many of the granules sometimes initially congregating around the cytoplasmic vacuoles before migrating to the proximity of the oocyte cell membrane. During fertilisation the spermatozoon binds to the zona pellucida that surrounds the oocyte with its tail lying parallel to the long axis of the head. As the spermatozoon passes through the zona pellucida it is surrounded by the zona matrix and, when entering the egg cytoplasm, a localised area of elevated ooplasm occurs around the spermatozoon. Also, unlike a recent claim to the contrary, the head of the spermatozoon travels a considerable distance into the egg cytoplasm before chromatin decondensation occurs, an event that probably minimises the chances of chromatin disruption by the sperm tail at the time of its incorporation.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

Opposing functions of the Hda1 complex and histone H2B mono-ubiquitylation in regulating cryptic transcription in Saccharomyces cerevisiae.

Maintenance of chromatin structure under the disruptive force of transcription requires cooperation among numerous regulatory factors. Histone post-translational modifications can regulate nucleosome stability and influence the disassembly and reassembly of nucleosomes during transcription elongation. The Paf1 transcription elongation complex, Paf1C, is required for several transcription-coupled histone modifications, including the mono-ubiquitylation of H2B. In Saccharomyces cerevisiae, amino acid substitutions in the Rtf1 subunit of Paf1C greatly diminish H2B ubiquitylation and cause transcription to initiate at a cryptic promoter within the coding region of the FLO8 gene, an indicator of chromatin disruption. In a genetic screen to identify factors that functionally interact with Paf1C, we identified mutations in HDA3, a gene encoding a subunit of the Hda1C histone deacetylase (HDAC), as suppressors of an rtf1 mutation. Absence of Hda1C also suppresses the cryptic initiation phenotype of other mutants defective in H2B ubiquitylation. The genetic interactions between Hda1C and the H2B ubiquitylation pathway appear specific: loss of Hda1C does not suppress the cryptic initiation phenotypes of other chromatin mutants and absence of other HDACs does not suppress the absence of H2B ubiquitylation. Providing further support for an appropriate balance of histone acetylation in regulating cryptic initiation, absence of the Sas3 histone acetyltransferase elevates cryptic initiation in rtf1 mutants. Our data suggest that the H2B ubiquitylation pathway and Hda1C coordinately regulate chromatin structure during transcription elongation and point to a potential role for a HDAC in supporting chromatin accessibility.

Cellular senescence as a response to multiwalled carbon nanotube (MWCNT) exposure in human mesothelial cells

Cellular senescence is a stable cell cycle arrest induced by diverse triggers, including replicative exhaustion, DNA damaging agents, oncogene activation, oxidative stress, and chromatin disruption. With important roles in aging and tumor suppression, cellular senescence has been implicated also in tumor promotion. Here we show that certain multiwalled carbon nanotubes (MWCNTs), as fiber-like nanomaterials, can trigger cellular senescence in primary human mesothelial cells. Using in vitro approaches, we found manifestation of several markers of cellular senescence, especially after exposure to a long and straight MWCNT. These included inhibition of cell division, senescence-associated heterochromatin foci, senescence-associated distension of satellites, LMNB1 depletion, γH2A.X nuclear panstaining, and enlarged cells exhibiting senescence-associated β-galactosidase activity. Furthermore, genome-wide transcriptome analysis revealed many differentially expressed genes, among which were genes encoding for a senescence-associated secretory phenotype. Our results clearly demonstrate the potential of long and straight MWCNTs to induce premature cellular senescence. This finding may find relevance in risk assessment of workplace safety, and in evaluating MWCNT’s use in medicine such as drug carrier, due to exposure effects that might prompt onset of age-related diseases, or even carcinogenesis.

The role of FACT in managing chromatin:disruption, assembly, or repair?

FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.

Interplay among ATP-Dependent Chromatin Remodelers Determines Chromatin Organisation in Yeast.

Cellular DNA is packaged into chromatin, which is composed of regularly-spaced nucleosomes with occasional gaps corresponding to active regulatory elements, such as promoters and enhancers, called nucleosome-depleted regions (NDRs). This chromatin organisation is primarily determined by the activities of a set of ATP-dependent remodeling enzymes that are capable of moving nucleosomes along DNA, or of evicting nucleosomes altogether. In yeast, the nucleosome-spacing enzymes are ISW1 (Imitation SWitch protein 1), Chromodomain-Helicase-DNA-binding (CHD)1, ISW2 (Imitation SWitch protein 2) and INOsitol-requiring 80 (INO80); the nucleosome eviction enzymes are the SWItching/Sucrose Non-Fermenting (SWI/SNF) family, the Remodeling the Structure of Chromatin (RSC) complexes and INO80. We discuss the contributions of each set of enzymes to chromatin organisation. ISW1 and CHD1 are the major spacing enzymes; loss of both enzymes results in major chromatin disruption, partly due to the appearance of close-packed di-nucleosomes. ISW1 and CHD1 compete to set nucleosome spacing on most genes. ISW1 is dominant, setting wild type spacing, whereas CHD1 sets short spacing and may dominate on highly-transcribed genes. We propose that the competing remodelers regulate spacing, which in turn controls the binding of linker histone (H1) and therefore the degree of chromatin folding. Thus, genes with long spacing bind more H1, resulting in increased chromatin compaction. RSC, SWI/SNF and INO80 are involved in NDR formation, either directly by nucleosome eviction or repositioning, or indirectly by affecting the size of the complex that resides in the NDR. The nature of this complex is controversial: some suggest that it is a RSC-bound “fragile nucleosome”, whereas we propose that it is a non-histone transcription complex. In either case, this complex appears to serve as a barrier to nucleosome formation, resulting in the formation of phased nucleosomal arrays on both sides.

An Epigenetics-Based Hypothesis of Autoantigen Development in Systemic Lupus Erythematosus.

Currently, we have a limited understanding of mechanisms leading to systemic lupus erythematosus, but we know that genetics, environmental factors, and epigenetics contribute to the disease. One common aspect of the various environmental triggers is that they can cause cellular stress. When extraordinary stress occurs, such as viral activation, a cell’s response can include increased nucleolar volume and activity to produce more machinery (e.g., ribosomes) to help the cell recover. However, nucleolar expansion can disrupt the epigenetic control in neighboring heterochromatin that comprises the nucleolar shell. This disruption can open underlying vulnerabilities that provoke an autoimmune reaction. Here, we review the “X chromosome-nucleolus nexus” hypothesis, which explains how nucleolar stress can disrupt epigenetically silenced chromatin, especially the neighboring inactive X chromosome (aka the nucleolar satellite). Chromatin disruption can lead to the expression of sequestered DNA, such as Alu elements and fully functional LINE-1 reverse transcriptase genes. In addition, Alu transcripts can disrupt the nucleolar structural integrity, leading to nucleolar disintegration. Such disintegration can leave nucleolar components and products in autoantigenic forms, such as abnormal conformations or incomplete macromolecular assemblies. Recent research on DNA sensing pathways can now be incorporated into the hypothesis to provide further details explaining how autoantibodies to endogenous nucleic acids arise.

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

Genomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the in-situ higher-order folding of chromatin structure during malignant transformation remains largely unknown. Here, using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncover a gradual decompaction and fragmentation of higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, and prior to tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal functional consequences in enhanced transcription activities and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin disruption to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.