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.

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.

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.

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.

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.

Copy number variation and selection during reprogramming to pluripotency.

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

Living without 30nm chromatin fibers.

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. According to the current paradigm, strings of nucleosomes, termed 10nm chromatin fibers, constitute the template of transcriptionally active genomic material. The majority of the genome is maintained in a silenced state through higher-order chromatin assemblies, based on the 30nm chromatin fiber, which excludes activating regulatory factors. New experimental approaches, however, including chromatin conformation capture and cryo-electron microscopy, call into question the in situ evidence for the 30nm chromatin fiber. We suggest that the organization of the genome based on 10nm chromatin fibers is sufficient to describe the complexities of nuclear organization and gene regulation.

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.

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.

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.

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.

Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.

Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR.

The promyelocytic leukemia (PML) nuclear body (NB) is a dynamic subnuclear compartment that is implicated in tumor suppression, as well as in the transcription, replication, and repair of DNA. PML NB number can change during the cell cycle, increasing in S phase and in response to cellular stress, including DNA damage. Although topological changes in chromatin after DNA damage may affect the integrity of PML NBs, the molecular or structural basis for an increase in PML NB number has not been elucidated. We demonstrate that after DNA double-strand break induction, the increase in PML NB number is based on a biophysical process, as well as ongoing cell cycle progression and DNA repair. PML NBs increase in number by a supramolecular fission mechanism similar to that observed in S-phase cells, and which is delayed or inhibited by the loss of function of NBS1, ATM, Chk2, and ATR kinase. Therefore, an increase in PML NB number is an intrinsic element of the cellular response to DNA damage.

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings.

The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors.

Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.

Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

Probing intranuclear environments at the single-molecule level.

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

A nonribosomal landscape in the nucleolus revealed by the stem cell protein nucleostemin.

Nucleostemin is a p53-interactive cell cycle progression factor that shuttles between the nucleolus and nucleoplasm, but it has no known involvement in ribosome synthesis. We found the dynamic properties of nucleostemin differed strikingly from fibrillarin (a protein directly involved in rRNA processing) both in response to rRNA transcription inhibition and in the schedule of reentry into daughter nuclei and the nucleolus during late telophase/early G1. Furthermore, nucleostemin was excluded from the nucleolar domains in which ribosomes are born--the fibrillar centers and dense fibrillar component. Instead it was concentrated in rRNA-deficient sites within the nucleolar granular component. This finding suggests that the nucleolus may be more subcompartmentalized than previously thought. In support of this concept, electron spectroscopic imaging studies of the nitrogen and phosphorus distribution in the nucleolar granular component revealed regions that are very rich in protein and yet devoid of nucleic acid. Together, these results suggest that the ultrastructural texture of the nucleolar granular component represents not only ribosomal particles but also RNA-free zones populated by proteins or protein complexes that likely serve other functions.

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo.

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.