Project description:Using a genetic model, we present a high resolution chromatin fibre analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30-nm chromatin fibre structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30-nm fibre and transcription does not alter bulk chromatin fibre structures. However, large scale chromatin structures on Xa are decondensed compared to the Xi and transcription inhibition is sufficient to promote large scale chromatin compaction. We show a link between transcription and large scale chromatin packaging independent of the bulk 30-nm chromatin fibre and propose that transcription, not the global compaction of 30-nm chromatin fibres, determines the cytological appearance of large scale chromatin structures. This SuperSeries is composed of the SubSeries listed below.
Project description:The genome is highly organized, yet whether DNA structures, such as A/B compartments, represent locations that are permissive/impermissive for transcription or whether these structures are simply correlated with locations of active/inactive genes remains unknown. This is because current methods cannot simultaneously measure DNA organization and transcription. Recently, we developed RNA&DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of DNA and RNA. Here, we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and their spatial contacts within the nucleus. We find that transcription occurs within B compartments – with multiple, simultaneously active genes colocalizing within them – and at genes localized proximal to nucleoli. Together, these results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.
Project description:The three-dimensional organization of chromosomes is tightly related to their biological function. Both imaging and chromosome conformation capture studies have revealed several layers of organization, including segregation into active and inactive compartments at the megabase scale, and partitioning into domains (TADs) and associated loops at the sub-megabase scale. Yet, it remains unclear how these layers of genome organization form, interact with one another, and contribute to or result from genome activities. TADs seem to have critical roles in regulating gene expression by promoting or preventing interactions between promoters and distant cis-acting regulatory elements, and different architectural proteins, including cohesin, have been proposed to play central roles in their formation. But so far, experimental depletions of these proteins have resulted in marginal changes in chromosome organization. Here, we show that deletion of the cohesin-loading factor, Nipbl, leads to loss of chromosome-associated cohesin and results in dramatic genome reorganization. TADs and associated loops vanish globally, even in the absence of transcriptional changes. In contrast, segregation into compartments is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the 3D organization of the genome results from the independent action of two distinct mechanisms: 1) cohesin-independent segregation of the genome into fine-scale compartment regions, defined by the underlying chromatin state; and 2) cohesin-dependent formation of TADs possibly by the recently proposed loop extrusion mechanism, enabling long-range and target-specific activity of promiscuous enhancers. The interplay between these mechanisms creates an architecture that is more complex than a simple structural hierarchy and can be central to guiding normal development.
Project description:Whereas spatial genome organization at large scale of compartments and topologically associating domains (TADs) is relatively well studied, the spatial organization of regulatory elements at finer scales is poorly understood in plants. Here, using high-resolution chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) approach, we mapped histone modification-marked DNA elements-associated and RNA polymerase II (RNAPII)-tethered chromatin interactions involving in transcriptionally active, inactive and Polycomb-repressed states in Arabidopsis. Analysis of the regulatory repertoire showed that both active proximal and distal cis-regulatory elements (CREs) promote the transcription of their nearest and long-range connecting genes; poised CREs act as transcriptional repressors to repress their interacting genes’ expression, indicating that the linear juxtaposition is not the only guiding principle modulating gene transcription. Chromatin connectivity networks revealed that genes implicating in flowering-time control are functionally compartmentalized into separate subnuclear domains, linking active mark- and H3K27me3-associated chromatin conformation to coordinated gene expression. Our study uncovers fine-scale Arabidopsis genome organization and their roles in orchestrating transcription and development.
Project description:To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programs within them. Hi-C, ChIP-Seq and RNA-Seq experiments were conducted in mouse neural stem cells and mouse astrocytes
Project description:To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programs within them. Hi-C, ChIP-Seq and RNA-Seq experiments were conducted in mouse neural stem cells and mouse astrocytes
Project description:To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programs within them. Hi-C, ChIP-Seq and RNA-Seq experiments were conducted in mouse neural stem cells and mouse astrocytes
Project description:Whereas folding of mammalian genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well-understood, how chromatin is folded below this scale remains largely unexplored in mammals. Here, we overcome this limitation using a high-resolution 3C-based method, Micro-C, and probe the links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into various finer-scale structures with distinct regulatory features (i.e. stripes, dots, and domains linking promoter-promoter (P-P) or enhancer-promoter (E-P), and bundle contacts between Polycomb regions). E-P stripes extending from the edge of domains predominantly link co-expressed loci, often independently of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Analysis of ligation events sheds light on both the putative loop extrusion model and the “two-start” zig-zag 30-nanometer model of the chromatin fiber. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.
Project description:Three-dimensional (3D) genome organization is thought to be important for regulation of gene expression. Chromosome conformation capture-based studies have uncovered ensemble organizational principles such as active (A) and inactive (B) compartmentalization. In addition, large inactive regions of the genome associate with the nuclear lamina, the Lamina Associated Domains (LADs). Here we investigate the dynamic relationship between A/B-compartment organization and the 3D organization of LADs. Using refined algorithms to identify active (A) and inactive (B) compartments from Hi-C data and to define LADs from DamID, we confirm that the LADs correspond to the B-compartment. Using specialized chromosome conformation paints, we show that LAD and A/B-compartment organization are dependent upon chromatin state and A-type lamins. By integrating single-cell Hi-C data with live cell imaging and chromosome conformation paints, we demonstrate that self-organization of the B-compartment within a chromosome is an early event post-mitosis and occurs prior to organization of these domains to the nuclear lamina.
Project description:Three-dimensional (3D) genome organization is thought to be important for regulation of gene expression. Chromosome conformation capture-based studies have uncovered ensemble organizational principles such as active (A) and inactive (B) compartmentalization. In addition, large inactive regions of the genome associate with the nuclear lamina, the Lamina Associated Domains (LADs). Here we investigate the dynamic relationship between A/B-compartment organization and the 3D organization of LADs. Using refined algorithms to identify active (A) and inactive (B) compartments from Hi-C data and to define LADs from DamID, we confirm that the LADs correspond to the B-compartment. Using specialized chromosome conformation paints, we show that LAD and A/B-compartment organization are dependent upon chromatin state and A-type lamins. By integrating single-cell Hi-C data with live cell imaging and chromosome conformation paints, we demonstrate that self-organization of the B-compartment within a chromosome is an early event post-mitosis and occurs prior to organization of these domains to the nuclear lamina.