Evolutionary diversity and developmental regulation of X-chromosome inactivation

X-chromosome inactivation (XCI) results in the transcriptional silencing of one X-chromosome in females to attain gene dosage parity between XX female and XY male mammals. Mammals appear to have developed rather diverse strategies to initiate XCI in early development. In placental mammals XCI depends on the regulatory noncoding RNA X-inactive specific transcript (Xist), which is absent in marsupials and monotremes. Surprisingly, even placental mammals show differences in the initiation of XCI in terms of Xist regulation and the timing to acquire dosage compensation. Despite this, all placental mammals achieve chromosome-wide gene silencing at some point in development, and this is maintained by epigenetic marks such as chromatin modifications and DNA methylation. In this review, we will summarise recent findings concerning the events that occur downstream of Xist RNA coating of the inactive X-chromosome (Xi) to ensure its heterochromatinization and the maintenance of the inactive state in the mouse and highlight similarities and differences between mammals

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

New image colocalization coefficient for fluorescence microscopy to quantify (bio-)molecular interactions.

The spatial relationship, or degree of colocalization, between two or more types of molecules in live cells is commonly detected using fluorescence microscopy. This spatial distribution can be used to estimate the interaction between fluorescently labelled molecules. These interactions are usually quantified by analysing the correlation and/or the overlap between images, using the Pearson's and Manders' coefficients, respectively. However, the correlation and overlap coefficients are parameters not designed to quantify molecular interactions. Here we propose a new colocalization coefficient specifically designed to quantify the interactions between molecules. In well-defined thermodynamic ensembles, this coefficient can in principle be used to calculate relevant statistical thermodynamic quantities such as binding free energies

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase

Epigenetic modifications in sex heterochromatin of vole rodents.

The genome of some vole rodents contains large blocks of heterochromatin coupled to the sex chromosomes. While the DNA content of these heterochromatic blocks has been extensively analyzed, little is known about the epigenetic modifications controlling their structure and dynamics. To better understand its organization and functions within the nucleus, we have compared the distribution pattern of several epigenetic marks in cells from two species, Microtus agrestis and Microtus cabrerae. We first could show that the heterochromatic blocks are identifiable within the nuclei due to their AT enrichment detectable by DAPI staining. By immunostaining analyses, we demonstrated that enrichment in H3K9me3 and HP1, depletion of DNA methylation as well as H4K8ac and H3K4me2, are major conserved epigenetic features of this heterochromatin in both sex chromosomes. Furthermore, we provide evidence of transcriptional activity for some repeated DNAs in cultivated cells. These transcripts are partially polyadenylated and their levels are not altered during mitotic arrest. In summary, we show here that enrichment in H3K9me3 and HP1, DNA demethylation, and transcriptional activity are major epigenetic features of sex heterochromatin in vole rodents

MeCP2-induced chromatin unfolding is independent of cell-cycle stage.

<p>(A) The images show 2-6-3 cells (U2OS derived clone containing a 200 copy chromosomal array of 256 lacO repeats and a reporter gene harboring 24 repeats of the MS2 bacteriophage) that were co-transfected with mCherry-tagged (red signal) or EGFP-tagged (green signal) lacR-MeCP2 and EGFP-tagged (green signal) or mRFP-tagged (red signal) PCNA. PCNA localizes at replication foci during S phase. The images represent individual optical sections of fixed cells. Bar  = 5 μm. (B) The histogram shows quantification of the number of lacR-MeCP2 transfected cells showing a condensed (grey bar) or decondensed (white bar) chromosomal array in either S or non-S phase based on PCNA expression or immunolabeling (on X-axis noted as immunolabeling, n = 74 and cotransfection, n = 68). A χ² test was performed on PCNA immunolabeled (p = 0.25) and cotransfected cells (p = 0.81). Random variation probabilities show a high random variation between the decondensation and the cell cycle phase.</p

The presence of MeCP2 associated factors at the amplified chromosomal array upon MeCP2 targeting.

<p>Various factors were assayed at light microscopical level for their presence at the amplified chromosomal array in the AO3_1 clone (CHO derived clone containing an amplified chromosomal region consisting of the DHFR cDNA transgene and 256 lac operator repeats). Immunolabeling or co-transfection were performed upon expressing EGFP-lacR (control) or EGFP-lacR-MeCP2. Localization at the array is scored as (+) present, (+/−) infrequently present or (−) absent.</p

Interference with the binding of HP1γ and separate HP1γ domains.

<p>2-6-3 clone (U2OS-derived clone containing a 200 copy chromosomal array of 256 lacO repeats and a reporter gene harboring 24 repeats of the MS2 bacteriophage) were co-transfected with YFP-tagged HP1γ, HP1γ CD (1–75) or HP1γ CSD (92–173) (green signal) and mCherry-tagged lacR, lacR-MeCP2 or lacR-VP16 (red signal). (A–C) Pictures show 3D images that were recorded of living cells (A–C). The images represent individual optical sections and nuclei have the same scale. Bar  = 5 μm. (D) The graphs show FLIP (Fluorescent loss after photobleaching) curves of YFP-HP1γ in the presence of mCherry-lacR (control, blue line), mCherry-lacR-MeCP2 (green line) or mCherry-lacR-VP16 (red line). (E) The graphs show FLIP curves of YFP-HP1γ CSD in the presence of mCherry-lacR (control, blue line) or mCherry-lacR-MeCP2 (red line).</p

Statistical evaluation of the chromatin structural analysis.

<p>Statistical evaluation of the differences in chromatin structure after targeting EGFP-lacR tagged constructs. Rows 1 through 7 show the comparison between control cells transfected with EGFP-lacR and cells transfected with EGFP-lacR-tagged full-length MeCP2, EGFP-lacR-tagged VP16 and EGFP-lacR-tagged separate MeCP2 domains (i.e. C-terminus, MBD, TRD, ΔC-terminus, R133C Rett syndrome mutation). Since the data are not normally distributed and do not have a shared variance, we used Wilcoxon nonparametric statistical testing corrected for multiple testing (Bonferoni) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069347#pone.0069347-Sokal1" target="_blank">[48]</a>. The p-values are shown, indicating the probabilities that two populations are different choosing a cut-off value of p = 0.007. Based on this analysis the EGFP-lacR tagged VP16, MeCP2 (full length) and the R133C population are significantly different from EGFP-lacR control, whereas EGFP-lacR tagged MBD, TRD, C-terminus and ΔC-terminus, are not significantly different from EGFP-lacR.</p