Intrinsically disordered sequences in the Polycomb protein Polyhomeotic regulate condensate formation

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains a polymerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph), forms phase separated condensates with DNA or chromatin in vitro, suggesting PcG bodies may form by phase separation. In cells, Ph forms multiple condensates, while mini-Ph forms a single large nuclear condensate. We therefore hypothesize that sequences outside of mini-Ph are required for proper condensate formation. We identified three distinct Intrinsically Disordered Regions (IDRs) in Ph based on sequence composition and complexity. We tested the role of each IDR in Ph condensates using live imaging of transfected Drosophila S2 cells. We find that each IDR uniquely affects Ph SAM-dependent condensate size, number, and morphology.

Intrinsically disordered sequences in the Polycomb protein Polyhomeotic regulate condensate formation

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains a polymerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph), forms phase separated condensates with DNA or chromatin in vitro, suggesting PcG bodies may form by phase separation. In cells, Ph forms multiple condensates, while mini-Ph forms a single large nuclear condensate. We therefore hypothesize that sequences outside of mini-Ph are required for proper condensate formation. We identified three distinct Intrinsically Disordered Regions (IDRs) in Ph based on sequence composition and complexity. We tested the role of each IDR in Ph condensates using live imaging of transfected Drosophila S2 cells. We find that each IDR uniquely affects Ph SAM-dependent condensate size, number, and morphology.

SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development. Both proteins localize to nuclear bodies and are modified by the small ubiquitin-like modifier (SUMO). Our results show that CBX4 and SALL1 interact in the nucleoplasm and that increased SALL1 expression reduces ubiquitination of CBX4, enhancing its stability. This is accompanied by an increase in the number and size of CBX4-containing Polycomb bodies, and by a greater repression of CBX4 target genes. Thus, our findings uncover a new way of SALL1-mediated regulation of Polycomb bodies through modulation of CBX4 stability, with consequences in the regulation of its target genes, which could have an impact in cell differentiation and development.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana

The 3D genome is characterized by a complex organization made of genomic and epigenomic layers with profound implications on gene regulation and cell function. However, the understanding of the fundamental mechanisms driving the crosstalk between nuclear architecture and (epi)genomic information is still lacking. The plant Arabidopsis thaliana is a powerful model organism to address these questions owing to its compact genome for which we have a rich collection of microscopy, chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymer modelling, we investigate the roles of nucleolus formation and epigenomics-driven interactions in shaping the 3D genome of A. thaliana. By validation of several predictions with published data, we demonstrate that self-attracting nucleolar organizing regions and repulsive constitutive heterochromatin are major mechanisms to regulate the organization of chromosomes. Simulations also suggest that interphase chromosomes maintain a partial structural memory of the V-shapes, typical of (sub)metacentric chromosomes in anaphase. Additionally, self-attraction between facultative heterochromatin regions facilitates the formation of Polycomb bodies hosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin are highly-conserved in eukaryotic cells, our findings pave the way for a comprehensive characterization of the generic principles that are likely to shape and regulate the 3D genome in many species.

A nervous system-specific subnuclear organelle in Caenorhabditis elegans

We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4–10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana

ABSTRACTThe 3D genome is characterized by a complex organization made of genomic and epigenomic layers with profound implications on gene regulation and cell function. However, the understanding of the fundamental mechanisms driving the crosstalk between nuclear architecture and (epi)genomic information is still lacking. The plant Arabidopsis thaliana is a powerful model organism to address these questions owing to its compact genome for which we have a rich collection of microscopy, Chromosome Conformation Capture (Hi-C), and ChIP-seq experiments. Using polymer modelling, we investigate the roles of nucleolus formation and epigenomics-driven interactions in shaping the 3D genome of A. thaliana. By validation of several predictions with published data, we demonstrate that self-attracting nucleolar organizing regions and repulsive constitutive heterochromatin are major mechanisms to regulate the organization of chromosomes. Simulations also suggest that interphase chromosomes maintain a partial structural memory of the V-shapes, typical of (sub)metacentric chromosomes in anaphase. Additionally, self-attraction between facultative heterochromatin regions facilitates the formation of Polycomb bodies hosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin are highly-conserved in eukaryotic cells, our findings pave the way for a comprehensive characterization of the generic principles that are likely to shape and regulate the 3D genome in many species.

Interaction between Polycomb and SSX Proteins in Pericentromeric Heterochromatin Function and Its Implication in Cancer

The stability of pericentromeric heterochromatin is maintained by repressive epigenetic control mechanisms, and failure to maintain this stability may cause severe diseases such as immune deficiency and cancer. Thus, deeper insight into the epigenetic regulation and deregulation of pericentromeric heterochromatin is of high priority. We and others have recently demonstrated that pericentromeric heterochromatin domains are often epigenetically reprogrammed by Polycomb proteins in premalignant and malignant cells to form large subnuclear structures known as Polycomb bodies. This may affect the regulation and stability of pericentromeric heterochromatin domains and/or the distribution of Polycomb factors to support tumorigeneses. Importantly, Polycomb bodies in cancer cells may be targeted by the cancer/testis-related SSX proteins to cause derepression and genomic instability of pericentromeric heterochromatin. This review will discuss the interplay between SSX and Polycomb factors in the repression and stability of pericentromeric heterochromatin and its possible implications for tumor biology.

Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins

Rearrangement of the 1q12 pericentromeric heterochromatin and subsequent amplification of the 1q arm is commonly associated with cancer development and progression and may result from epigenetic deregulation. In many premalignant and malignant cells, loss of 1q12 satellite DNA methylation causes the deposition of polycomb factors and formation of large polycomb aggregates referred to as polycomb bodies. Here, we show that SSX proteins can destabilize 1q12 pericentromeric heterochromatin in melanoma cells when it is present in the context of polycomb bodies. We found that SSX proteins deplete polycomb bodies and promote the unfolding and derepression of 1q12 heterochromatin during replication. This further leads to segregation abnormalities during anaphase and generation of micronuclei. The structural rearrangement of 1q12 pericentromeric heterochromatin triggered by SSX2 is associated with loss of polycomb factors, but is not mediated by diminished polycomb repression. Instead, our studies suggest a direct effect of SSX proteins facilitated though a DNA/chromatin binding, zinc finger-like domain and a KRAB-like domain that may recruit chromatin modifiers or activate satellite transcription. Our results demonstrate a novel mechanism for generation of 1q12-associated genomic instability in cancer cells.

Phase separation and nucleosome compaction are governed by the same domain of Polycomb Repressive Complex 1

SummaryMammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb Repressive Complex 1 (PRC1) family complexes are central to maintaining this repression1. These include a set of canonical PRC1 complexes that each contain four core proteins, including one from the CBX family. These complexes have previously been shown to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus2,3. We show here that reconstituted PRC1 readily phase separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of CBX2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells, and have previously been shown to eliminate nucleosome compaction in vitro4 and to generate axial patterning defects in mice5. Thus, a single domain in CBX2 is required for phase separation and nucleosome compaction, a finding that relates these functions to each other and to proper development.