Myogenic differentiation triggers PML nuclear body loss and DAXX relocalization to chromocentres.

The promyelocytic leukemia protein (PML) is expressed in most normal human tissues and forms nuclear bodies (NBs) that have roles in gene regulation and cellular processes such as DNA repair, cell cycle control, and cell fate decisions. Using murine C2C12 myoblasts, we demonstrate that activation of skeletal muscle differentiation results in loss of PML and PML NBs prior to myotube fusion. Myotube formation was associated with marked chromatin reorganization and the relocalization of DAXX from PML NBs to chromocentres. MyoD expression was sufficient to cause PML NB loss, and silencing of PML induced DAXX relocalization. Fusion of C2C12 cells using the reptilian reovirus p14 fusogenic protein failed to disrupt PML NBs yet still promoted DAXX redistribution and loss; whereas ectopic expression of PML in differentiated cells only partially restored PML NB formation and DAXX localization at NBs. Finally, we determined that the C-terminal SUMO-interacting motif of DAXX is required for its colocalization with ATRX in heterochromatin domains during myotube formation. These data support a model in which activation of myogenic differentiation results in PML NB loss, chromatin reorganization and DAXX relocalization, and provides a paradigm for understanding the consequence of PML loss in other cellular contexts, such as during cancer development and progression.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

FANCD2 limits BLM-dependent telomere instability in the alternative lengthening of telomeres pathway.

Fanconi anemia and Bloom syndrome are genomic instability syndromes caused by mutations in proteins that participate in overlapping DNA repair and replication pathways. Here, we show that the monoubiquitinated form of the Fanconi Anemia protein FANCD2 acts in opposition to the BLM DNA helicase to restrain telomere replication and recombination in human cells that utilize the Alternative Lengthening of Telomeres (ALT) pathway. ALT relies on exchanges of telomeric DNA to maintain telomeres, a process that we show FANCD2 suppresses. Depletion of FANCD2 results in a hyper-ALT phenotype, including an increase in extrachromosomal telomeric repeat DNAs, putative recombinational byproducts that we show exist as intertwined complexes forming the nucleic acid component of ALT-associated PML bodies. Increases in telomeric DNA are suppressed by loss of BLM but not RAD51, occur without parallel upregulation of shelterin proteins TRF1 and TRF2, and are associated with increased frequencies of deprotected and fragile telomeres. Inactivation of the FA pathway does not trigger ALT, as FANCD2 depleted telomerase positive cells do not acquire ALT-like phenotypes. We observe frequent fragile telomeres in ALT cells, suggesting that telomere sequences are prone to replication problems. We propose that, in ALT cells, FANCD2 promotes intramolecular resolution of stalled replication forks in telomeric DNA while BLM facilitates their resection and subsequent involvement in the intermolecular exchanges that drive ALT.

A novel single cell method to identify the genetic composition at a single nuclear body.

Gene loci make specific associations with compartments of the nucleus (e.g. the nuclear envelope, nucleolus, and transcription factories) and this association may determine or reflect a mechanism of genetic control. With current methods, it is not possible to identify sets of genes that converge to form a "gene hub" as there is a reliance on loci-specific probes, or immunoprecipitation of a particular protein from bulk cells. We introduce a method that will allow for the identification of loci contained within the vicinity of a single nuclear body in a single cell. For the first time, we demonstrate that the DNA sequences originating from a single sub-nuclear structure in a single cell targeted by two-photon irradiation can be determined, and mapped to a particular locus. Its application to single PML nuclear bodies reveals ontologically related loci that frequently associate with each other and with PML bodies in a population of cells, and a possible nuclear body targeting role for specific transcription factor binding sites.

The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.

Coming to terms with chromatin structure.

Chromatin, once thought to serve only as a means to package DNA, is now recognized as a major regulator of gene activity. As a result of the wide range of methods used to describe the numerous levels of chromatin organization, the terminology that has emerged to describe these organizational states is often imprecise and sometimes misleading. In this review, we discuss our current understanding of chromatin architecture and propose terms to describe the various biochemical and structural states of chromatin.

MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin.

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

The histone chaperone DAXX maintains the structural organization of heterochromatin domains.

BACKGROUND: The death domain-associated protein (DAXX) collaborates with accessory proteins to deposit the histone variant H3.3 into mouse telomeric and pericentromeric repeat DNA. Pericentromeric repeats are the main genetic contributor to spatially discrete, compact, constitutive heterochromatic structures called chromocentres. Chromocentres are enriched in the H3K9me3 histone modification and serve as integral, functionally important components of nuclear organization. To date, the role of DAXX as an H3.3-specific histone chaperone has been investigated primarily using biochemical approaches which provide genome-wide views on cell populations and information on changes in local chromatin structures. However, the global chromatin and subnuclear reorganization events that coincide with these changes remain to be investigated. RESULTS: Using electron spectroscopic imagine (ESI), a specialized form of energy-filtered transmission electron microscopy that allows us to visualize chromatin domains in situ with high contrast and spatial resolution, we show that in the absence of DAXX, H3K9me3-enriched domains are structurally altered and become uncoupled from major satellite DNA. In addition, the structural integrity of nucleoli and the organization of ribosomal DNA (rDNA) are disrupted. Moreover, the absence of DAXX leads to chromatin that is more sensitive, on a global level, to micrococcal nuclease digestion. CONCLUSIONS: We identify a novel role of DAXX as a major regulator of subnuclear organization through the maintenance of the global heterochromatin structural landscape. As well, we show, for the first time, that the loss of a histone chaperone can have severe consequences for global nuclear organization.

Unfolding the story of chromatin organization in senescent cells.

Cell senescence, the permanent withdrawal of a cell from the cell cycle, is characterized by dramatic, cytological scale changes to DNA condensation throughout the genome. While prior emphasis has been placed on increases in heterochromatin, such as the formation of compact Senescent Associated Heterochromatin Foci (SAHF) structures, our recent findings showed that SAHF formation is preceded by the unravelling of constitutive heterochromatin into visibly extended structures, which we have termed Senescent Associated Distension of Satellites or SADS. Interestingly, neither of these marked changes in DNA condensation appear to be mediated by changes in canonical, heterochromatin-associated histone modifications. Rather, several observations suggest that these events may be facilitated by changes in LaminB1 levels and/or other factors that control higher-order chromatin architecture. Here, we review what is known about senescence-associated chromatin reorganization and present preliminary results using high-resolution microscopy techniques to show that each peri/centromeric satellite in senescent cells is comprised of several condensed domains connected by thin fibrils of satellite DNA. We then discuss the potential importance of these striking changes in chromatin condensation for cell senescence, and also as a model to provide a needed window into the higher-order packaging of the genome.

Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

Identification of c-MYC SUMOylation by mass spectrometry.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation.

Nano-dissection and sequencing of DNA at single sub-nuclear structures.

The relative positioning of gene loci within a mammalian nucleus is non-random and plays a role in gene regulation. Some sub-nuclear structures may represent "hubs" that bring specific genetic loci into close proximity where co-regulatory mechanisms can operate. The identification of loci in proximity to a shared sub-nuclear structure can provide insights into the function of the associated structure, and reveal relationships between the loci sharing a common association. A technique is introduced based on the nano-dissection of DNA from thin sections of cells by high-precision nano-tools operated inside a scanning electron microscope. The ability to dissect and identify gene loci occupying a shared site at a single sub-nuclear structure is demonstrated here for the first time. The technique is applied to the nano-dissection of DNA in vicinity of a single promyelocytic leukemia nuclear body (PML NB), and reveals novel loci from several chromosomes that are confirmed to associate at PML NBs with statistical significance in a cell population. Furthermore, it is demonstrated that pairs of loci from different chromosomes congregate at the same nuclear body. It is proposed that this technique is the first that allows the de novo determination of gene loci associations with single nuclear sub-structures.

Electron spectroscopic tomography of specific chromatin domains.

The eukaryotic genome is packaged within the nucleus as poly-nucleosome 10 nm chromatin fibres. The nucleosome core particle, the fundamental chromatin subunit, consists of a DNA molecule wrapped around a histone octamer. Biochemical modifications of both the DNA and histone proteins have been characterized that influence chromatin structure and function. These modifications include DNA methylation, histone variants and posttranslational modifications of the core histone protein tails. An outstanding area for investigation in the field of nuclear cell biology is the characterization of the functional relation between these biochemical modifications and the underlying chromatin structure and nuclear sub-compartmentalization. Electron spectroscopic tomography is a high-resolution microscopy technique that facilitates visualization of individual 10 nm chromatin fibres in three dimensions. The method, therefore, has a role to play in exploring the relationships of the epigenome and nuclear organization. Correlating immunofluorescence microscopy with electron spectroscopic tomography provides a powerful approach to relate epigenetic marks with high resolution chromatin organization.

CEP120 and SPICE1 cooperate with CPAP in centriole elongation.

Centrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins. Centrioles are barrel-shaped structures composed of MTs, and as basal bodies they template the formation of cilia/flagella. Defects in centriole assembly can lead to ciliopathies and genome instability. The assembly of procentrioles requires a set of conserved proteins. It is initiated at the G1-to-S transition by PLK4 and CEP152, which help recruit SASS6 and STIL to the vicinity of the mother centriole to organize the cartwheel. Subsequently, CPAP promotes centriolar MT assembly and elongation in G2. While centriole integrity is maintained by CEP135 and POC1 through MT stabilization, centriole elongation requires POC5 and is restricted by CP110 and CEP97. How strict control of centriole length is achieved remains unclear. Here, we show that CEP120 and SPICE1 are required to localize CEP135 (but not SASS6, STIL, or CPAP) to procentrioles. CEP120 associates with SPICE1 and CPAP, and depletion of any of these proteins results in short procentrioles. Furthermore, CEP120 or CPAP overexpression results in excessive centriole elongation, a process dependent on CEP120, SPICE1, and CPAP. Our findings identify a shared function for these proteins in centriole length control.

Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations.

The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.

Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP.

Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations. Using promyelocytic leukemia nuclear bodies (PML NBs) as a model, we used immuno-TRAP to determine if specific genes localize within molecular dimensions with these bodies. Although we confirmed a TP53 gene-PML NB association, immuno-TRAP allowed us to uncover novel locus-PML NB associations, including the ABCA7 and TFF1 loci and, most surprisingly, the PML locus itself. These associations were cell type specific and reflected the cell's physiological state. Combined with microarrays or deep sequencing, immuno-TRAP provides powerful opportunities for identifying gene locus associations with potentially any nuclear subcompartment.

Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres.

The mammalian genome is compacted to fit within the confines of the cell nucleus. DNA is wrapped around nucleosomes, forming the classic "beads-on-a-string" 10-nm chromatin fibre. Ten-nanometre chromatin fibres are thought to condense into 30-nm fibres. This structural reorganization is widely assumed to correspond to transitions between active and repressed chromatin, thereby representing a chief regulatory event. Here, by combining electron spectroscopic imaging with tomography, three-dimensional images are generated, revealing that both open and closed chromatin domains in mouse somatic cells comprise 10-nm fibres. These findings indicate that the 30-nm chromatin model does not reflect the true regulatory structure in vivo.

Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation.

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

A view of the chromatin landscape.

The microscope has been indispensable to the last century of chromatin structure research. Microscopy techniques have revealed that the three-dimensional location of chromatin is not random but represents a further manifestation of a highly compartmentalized cell nucleus. Moreover, the structure and location of genetic loci display cell type-specific differences and relate directly to the state of differentiation. Advances to bridge imaging with genetic, molecular and biochemical approaches have greatly enhanced our understanding of the interdependence of chromatin structure and nuclear function in mammalian cells. In this review we discuss the current state of chromatin structure research in relationship to the variety of microscopy techniques that have contributed to this field.

Constitutive heterochromatin reorganization during somatic cell reprogramming.

Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by erasing and establishing repressive epigenetic marks. In contrast to loci-specific epigenetic changes, heterochromatin domains undergo epigenetic resetting during the reprogramming process, but the effect on the heterochromatin ultrastructure is not known. Here, we characterize the physical structure of heterochromatin domains in full and partial mouse iPS cells by correlative electron spectroscopic imaging. In somatic and partial iPS cells, constitutive heterochromatin marked by H3K9me3 is highly compartmentalized into chromocentre structures of densely packed chromatin fibres. In contrast, chromocentre boundaries are poorly defined in pluripotent embryonic stem and full iPS cells, and are characterized by unusually dispersed 10 nm heterochromatin fibres in high Nanog-expressing cells, including pluripotent cells of the mouse blastocyst before differentiation. This heterochromatin reorganization accompanies retroviral silencing during conversion of partial iPS cells by MEK/GSK3 2i inhibitor treatment. Thus, constitutive heterochromatin is compacted in partial iPS cells but reorganizes into dispersed 10 nm chromatin fibres as the fully reprogrammed iPS cell state is acquired.