Nucleolar-based Dux repression is essential for embryonic two-cell stage exit.

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Altered nuclear architecture in blood cells from Huntington's disease patients.

BACKGROUND: Cell nuclear architecture has been explored in cancer and laminopathies but not in neurodegenerative disorders. Huntington's disease (HD) is a neurodegenerative disorder that leads to neuronal death. Chromosome-wide changes in gene expression have been reported in HD, not only in the brain but also in peripheral blood cells, but whether this translates into nuclear and chromosome architecture alterations has not yet been studied. METHODS: We investigate nuclear structure and chromosome organization in HD blood cells using fluorescence in situ hybridization in ultrathin cryosections (cryoFISH), coupled with machine learning image analysis to evaluate size, distribution, and morphology of nuclei and chromosomes. Four chromosomes were analyzed based on up- or downregulation of gene expression in HD. RESULTS: We show that blood cells from HD patients display increased nuclear size and filamentary shape, increased size of gene-rich chromosome 19, decreased filamentary shape of gene-rich chromosome 22, and a more radially centralized position for chromosome 19, whereas chromosomes 4 and 5 do not show detectable differences. CONCLUSIONS: We identify gross changes in nuclear architecture and chromosome organization associated with HD in blood. This adds a new layer of information onto disrupting mechanisms in HD and increases the potential of using blood to survey HD.

Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

Poised transcription factories prime silent uPA gene prior to activation.

The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs) and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA) gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+)S2p(-)) factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+)S2p(+)) factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.

Identification of Two Subsets of Subcompartment A1 Associated with High Transcriptional Activity and Frequent Loop Extrusion.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Splicing speckles are not reservoirs of RNA polymerase II, but contain an inactive form, phosphorylated on serine2 residues of the C-terminal domain.

"Splicing speckles" are major nuclear domains rich in components of the splicing machinery and polyA(+) RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes but that they contain a stable pool of polymerase II phosphorylated on serine(2) residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells.

Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.

Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs.

Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p(+)S7p(-)S2p(-)) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p(+)S7p(+)S2p(+)); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.

CryoFISH: fluorescence in situ hybridization on ultrathin cryosections.

The visualization of cellular structures and components has become an invaluable tool in biological and medical sciences. Imaging subcellular compartments and single molecules within a cell has prompted the development of a wide range of sample preparation techniques as well as various microscope devices to obtain images with increased spatial resolution. Here, we present cryoFISH, a method for fluorescence in situ hybridization (FISH) on thin ( approximately 150 nm thick) cryosections from sucrose-embedded fixed cells or tissues. CryoFISH can be used in combination with immunodetection (IF) of other cellular components. The main advantages of cryoFISH and cryoIF over whole-cell labeling methods are increased spatial resolution with confocal microscopy, greater sensitivity of detection due to increased probe accessibility, and better image contrast. CryoFISH and cryoIF methods typically used on samples fixed in conditions that preserve ultrastructure, are compatible with the labeling of cells in their tissue context and are ideal for correlative studies that compare fluorescence with electron microscopy.

The molecular basis for stability of heterochromatin-mediated silencing in mammals.

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.

The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells.

RNA polymerase II (pol II) transcribes the most varied group of genes and is present in hypo- and hyperphosphorylated forms, with residues Ser(2) and Ser(5) of the C-terminal domain (CTD) of the largest subunit as main targets of phosphorylation. The elongating (active) form is phosphorylated on Ser(2) and can be specifically recognized with the H5 antibody. It has been found in different nuclear distributions: in discrete sites throughout the nucleoplasm, consistent with a role in transcription, and/or concentrated in "splicing speckles", a nuclear compartment mostly devoid of transcriptional activity. Here, we assess the effects of cell fixation and permeabilization on the distribution of polymerase II and correlate its distribution with the preservation of cellular ultrastructure. We show that phospho-Ser(2) polymerase II can redistribute to, or be differentially retained in, "speckles" in conditions that do not preserve cellular ultrastructure. The fixation protocols that disrupt polymerase II distribution also cause partial or total loss of TATA-binding protein, Sm antigen and PML staining in PML bodies, and have no noticeable effect in the labeling of SC35 in "splicing speckles" or coilin in Cajal bodies. When nuclear ultrastructure is preserved, phospho-Ser(2) polymerase II is found in discrete sites throughout the nucleoplasm, without visible enrichment within splicing speckles. A minor proportion of the total amount of the phospho-Ser(2) form is present in these domains.