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2 Dual immunofluorescence combined with RNA FISH in differentiated mouse female ES cells. RNA FISH detects Xist RNA coating of the X chromosome undergoing inactivation ( green ), combined with dual IF showing the specific enrichment in histone H3 tri-methylated on lysine 27 (H3K27me3) ( red ) and the exclusion of RNA polymerase II (Pol II; blue) on the inactive X chromosome. DAPI is shown in gray. Bar, 5 μ m. To view this figure in color, see COLOR PLATE 15
Source publication
Epigenetic mechanisms lead to the stable regulation of gene expression without alteration of DNA and trigger initiation and/or maintenance of cell-type-specific transcriptional profiles. Indeed, modulation of chromatin structure and the global 3D organization of the genome and nuclear architecture participate in the precise control of transcription...
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Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1–3, the loss of which has been associated with deficient...
Citations
... RNA FISH was performed using established protocols (Chaumeil et al., 2008;Souyris et al., 2018). Probes were prepared by PCR amplification of human genomic DNA fragments using primer sets, targeting exon 1 of XIST (Mendelian inheritance in men [MIM] *314670), and both exon and intron regions of TLR7 (Xp22.2; ...
Systemic sclerosis (SSc) is an autoimmune disease that has a strong female predominance. Both the X-linked TLR7 and TLR8 can induce type I IFN (IFN-I) by plasmacytoid DCs (pDCs), which can promote fibrosis. We identified five subclusters of pDCs, including ISGhigh clusters that were over-represented in SSc patients. We observed that both TLR7 and TLR8 genes escape from X chromosome inactivation (XCI) at higher frequency in pDCs of SSc patients, which was associated with changes in TLR7 protein profile. Combined DNA/RNA FISH analysis revealed that the TLR7/8 locus is preferentially located outside of the inactive X (Xi) territory when TLR7 is expressed, suggesting that higher-order loop formation is linked to TLR7/8 expression from the Xi. Furthermore, the expression levels of XIST and the transcriptional repressor SPEN were reduced in SSc pDCs. Hence, our data revealed the heterogeneity of pDCs in SSc and suggested that altered XCI at the TLR7/8 locus may contribute to the chronic IFN-I activity of pDCs in female SSc patients.
... DNA 3D-FISH was adapted from previously established methods [96][97][98] . Fosmid clones from the WIBR-1 library were purchased from the BAC-PAC Resources Center (for coordinates and names, see Supplementary Table 4) and isolated using the Large-Construct Kit (Qiagen). ...
Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer–promoter and enhancer–enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer–promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer–promoter physical proximity for developmental gene activation in mammals.
... RNA-FISH on NPCs and preimplantation embryos was performed as previously described 72,73 . NPCs were dissociated using Accutase (Invitrogen), and attached to Poly-L-Lysine (Sigma) coated coverslips for 10 min. ...
In placental females, one copy of the two X chromosomes is largely silenced during a narrow developmental time window, in a process mediated by the non-coding RNA Xist ¹ . Here, we demonstrate that Xist can initiate X-chromosome inactivation (XCI) well beyond early embryogenesis. By modifying its endogenous level, we show that Xist has the capacity to actively silence genes that escape XCI both in neuronal progenitor cells (NPCs) and in vivo , in mouse embryos. We also show that Xist plays a direct role in eliminating TAD-like structures associated with clusters of escapee genes on the inactive X chromosome, and that this is dependent on Xist’s XCI initiation partner, SPEN ² . We further demonstrate that Xist’s function in suppressing gene expression of escapees and topological domain formation is reversible for up to seven days post-induction, but that sustained Xist up-regulation leads to progressively irreversible silencing and CpG island DNA methylation of facultative escapees. Thus, the distinctive transcriptional and regulatory topologies of the silenced X chromosome is actively, directly - and reversibly - controlled by Xist RNA throughout life.
... Coverslips were then rinsed 3 times in 70% EtOH and stored at -20C. RNA FISH was performed essentially as previously described (Chaumeil et al. 2008). Briefly, coverslips, stored in 70% EtOH, were dehydrated in 80%, 95% and 100% EtOH (5 min each) and then allowed to air-dry. ...
Long interspersed element-1 (LINE-1 or L1) retrotransposons constitute the largest transposable element (TE) family in mammalian genomes and contribute prominently to inter- and intra-individual genetic variation. Although most L1 elements are inactive, some evolutionary younger elements remain intact and genetically competent for transcription and occasionally retrotransposition. Despite being generally more abundant in gene-poor regions, intact or full-length L1s (FL-L1) are also enriched around specific classes of genes and on the eutherian X chromosome. How proximal FL-L1 may affect nearby gene expression remains unclear. In this study, we aim to examine this in a systematic manner using engineered mouse embryonic stem cells (ESCs) where the expression of one representative active L1 subfamily is specifically perturbed. We found that ∼1,024 genes are misregulated following FL-L1 activation and to a lesser extent (∼81 genes), following their repression. In most cases (68%), misexpressed genes contain an intronic FL-L1 or lie near a FL-L1 (<260 kb). Gene ontology analysis shows that upon L1 activation, up-regulated genes are enriched for neuronal function-related terms, suggesting that some L1 elements may have evolved to control neuronal gene networks. These results illustrate the cis -regulatory impact of FL-L1 elements and suggest a broader role for L1s than originally anticipated.
... RNA-FISH was performed using a bacterial artificial chromosome (BAC) probe against human XACT (BAC RP11-35D3) and a plasmid (vi.34) probe containing the human XIST cDNA sequence (73) as described previously with minor modifications (74). The XACT and XIST probes were labeled by nick translation (Abbot) using deoxyuridine triphosphate (dUTP)-Atto550 (Jena Bioscience) and Cy5-dUTP (Cytiva). ...
Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.
... RNA FISH was performed as described in our earlier reports [48,59]. Briefly, spreads of monocytes or T lymphocytes on poly-L-lysine-coated coverslips were fixed for 10 min with 3% paraformaldehyde at room temperature, and permeabilized for 7 min in ice-cold cytoskeletal buffer containing 0.5% Triton X-100 and 2 mM vanadylribonucleoside complex (New England Biolabs). ...
Background
Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are, respectively, encoded by adjacent X-linked genes. We previously established that TLR7 evades X chromosome inactivation (XCI) in female immune cells. Whether TLR8 also evades XCI, however, has not yet been explored.
Method
In the current study, we used RNA fluorescence in situ hybridization (RNA FISH) to directly visualize, on a single-cell basis, primary transcripts of TLR7 and TLR8 relative to X chromosome territories in CD14 ⁺ monocytes and CD4 ⁺ T lymphocytes from women, Klinefelter syndrome (KS) men, and euploid men. To assign X chromosome territories in cells lacking robust expression of a XIST compartment, we designed probes specific for X-linked genes that do not escape XCI and therefore robustly label the active X chromosome. We also assessed whether XCI escape of TLR8 was associated with sexual dimorphism in TLR8 protein expression by western blot and flow cytometry.
Results
Using RNA FISH, we show that TLR8 , like TLR7 , evades XCI in immune cells, and that cells harboring simultaneously TLR7 and TLR8 transcript foci are more frequent in women and KS men than in euploid men, resulting in a sevenfold difference in frequency. This transcriptional bias was again observable when comparing the single X of XY males with the active X of cells from females or KS males. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in male cells.
Conclusions
TLR8 , mirroring TLR7 , escapes XCI in human monocytes and CD4 ⁺ T cells. Co-dependent transcription from the active X chromosome and escape from XCI could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases.
... Imaging the plant nucleus is further encumbered by the presence of the cell wall, which blocks the access of protein antibodies and/or labelled probes within the cell. The major challenge in combining immunodetection with FISH lies in the apparently contradictory requirement to preserve the antigen epitope(s) detected by the antibody as well as the 3D structure of the nucleus but also to allow the penetration of the DNA probe to detect gene loci or chromosomal sub-regions [13]. ...
... Two of these combinations, immunoFISH and immunoFISH-GISH, have been presented here and applied to somatic nuclei. Combining immunolabelling with 3D FISH and/or GISH creates a unique tool to assess the specific interactions and associations between proteins and nucleic acid sequences within the nucleus and this at the single cell level [13]. Thus, immunoFISH-GISH combined with the nuclei preparation procedure will facilitate more refined studies of chromatin dynamics, packaging, and stability throughout the cell cycle. ...
Background
Though multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity.
The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).
Results
The mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.
Conclusion
A simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.
... Imaging the plant nucleus is further encumbered by the presence of the cell wall, which blocks the access of protein antibodies and/or labelled probes within the cell. The major challenge in combining immunodetection with FISH lies in the apparently contradictory requirement to preserve the antigen epitope(s) detected by the antibody as well as the 3D structure of the nucleus but also to allow the penetration of the DNA probe to detect gene loci or chromosomal sub-regions [13]. ...
... Two of these combinations, immunoFISH and immunoFISH-GISH, have been presented here and applied to somatic nuclei. Combining immunolabelling with 3D FISH and/or GISH creates a unique tool to assess the speci c interactions and associations between proteins and nucleic acid sequences within the nucleus and this at the single cell level [13]. Thus, immunoFISH-GISH combined with the nuclei preparation procedure will facilitate more re ned studies of chromatin dynamics, packaging, and stability throughout the cell cycle. ...
Background Though multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situdetection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity.
The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).
Results The mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.
Conclusion A simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.
... Our protocol provides reliable results of high-quality bright signal, low background staining and can be applied to several T cell subtypes. The protocol presented here describes the methodology for combinations of immunofluorescence with RNA and/or DNA FISH in mouse embryonic stem cells (mESC) (54)(55)(56)(57), whole thymocytes, naïve and differentiating T and B lymphocytes (25)(26)(27)(58)(59)(60)(61), activated T and B cells (60, 62) and leukemic cell lines (63). In our hands, the following protocol is also successfully working in both murine and human primary and immortalized cell lines such as HEK293T, RAW264.7 and mouse embryonic fibroblasts (MEFs). ...
... In our hands, the following protocol is also successfully working in both murine and human primary and immortalized cell lines such as HEK293T, RAW264.7 and mouse embryonic fibroblasts (MEFs). Although we have already published the protocols for the methodologies regarding 3D immuno-RNA FISH, 3D immuno-DNA FISH and 3D RNA/DNA FISH (55,57,58), the current protocol represents a step forward with the combination of the three stainings together (ie. 3D immuno-RNA/DNA FISH) including also chromosome painting. ...
Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture (“3C”-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.
... Imaging the recruitment of DSB repair factors to the inactive X chromosome. (A) RNA-FISH staining for XIST RNA on the inactive X chromosome in RPE-1 cells using the method described inChaumeil et al. (2008). The XIST cloud colocalizes with the most intense DAPI region in the nucleus. ...
The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vital role in the maintenance of genomic integrity and cellular homeostasis. It is thus of critical importance to decipher whether and how heterochromatin affects the DNA damage response (DDR) to understand how this chromatin state is preserved after DNA damage. Here, we present two laser micro-irradiation-based methods for imaging the DDR in heterochromatin domains in mammalian cells. These methods allow DNA damage targeting to specific subnuclear compartments, direct visualization of the DDR and image-based quantification of the repair response. We apply them to study DNA double-strand break repair pathways in facultative heterochromatin and the repair of UV photoproducts in constitutive heterochromatin. We discuss the advantages and limitations of these methods compared to other targeted approaches for DNA damage induction.
