Forced expression of MSR repeat transcripts above a threshold limit breaks heterochromatin organisation.

Mouse heterochromatin is characterised by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR RNA contributes to the integrity of heterochromatin. We establish an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can modulate MSR transcription through the targeting of a dCas9-Repressor or a dCas9-Activator. With this system, we can define a threshold limit of >300-fold deregulation of MSR transcript levels, above which the structural organisation of heterochromatin becomes disrupted.

Epigenetic dosage identifies two major and functionally distinct β cell subtypes.

The mechanisms that specify and stabilize cell subtypes remain poorly understood. Here, we identify two major subtypes of pancreatic β cells based on histone mark heterogeneity (β and β). β cells exhibit ∼4-fold higher levels of H3K27me3, distinct chromatin organization and compaction, and a specific transcriptional pattern.

m6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin.

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated.

Laser Targeted Oligo Ligation (LTOL) to Identify DNA Sequences in the Vicinity of a Single Subnuclear Structure in a Single Cell.

Gene loci are organized around nuclear substructures, forming gene hubs which provide a level of transcriptional control. To date, most techniques used to investigate the genes in these hubs have been based on using material from bulk cells. This makes identifying specific gene associations difficult.

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes.

A novel single cell method to identify the genetic composition at a single nuclear body.

Gene loci make specific associations with compartments of the nucleus (e.g. the nuclear envelope, nucleolus, and transcription factories) and this association may determine or reflect a mechanism of genetic control.

Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP.

Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations.

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.

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.

PML nuclear bodies as sites of epigenetic regulation.

The protein-based core of a promyelocytic leukemia nuclear body (PML NB) accumulates numerous factors involved in many nuclear processes, including transcription and DNA repair. We suggest that these proteins could act on chromatin in the vicinity of the bodies. The physical dependence of PML NB structure on the integrity of the surrounding DNA implies a functional connection between the bodies and chromatin.

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.

The number of PML nuclear bodies increases in early S phase by a fission mechanism.

Promyelocytic leukemia (PML) nuclear bodies have been implicated in a variety of cellular processes including apoptosis, tumour suppression, anti-viral response, DNA repair and transcriptional regulation. PML nuclear bodies are both positionally and structurally stable over extended periods of interphase. As demonstrated in this study, the structural stability is lost as cells enter S phase, evidenced both by distortions in shape and by fission and fusion events.

Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1.

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100.

PML bodies: a meeting place for genomic loci?

Promyelocytic leukemia (PML) bodies have been implicated in a variety of cellular processes, such as cell-cycle regulation, apoptosis, proteolysis, tumor suppression, DNA repair and transcription. Despite this, the function of PML bodies is still unknown. Direct and indirect evidence supports the hypothesis that PML bodies interact with specific genes or genomic loci.

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation.

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation.