FRET-FISH probes chromatin compaction at individual genomic loci in single cells

The density or compaction of chromatin throughout the cell nucleus is a key biophysical property that influences DNA replication, transcription, and repair. Chromatin accessibility is often used as a proxy for chromatin compaction or density, however it is not clear how these two properties relate to each other, given the lack of tools for directly probing compaction at defined genomic loci. To fill in this gap, here we developed FRET-FISH, a microscopy-based method combining fluorescence resonance energy transference (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at selected loci in single cells. We optimized FRET-FISH by testing different probe designs in situ in fixed cells, readily detecting FRET generated by DNA FISH probes. To validate FRET-FISH, we compared it with ATAC-seq and Hi-C, demonstrating that local chromatin compaction and accessibility are strongly correlated and that the frequency of intra-genic contacts measured by Hi-C may be an even better proxy for local chromatin density. To further validate FRET-FISH, we showed that it can detect expected differences in chromatin compaction along the nuclear radius, with peripheral loci being more compacted and central ones less compacted. Lastly, we assessed the sensitivity of FRET-FISH, demonstrating its ability to reproducibly detect differences in chromatin density (i) upon treatment of cells with drugs that perturb global chromatin condensation; (ii) during prolonged cell culture; and (iii) in different phases of the cell cycle. We conclude that FRET-FISH is a robust tool for probing chromatin compaction at selected loci in single cells and for studying inter-allelic and cell-to-cell variability in chromatin density.

2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.

Polycomb condensates can promote epigenetic marks but are not required for sustained chromatin compaction

Organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Phase-separation has been implicated in heterochromatin formation, but it is unclear how phase-separated condensates can contribute to stable repression, particularly for heritable epigenetic changes. Polycomb complex PRC1 is key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we show that PRC1 forms multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. We show that inducing Polycomb phase-separation can cause chromatin compaction, but polycomb condensates are dispensable for maintenance of the compacted state. Our data and simulations are consistent with a model in which the time integral of Polycomb phase-separation is progressively recorded in repressive histone marks, which subsequently drive compaction. These findings link the equilibrium thermodynamics of phase-separation with the fundamentally non-equilibrium concept of epigenetic memory.

Balance of osmotic pressures determines the volume of the cell nucleus

The volume of the cell nucleus varies across cell-types and species, and is commonly thought to be determined by the size of the genome and degree of chromatin compaction. However, this notion has been challenged over the years by multiple experimental evidence. Here, we consider the physical condition of mechanical force balance as a determining condition of the nuclear volume and use quantitative, order-of-magnitude analysis to estimate the forces from different sources of nuclear and cellular pressure. Our estimates suggest that the dominant pressure within the nucleus and cytoplasm originates from the osmotic pressure of proteins and RNA molecules that are localized to the nucleus or cytoplasm by out-of-equilibrium, active nucleocytoplasmic transport rather than from chromatin or its associated ions. This motivates us to formulate a physical model for the ratio of the cell and nuclear volumes in which osmotic pressures of localized proteins determine the relative volumes. In accordance with unexplained observations that are century-old, our model predicts that the ratio of the cell and nuclear volumes is a constant, robust to a wide variety of biochemical and biophysical manipulations, and is changed only if gene expression or nucleocytoplasmic transport are modulated.

Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

Hydrozoan sperm-specific H2B histone variants stabilize chromatin and block transcription without enhancing chromatin condensation

Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.

Temporally dynamic antagonism between transcription and chromatin compaction controls stochastic photoreceptor specification

Stochastic mechanisms diversify cell fates during development. How cells randomly choose between two or more fates remains poorly understood. In the Drosophila eye, the random mosaic of two R7 photoreceptor subtypes is determined by expression of the transcription factor Spineless (Ss). Here, we investigated how cis-regulatory elements and trans factors regulate nascent transcriptional activity and chromatin compaction at the ss gene locus during R7 development. We find that the ss locus is in a compact state in undifferentiated cells. An early enhancer drives ss transcription in all R7 precursors to open the ss locus. In differentiating cells, transcription ceases and the ss locus stochastically remains open or compacts. In SsON R7s, ss is open and competent for activation by a late enhancer, whereas in SsOFF R7s, ss is compact and repression prevents expression. Our results suggest that a temporally dynamic antagonism, in which transcription drives decompaction and then compaction represses transcription, controls stochastic cell fate specification.

Parental Conflicting Role Mediates Regulation of The Chromatin Structure in The Mouse Zygote

Parental pronuclei (PN) are asymmetrical in several points but the underlying mechanism for this is still unclear. Recently, a theory has been become broadly accepted that sperm are more than mere vehicles to carry the paternal haploid genome into oocytes. Here, in order to reveal the formation mechanisms for parental asymmetrically relaxed chromatin structure in zygotes, we investigated histone mobility in parthenogenetic-, androgenic-, ROSI-, ELSI-, tICSI-, and ICSI-zygotes with several numbers of PNs with the use of zygotic fluorescence recovery after photobleaching, a method previous established by our group. The results showed that sperm played a role to cause chromatin compaction in both parental PNs. Interestingly, during spermiogenesis, male germ cells acquired this ability and its resistance. On the other hand, oocytes harbored chromatin relaxation ability. Furthermore, the chromatin relaxation factor was competed for between PNs. Thus, these results indicated that the parental asymmetrically relaxed chromatin structure was established as a result of a competition between the PNs for the chromatin relaxation factor that opposed the chromatin compaction effect by sperm. Together, it was suggested that parental germ cells cooperated for their just arisen newborn zygotes by playing a distinct role in the regulation of chromatin structure.