Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

Differences in nanoscale organization of DNase I hypersensitive and insensitive chromatin in single human cells

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. By using a combination of high throughput super-resolution microscopy and coarse-grained modelling we investigated properties of active and inactive chromatin in interphase nuclei. Using DNase I hypersensitivity as a criterion, we have selected prototypic active and inactive regions from ENCODE data that are representative for K-562 and more than 150 other cell types. By using oligoFISH and automated STED microscopy we systematically measured physical distances of the endpoints of 5kb DNA segments in these regions. These measurements result in high-resolution distance distributions which are right-tailed and range from very compact to almost elongated configurations of more than 200 nm length for both the active and inactive regions. Coarse-grained modeling of the respective DNA segments suggests that in regions with high DNase I hypersensitivity cell-to-cell differences in nucleosome occupancy determine the histogram shape. Simulations of the inactive region cannot sufficiently describe the compaction measured by microscopy, although internucleosomal interactions were elevated and the linker histone H1 was included in the model. These findings hint at further organizational mechanisms while the microscopy-based distance distribution indicates high cell-to-cell differences also in inactive chromatin regions. The analysis of the distance distributions suggests that direct enhancer-promoter contacts, which most models of enhancer action assume, happen for proximal regulatory elements in a probabilistic manner due to chromatin flexibility.

Transcription organizes euchromatin via microphase separation

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

Exploring chromosomal structural heterogeneity across multiple cell lines

Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make two-state transitions between closed conformations and open dumbbell conformations. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.

Differentiating Drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. Our results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.

Studying RNA–DNA interactome by Red-C identifies noncoding RNAs associated with repressed chromatin compartment and reveals transcription dynamics

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. We found two microRNAs—MIR3648 and MIR3687 transcribed from the rRNA locus—that are associated with inactive chromatin genome wide. These miRNAs favor bulk heterochromatin over Polycomb-repressed chromatin and interact preferentially with late-replicating genomic regions. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading into euchromatic islands

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically downregulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some TE loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine (SAM) synthase MAT3, which is required for H3K9me2 deposition. Topoisomerase VI promotes MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a mechanistic insight into the essential role of Topoisomerase VI in the delimitation of chromatin domains.

A chromosome folding intermediate at the condensin-to-cohesin transition during telophase

SummaryChromosome folding is extensively modulated as cells progress through the cell cycle. During mitosis, condensin complexes fold chromosomes in helically arranged nested loop arrays. In interphase, the cohesin complex generates loops that can be stalled at CTCF sites leading to positioned loops and topologically associating domains (TADs), while a separate process of compartmentalization drives the spatial segregation of active and inactive chromatin domains. We used synchronized cell cultures to determine how the mitotic chromosome conformation is transformed into the interphase state. Using Hi-C, chromatin binding assays, and immunofluorescence we show that by telophase condensin-mediated loops are lost and a transient folding intermediate devoid of most loops forms. By late telophase, cohesin-mediated CTCF-CTCF loops and positions of TADs start to emerge rapidly. Compartment boundaries are also established in telophase, but long-range compartmentalization is a slow process and proceeds for several hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin and cohesin driven chromosome folding.