rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin.

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.

Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

The shifting shape of genomes: dynamics of heterochromatin interactions at the nuclear lamina.

The nucleus is a highly structured organelle with many chromatin and protein compartments that partition the genome into regulatory domains. One such a compartment within the mammalian nucleus is the microenvironment underlying the nuclear envelope (NE) where intermediate filament proteins, lamins, act as a link between cytoskeletal and inner nuclear membrane (INM) proteins, chromatin binders and modifiers, and heterochromatin. These dynamic interactions regulate many cellular processes and, when they are perturbed, can lead to genome dysregulation and disease.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens.

Entamoeba histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ∼20 h post induction, and normal nuclear morphology was restored by 72 h. Two types of cellular structures were visible by 16 h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20 h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

CENP-A Nucleosome is a Sensitive Allosteric Scaffold for DNA and Chromatin Factors.

Centromeric loci of chromosomes are defined by nucleosomes containing the histone H3 variant CENP-A, which bind their DNA termini more permissively than their canonical counterpart, a feature that is critical for the mitotic fidelity. A recent cryo-EM study demonstrated that the DNA termini of CENP-A nucleosomes, reconstituted with the Widom 601 DNA sequence, are asymmetrically flexible, meaning one terminus is more clearly resolved than the other. However, an earlier work claimed that both ends could be resolved in the presence of two stabilizing single chain variable fragment (scFv) antibodies per nucleosome, and thus are likely permanently bound to the histone octamer. This suggests that the binding of scFv antibodies to the histone octamer surface would be associated with CENP-A nucleosome conformational changes, including stable binding of the DNA termini. Here, we present computational evidence that allows to explain at atomistic level the structural rearrangements of CENP-A nucleosomes resulting from the antibody binding. The antibodies, while they only bind the octamer façades, are capable of altering the dynamics of the nucleosomal core, and indirectly also the surrounding DNA. This effect has more drastic implications for the structure and the dynamics of the CENP-A nucleosome in comparison to its canonical counterpart. Furthermore, we find evidence that the antibodies bind the left and the right octamer façades at different affinities, another manifestation of the DNA sequence. We speculate that the cells could use induction of similar allosteric effects to control centromere function.

Structures of Native-like Nucleosomes: One Step Closer toward Understanding the Structure and Function of Chromatin.

Genomic DNA in eukaryotes is organized into chromatin through association with core histone proteins to form nucleosomes. To understand the structure and function of chromatin, we must determine the structures of nucleosomes containing native DNA sequences. However, to date, our knowledge of nucleosome structures is mainly based on the crystallographic studies of the nucleosomes containing non-native DNA sequences. Here, we discuss the technical issues related to the determination of the nucleosome structures and review the few structural studies on native-like nucleosomes. We show how an antibody fragment-aided single-particle cryo-EM can be a useful method to determine the structures of nucleosomes containing genomic DNA. Finally, we provide a perspective for future structural studies of some native-like nucleosomes that play critical roles in chromatin functions.

Contributions of Histone Variants in Nucleosome Structure and Function.

Chromatin compacts genomic DNA in eukaryotes. The primary chromatin unit is the nucleosome core particle, composed of four pairs of the core histones, H2A, H2B, H3, and H4, and 145-147 base pairs of DNA. Since replication, recombination, repair, and transcription take place in chromatin, the structure and dynamics of the nucleosome must be versatile. These nucleosome characteristics underlie the epigenetic regulation of genomic DNA. In higher eukaryotes, many histone variants have been identified as non-allelic isoforms, which confer nucleosome diversity. In this article, we review the manifold types of nucleosomes produced by histone variants, which play important roles in the epigenetic regulation of chromatin.

Histone Variant H2A.J Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence.

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.

Nucleosome binding by the pioneer transcription factor OCT4.

Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.

Changes in relative histone abundance and heterochromatin in αA-crystallin and αB-crystallin knock-in mutant mouse lenses.

OBJECTIVE: Understanding the mechanisms of cataract formation is important for age-related and hereditary cataracts caused by mutations in lens protein genes. Lens proteins of the crystallin gene families α-, ß-, and γ-crystallin are the most abundant proteins in the lens. Single point mutations in crystallin genes cause autosomal dominant cataracts in multigenerational families. Our previous proteomic and RNAseq studies identified genes and proteins altered in the early stages of cataract formation in mouse models. Histones H2A, H2B, and H4 increase in abundance in αA- and αB-crystallin mutant mouse lenses and in cultured cells expressing the mutant form of αA-crystallin linked with hereditary cataracts. RESULTS: In this study of histones in mutant lenses, we extracted histones from adult mouse lenses from cryaa-R49C and cryab-R120G mutant knock-in mice. We characterized the histones using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometric analysis and gel electrophoresis and characterized the lens nucleus morphology using electron microscopy (EM). The relative abundance of histone H3 protein decreased in lenses from cryaa-R49C mutant mice and the relative abundance of histone H2 increased in these lenses. Electron microscopy of nuclei from cryaa-R49C-homozygous mutant mouse lenses revealed a pronounced alteration in the distribution of heterochromatin.

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation1,2. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.

Evolutionary Variability of W-Linked Repetitive Content in Lacertid Lizards.

Lacertid lizards are a widely radiated group of squamate reptiles with long-term stable ZZ/ZW sex chromosomes. Despite their family-wide homology of Z-specific gene content, previous cytogenetic studies revealed significant variability in the size, morphology, and heterochromatin distribution of their W chromosome. However, there is little evidence about the accumulation and distribution of repetitive content on lacertid chromosomes, especially on their W chromosome. In order to expand our knowledge of the evolution of sex chromosome repetitive content, we examined the topology of telomeric and microsatellite motifs that tend to often accumulate on the sex chromosomes of reptiles in the karyotypes of 15 species of lacertids by fluorescence in situ hybridization (FISH). The topology of the above-mentioned motifs was compared to the pattern of heterochromatin distribution, as revealed by C-banding. Our results show that the topologies of the examined motifs on the W chromosome do not seem to follow a strong phylogenetic signal, indicating independent and species-specific accumulations. In addition, the degeneration of the W chromosome can also affect the Z chromosome and potentially also other parts of the genome. Our study provides solid evidence that the repetitive content of the degenerated sex chromosomes is one of the most evolutionary dynamic parts of the genome.

Heterochromatin replication goes hand in hand with telomere protection.

Telomeres arose from the need to stabilize natural chromosome ends, resulting in terminal chromatin structures with specific protective functions. Their constituent proteins also execute general functions within heterochromatin, mediating late replication and facilitating fork progression. Emerging insights into the mechanisms governing heterochromatin replication suggest telomeres and heterochromatin act in concert during development and aging. They also suggest a common evolutionary origin for these two chromosome regions that arose during eukaryogenesis.

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.

Phase-separation in chromatin organization.

The organization of chromatin into different types of compact versus open states provides a means to fine tune gene regulation. Recent studies have suggested a role for phase-separation in chromatin compaction, raising new possibilities for regulating chromatin compartments. This perspective discusses some specific molecular mechanisms that could leverage such phase-separation processes to control the functions and organization of chromatin.

RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster.

Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes 'primes' post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.

A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation.

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.

Infrared nanospectroscopic mapping of a single metaphase chromosome.

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo.

Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.