FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2021894549> ?p ?o ?g. }

• W2021894549 endingPage "458" @default.
• W2021894549 startingPage "447" @default.
• W2021894549 abstract "Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation. Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation. Live-cell imaging reveals increased dynamics of genomic loci during differentiation X chromosome pairing initiates in this context and lasts for about 45 min Tsix shows transient monoallelic expression immediately after pairing This may provide a window of opportunity for monoallelic upregulation of Xist in cis X chromosome inactivation (XCI) ensures equal levels of X-linked gene products in females (XX) and males (XY) (Lyon, 1961Lyon M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2457) Google Scholar). XCI is initiated during early development via upregulation of the noncoding Xist transcript, which coats one X chromosome in cis and triggers its silencing. Once established, XCI is then maintained through propagation of epigenetic marks during cell divisions. A remarkable feature of XCI is that two identical chromosomes become differentially expressed in the same nucleoplasm. Germline imprinting provides one way of achieving asymmetric expression (see Okamoto and Heard, 2009Okamoto I. Heard E. Lessons from comparative analysis of X-chromosome inactivation in mammals.Chromosome Res. 2009; 17: 659-669Crossref PubMed Scopus (47) Google Scholar for review). However, in most eutherians, and in postimplantation mouse embryos, XCI is random, with either the paternal or maternal X being silenced (Lyon, 1961Lyon M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2457) Google Scholar). Random monoallelic gene expression has also been reported to occur at some autosomal loci, with potentially important implications for development and disease (Gimelbrant et al., 2007Gimelbrant A. Hutchinson J.N. Thompson B.R. Chess A. Widespread monoallelic expression on human autosomes.Science. 2007; 318: 1136-1140Crossref PubMed Scopus (421) Google Scholar). In the case of random XCI, the X-inactivation center (Xic), which includes the Xist gene and its antisense transcript Tsix, controls the initiation of this process (see Navarro and Avner, 2010Navarro P. Avner P. An embryonic story: analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells.Bioessays. 2010; 32: 581-588Crossref PubMed Scopus (26) Google Scholar for review). XCI takes place during the earliest stages of embryonic stem (ES) cell differentiation, at a time when other important developmental decisions are also being made. In undifferentiated female ES cells, Xist and Tsix are expressed at low levels, but upon differentiation, Xist becomes upregulated and Tsix downregulated on one of the two X chromosomes (Lee et al., 1999Lee J.T. Davidow L.S. Warshawsky D. Tsix, a gene antisense to Xist at the X-inactivation centre.Nat. Genet. 1999; 21: 400-404Crossref PubMed Scopus (619) Google Scholar, Debrand et al., 1999Debrand E. Chureau C. Arnaud D. Avner P. Heard E. Functional analysis of the DXPas34 locus, a 3′ regulator of Xist expression.Mol. Cell. Biol. 1999; 19: 8513-8525PubMed Google Scholar). Consistent with this inverse expression pattern, Tsix and its enhancer Xite (Lee et al., 1999Lee J.T. Davidow L.S. Warshawsky D. Tsix, a gene antisense to Xist at the X-inactivation centre.Nat. Genet. 1999; 21: 400-404Crossref PubMed Scopus (619) Google Scholar, Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar, Ogawa and Lee, 2003Ogawa Y. Lee J.T. Xite X-inactivation intergenic transcription elements that regulate the probability of choice.Mol. Cell. 2003; 11: 731-743Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) are known to repress Xist in cis. Understanding how Xist becomes asymmetrically upregulated during early differentiation is thus central to our understanding of how the two X chromosomes become differentially expressed during random XCI. Activation of Xist during ES cell differentiation depends on downregulation of pluripotency factors such as Oct4, Nanog, and Sox2 (Navarro et al., 2008Navarro P. Chambers I. Karwacki-Neisius V. Chureau C. Morey C. Rougeulle C. Avner P. Molecular coupling of Xist regulation and pluripotency.Science. 2008; 321: 1693-1695Crossref PubMed Scopus (251) Google Scholar), as well as the presence of XX-dosage-sensitive competence of sensing factors, such as the X-linked Rnf12 protein (Jonkers et al., 2009Jonkers I. Barakat T.S. Achame E.M. Monkhorst K. Kenter A. Rentmeester E. Grosveld F. Grootegoed J.A. Gribnau J. RNF12 is an X-encoded dose-dependent activator of X chromosome inactivation.Cell. 2009; 139: 999-1011Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), and possibly other loci (Xpr) or noncoding transcripts (Jpx, Ftx) located 5′ to Xist (Augui et al., 2007Augui S. Filion G.J. Huart S. Nora E. Guggiari M. Maresca M. Stewart A.F. Heard E. Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.Science. 2007; 318: 1632-1636Crossref PubMed Scopus (140) Google Scholar, Tian et al., 2010Tian D. Sun S. Lee J.T. The long noncoding RNA, Jpx is a molecular switch for X chromosome inactivation.Cell. 2010; 143: 390-403Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (158) Google Scholar). However, these sensing mechanisms do not readily explain why only one of the two Xist alleles is upregulated, not both. Stochastic Xist expression models might partly explain this (Monkhorst et al., 2008Monkhorst K. Jonkers I. Rentmeester E. Grosveld F. Gribnau J. X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator.Cell. 2008; 132: 410-421Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), but the surprisingly low frequency of biallelic Xist upregulation during the initiation of XCI in mice suggests that some other means of ensuring precise monoallelic regulation exists. Recently it was shown that the two Xic loci undergo transient homologous associations (pairing) during early differentiation, and it was proposed that this might play a role in the monoallelic regulation of Xist and Tsix during initiation of XCI (Bacher et al., 2006Bacher C.P. Guggiari M. Brors B. Augui S. Clerc P. Avner P. Eils R. Heard E. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.Nat. Cell Biol. 2006; 8: 293-299Crossref PubMed Scopus (263) Google Scholar, Xu et al., 2006Xu N. Tsai C.L. Lee J.T. Transient homologous chromosome pairing marks the onset of X inactivation.Science. 2006; 311: 1149-1152Crossref PubMed Scopus (312) Google Scholar, Xu et al., 2007Xu N. Donohoe M.E. Silva S.S. Lee J.T. Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein.Nat. Genet. 2007; 39: 1390-1396Crossref PubMed Scopus (152) Google Scholar, Augui et al., 2007Augui S. Filion G.J. Huart S. Nora E. Guggiari M. Maresca M. Stewart A.F. Heard E. Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.Science. 2007; 318: 1632-1636Crossref PubMed Scopus (140) Google Scholar). Associations between homologous chromosomal loci have been proposed to underlie the establishment of opposite states of transcriptional activity on homologous alleles in other situations, for example, during immunoglobulin recombination in B cell development (Hewitt et al., 2009Hewitt S.L. Yin B. Ji Y. Chaumeil J. Marszalek K. Tenthorey J. Salvagiotto G. Steinel N. Ramsey L.B. Ghysdael J. et al.RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.Nat. Immunol. 2009; 10: 655-664Crossref PubMed Scopus (110) Google Scholar). In the case of X inactivation, pairing via the Xpr locus (Figure 1A ) has been proposed to help bring together and facilitate pairing at the Tsix loci (Augui et al., 2007Augui S. Filion G.J. Huart S. Nora E. Guggiari M. Maresca M. Stewart A.F. Heard E. Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.Science. 2007; 318: 1632-1636Crossref PubMed Scopus (140) Google Scholar), which in turn is proposed to enable coordination of monoallelic Tsix expression and reciprocal Xist expression (Xu et al., 2007Xu N. Donohoe M.E. Silva S.S. Lee J.T. Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein.Nat. Genet. 2007; 39: 1390-1396Crossref PubMed Scopus (152) Google Scholar, Scialdone and Nicodemi, 2008Scialdone A. Nicodemi M. Mechanics and dynamics of X-chromosome pairing at X inactivation.PLoS Comput. Biol. 2008; 4: e1000244Crossref PubMed Scopus (16) Google Scholar). In support of this, deletion of both alleles of Tsix in females results in chaotic XCI, with biallelic or no Xist upregulation in a significant proportion of cells (Lee, 2005Lee J.T. Regulation of X-chromosome counting by Tsix and Xite sequences.Science. 2005; 309: 768-771Crossref PubMed Scopus (114) Google Scholar). However, the coordinating role of Tsix pairing in monoallelic XCI has never been tested experimentally, and the actual relationship between Xic pairing and Xist/Tsix regulation has remained unclear, partly because of the asynchronous nature and heterogeneity of early differentiating ES cells, which renders the precise ordering of events impossible in fixed cells, where only snapshots of dynamic events can be obtained. In this paper, we set out to examine the dynamics of X chromosome pairing and its possible outcome, using the Tet operator/Tet repressor (TetO/TetR) tagging system in living ES cells. Both the Xic loci as well as other autosomal regions were visualized in real time in this way. We find a general increase in the dynamics of loci during early ES cell differentiation. This could have important implications for the multiple developmental decisions being taken during this time. We show that Tsix pairing is a transient event, lasting approximately 45 min, and that during pairing, the Tsix loci show reduced mobility, suggesting some form of tethering to each other. We also demonstrate that the outcome of pairing is often monoallelic Tsix expression, which could in turn lead to monoallelic Xist upregulation. We thus present one of the first live-cell investigations of the dynamics of genomic loci during early XX ES cell differentiation and demonstrate that transient homologous associations can provide an efficient means of generating asymmetric expression states. To visualize the Xic loci and other genomic loci in living ES cells, we exploited the TetO/TetR system (Michaelis et al., 1997Michaelis C. Ciosk R. Nasmyth K. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids.Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar). We first targeted a single TetO array into a site located 65 kb downstream of Xist's 3′ end and 35 kb away from Xite (Figure 1A and Figure S1A available online), in order to avoid any deleterious effects on Xist/Tsix/Xite regulation and initiation of XCI. The homologous recombination construct harboring the 224-repeat TetO array (11.2 kb) and a neomycin resistance gene was targeted into one of the two Xic loci in the PGK12.1 mouse female ES cell line (Penny et al., 1996Penny G.D. Kay G.F. Sheardown S.A. Rastan S. Brockdorff N. Requirement for Xist in X chromosome inactivation.Nature. 1996; 379: 131-137Crossref PubMed Scopus (934) Google Scholar) (Figure 1B and Figure S1A). Two correctly targeted clones out of 310 screened were obtained and one of them was used for further analysis (Figure S1B). Insertion of the TetO into the Xic did not affect the onset of XCI or the choice of X chromosome for XCI. The hemizygous clone (PGKT1) was differentiated in vitro and analyzed for random XCI by Xist RNA FISH accompanied by TetO DNA FISH to distinguish the TetO-tagged allele. Normal kinetics of Xist upregulation and similar frequencies of XCI were observed for both the XicTetO and untagged Xic alleles (Figure 1C and data not shown). This hemizygous PGKT1 clone was then used to generate ES cells in which both Xic loci were tagged for visualization. Initially, targeting of the second Xic locus was attempted using a targeting construct containing a Lac operator (LacO) array. However, this failed despite repeated attempts. Instead, we generated XicTetO homozygous cells by treating the hemizygous PGKT1 cells using increased G418 selection (Figure 1B) (Mortensen et al., 1992Mortensen R.M. Conner D.A. Chao S. Geisterfer-Lowrance A.A. Seidman J.G. Production of homozygous mutant ES cells with a single targeting construct.Mol. Cell. Biol. 1992; 12: 2391-2395Crossref PubMed Scopus (348) Google Scholar). One of the clones obtained (PGKT2) was found to have two X chromosomes, each of which carried a TetO-tagged Xic locus based on Southern blotting and DNA FISH on metaphase spreads (Figures S1B–S1D). A TetR-mCherry fusion protein construct was introduced into the PGKT2 cell line as a stable transgene to enable visualization of the two XicTetO loci (Figure 1B). In this clone (PGKT2-TetR), two TetR-mCherry foci could be readily detected by fluorescence microscopy (Figure S1E and Movie S1). The two TetR-mCherry foci corresponded to the TetO-tagged Xics as they systematically colocalized with punctate Xist/Tsix RNA FISH signals in undifferentiated ES cells (Figure S1E). Upon differentiation of PGKT2-TetR cells, monoallelic Xist RNA accumulation was observed with similar kinetics to the parental line (Figure 1D). We conclude that the presence of the TetO tag within each of the Xic loci and the expression of the TetR-mCherry protein in PGKT2-TetR cells did not interfere with normal XCI induction and kinetics. The PGKT2-TetR cell line was therefore used for subsequent experiments. In the course of this work, we also generated a series of ES cell lines carrying randomly inserted, autosomal TetO arrays, which we were able to use to assess general chromosome mobility, as will be described later. We next established conditions for live-cell imaging of TetO-tagged Xic loci in PGKT2-TetR ES cells (Figure S2A ; see also Experimental Procedures). Following deconvolution of three-dimensional (3D) image stacks, more than 80% of nuclei showed two clear XicTetO spots. In some cases, the TetR-bound locus appeared as a doublet, presumably due to sister chromatid separation (Figure 2A and Movie S1 and Movie S2). In order to determine the time window in which Xic pairing most often occurred, we assessed Xic pairing frequencies at different stages of differentiation, by measuring 3D distances between the centers of mass of the two XicTetO spots in snapshots of living cells (Figures 2A and 2B). Based on our previous criteria defined in fixed cells, pairing in living cells was defined as Xic-Xic distances of ≤2 μm (Augui et al., 2007Augui S. Filion G.J. Huart S. Nora E. Guggiari M. Maresca M. Stewart A.F. Heard E. Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.Science. 2007; 318: 1632-1636Crossref PubMed Scopus (140) Google Scholar). A peak in XicTetO pairing frequencies was found at day 1 of differentiation, as indicated by a marked shift to shorter TetO-TetO distances when compared to undifferentiated cells or late differentiated cells (day 8) (Figures 2B and 2C and Figure S2B). We reproducibly observed a peak of approximately 5% to 10% cells showing XicTetO pairing at around day 1 of differentiation in three independent experiments (Figures 2B and 2C and data not shown). These results agree with previous DNA FISH studies, where Tsix pairing was significantly enriched at day 1 or 2 of differentiation (Bacher et al., 2006Bacher C.P. Guggiari M. Brors B. Augui S. Clerc P. Avner P. Eils R. Heard E. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.Nat. Cell Biol. 2006; 8: 293-299Crossref PubMed Scopus (263) Google Scholar, Augui et al., 2007Augui S. Filion G.J. Huart S. Nora E. Guggiari M. Maresca M. Stewart A.F. Heard E. Sensing X chromosome pairs before X inactivation via a novel X-pairing region of the Xic.Science. 2007; 318: 1632-1636Crossref PubMed Scopus (140) Google Scholar), and show that TetO-tagging of Xic loci does not interfere with normal pairing kinetics.Figure 2Analysis of XicTetO Dynamics by Live-Cell ImagingShow full caption(A) An example of a projected deconvolved image of day 1 differentiated living PGKT2-TetR cells. Indicated values correspond to the 3D distance between two XicTetO spots. Xic pairing cells are marked with white circles.(B) Detailed distribution of 3D distances between two XicTetO spots for each differentiation stage shown in (C). The black solid bars indicate the “Xic pairing” population (d ≤ 2 μm).(C) Summary of distribution of 3D distances between two XicTetO spots for each differentiation stage shown in (B).See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) An example of a projected deconvolved image of day 1 differentiated living PGKT2-TetR cells. Indicated values correspond to the 3D distance between two XicTetO spots. Xic pairing cells are marked with white circles. (B) Detailed distribution of 3D distances between two XicTetO spots for each differentiation stage shown in (C). The black solid bars indicate the “Xic pairing” population (d ≤ 2 μm). (C) Summary of distribution of 3D distances between two XicTetO spots for each differentiation stage shown in (B). See also Figure S2. In order to determine the general context in which Xic pairing events occur, we wished to assess the dynamics of the Xics and of other genomic loci during ES cell differentiation. First, time-lapse 3D imaging was performed at various stages of differentiation of PGKT2-TetR cells (Figure 2A). Using imaging conditions that minimize phototoxicity in differentiating ES cells (see Experimental Procedures), we first carried out experiments using 1 min time intervals (Δt = 1 min), over a 30 min period, in undifferentiated and differentiated ES cells (from days 1 to 4) (illustrated in Movie S1 for undifferentiated cells and Movie S2 for day 1 differentiated cells). The distances (d) between two loci were used to assess mobility, as this avoids issues of nuclear rotation (Marshall et al., 1997Marshall W.F. Straight A. Marko J.F. Swedlow J. Dernburg A. Belmont A. Murray A.W. Agard D.A. Sedat J.W. Interphase chromosomes undergo constrained diffusional motion in living cells.Curr. Biol. 1997; 7: 930-939Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Measuring 3D distances at each time point (n > 50 cells imaged, at each day of differentiation), we noted that distances between the two XicTetO loci appeared to be more variable during early differentiation as compared to undifferentiated ES cells, indicating a possible increase in mobility. This is illustrated when the change in TetO-TetO distances is plotted over time for multiple cells (Figure S2C, compare cells differentiated for 1 or 2 days to undifferentiated cells). To quantify whether there was a difference in mobility and assess whether the movements observed were random (diffusive) or directional, we plotted the mean square displacement (MSD, < Δd2 >) of the loci over time (Marshall et al., 1997Marshall W.F. Straight A. Marko J.F. Swedlow J. Dernburg A. Belmont A. Murray A.W. Agard D.A. Sedat J.W. Interphase chromosomes undergo constrained diffusional motion in living cells.Curr. Biol. 1997; 7: 930-939Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, Carmo-Fonseca et al., 2002Carmo-Fonseca M. Platani M. Swedlow J.R. Macromolecular mobility inside the cell nucleus.Trends Cell Biol. 2002; 12: 491-495Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The slope of the MSD is proportional to the diffusion coefficient (D) of the loci (see Experimental Procedures). The more mobile two loci are, the faster the < Δd2 > value increases over time, and the steeper the MSD slope. We measured the MSD for ≥40 cells at each stage of differentiation. For short time experiments, a linear increase of < Δd2 > with increasing time intervals (Δt) was observed in all cases, suggesting that XicTetO loci undergo diffusive rather than directional motion over a timescale of several to tens of minutes (Figure 3A ). However, the MSD < Δd2 > value increased more rapidly in differentiating cells (days 1, 2, 3, and 4) compared to undifferentiated cells (Figure 3A). This increased mobility could be seen even in day 8 differentiated cells (data not shown). At day 1 of differentiation, 47% of cells (n = 62) showed a change in TetO-TetO distances of >2 μm over 1 hr, in contrast to only 14% of undifferentiated cells (n = 21). MSD values calculated from data points near the beginning versus the ends of extended observation periods did not change in any significant way indicating that the observed increase in mobility was not due to variable phototoxicity effects in differentiated versus undifferentiated cells (Figure S3A ). We could also exclude that this increase in mobility was due to increased nuclear volume during differentiation, as we found no significant change in nuclear volume in day 1 differentiated cells compared to undifferentiated cells (data not shown), similarly to a previous study (Bacher et al., 2006Bacher C.P. Guggiari M. Brors B. Augui S. Clerc P. Avner P. Eils R. Heard E. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.Nat. Cell Biol. 2006; 8: 293-299Crossref PubMed Scopus (263) Google Scholar), even though mobility changed substantially between these two time points. At sufficiently longer times, the plateau of an MSD curve reflects constraints on long-range chromatin mobility imposed by chromosome and/or nuclear structure (see legend to Figure 3B). Measurements over longer time periods (≥120 min) revealed radii of “constrained diffusion” of ∼1.7 μm (square root of 3 μm2 plateau) for both days 0 and 1. This is in the range of chromosome territory size and well below nuclear size (Figure 3B).Figure S3Mean Square Displacement Analyses, Related to Figure 3Show full caption(A) Assessing potential phototoxicity effects on XicTetO dynamics. Averaged MSDs curves for different stages of differentiation calculated on sequential part of trajectories. Thirty minute movies were divided into three temporal intervals: the first 10 min (green), the 10 following (blue), and the 10 last ones (red). This enables the comparison of mobility at the beginning of acquisitions (when presumably phototoxicity would be minimal) and at the end (when presumably phototoxicity would be maximal). Averaged MSDs on these truncated trajectories show that there are no significant differences of MSD mobility (slope) with time for each differentiation stage.(B) Individual MSD curves for all day 1 pairing cells (left) and day 1 differentiated cells (total population) (right). For visibility, error bars do not appear. Inset: to avoid points tainted with statistical variations, we cut off the individual MSDs at a maximum lag time of one-quarter of the total number of time steps: Δt < 8 min (Saxton, 1997Saxton M.J. Single-particle tracking: the distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (442) Google Scholar). For these statistically significant time points, MSD curves depend linearly on time: 3D diffusion constants D are deduced from the slope of the curve.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Assessing potential phototoxicity effects on XicTetO dynamics. Averaged MSDs curves for different stages of differentiation calculated on sequential part of trajectories. Thirty minute movies were divided into three temporal intervals: the first 10 min (green), the 10 following (blue), and the 10 last ones (red). This enables the comparison of mobility at the beginning of acquisitions (when presumably phototoxicity would be minimal) and at the end (when presumably phototoxicity would be maximal). Averaged MSDs on these truncated trajectories show that there are no significant differences of MSD mobility (slope) with time for each differentiation stage. (B) Individual MSD curves for all day 1 pairing cells (left) and day 1 differentiated cells (total population) (right). For visibility, error bars do not appear. Inset: to avoid points tainted with statistical variations, we cut off the individual MSDs at a maximum lag time of one-quarter of the total number of time steps: Δt < 8 min (Saxton, 1997Saxton M.J. Single-particle tracking: the distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (442) Google Scholar). For these statistically significant time points, MSD curves depend linearly on time: 3D diffusion constants D are deduced from the slope of the curve. We next evaluated whether the increase in mobility we observed during early differentiation was specific to the Xic locus or was a more general feature of the genome in early differentiating ES cells. To this end, different ES cell lines in which two independent autosomal loci had been tagged by random integration of TetO arrays and also expressed a TetR-mCherry transgene to enable visualization, were examined (Figure 3C and Movie S3). The TetO-TetO distances in these lines (PGK28 and PGK134) were measured in living cells (in 3D, over time) at different stages of differentiation (n ≥ 39 cells for each differentiation day) and MSDs were calculated. We observed a general increase in locus mobility upon differentiation, similar to that observed for the Xics, in both cell lines (Figure 3D). Although the kinetics of this increase differed in each case, the trend was the same, implying a general increase in genome mobility during early differentiation. In the case of the Xics, this greater mobility during early differentiation might facilitate the onset of homologous Xic pairing through an increase in the frequency of collisions between loci. On the other hand, it might also inhibit prolonged interactions during pairing. We therefore examined the dynamics of Xic loci during the pairing process in more detail. Based on previous fixed cell data, it has remained unclear whether Xic pairing is a transient but frequent event, or else a prolonged but rare event. To assess the frequency and duration of pairing, and the mobility of the Xic loci during this process, live-cell imaging experiments were performed on PGKT2-TetR cells at day 1 of differentiation, which show the highest frequencies of Xic pairing (Figures 2B and 2C). This also corresponds to the time when reciprocal Xist/Tsix expression patterns are first seen. Time-lapse imaging was performed on cells for 30 min periods, with 1 min intervals. We noted that in the majority of cells where Xic pairing (distances ≤ 2 μm) was detected, both XicTetO loci" @default.
• W2021894549 created "2016-06-24" @default.
• W2021894549 creator A5007435716 @default.
• W2021894549 creator A5018287271 @default.
• W2021894549 creator A5030078016 @default.
• W2021894549 creator A5032144337 @default.
• W2021894549 creator A5038201165 @default.
• W2021894549 creator A5062067186 @default.
• W2021894549 creator A5062217480 @default.
• W2021894549 creator A5063035597 @default.
• W2021894549 creator A5072711950 @default.
• W2021894549 creator A5087714116 @default.
• W2021894549 date "2011-04-01" @default.
• W2021894549 modified "2023-10-16" @default.
• W2021894549 title "Live-Cell Chromosome Dynamics and Outcome of X Chromosome Pairing Events during ES Cell Differentiation" @default.
• W2021894549 cites W1535914077 @default.
• W2021894549 cites W1556351111 @default.
• W2021894549 cites W1979245454 @default.
• W2021894549 cites W1984479378 @default.
• W2021894549 cites W1986004274 @default.
• W2021894549 cites W1986490461 @default.
• W2021894549 cites W1986571517 @default.
• W2021894549 cites W1987362169 @default.
• W2021894549 cites W1993356393 @default.
• W2021894549 cites W2000580034 @default.
• W2021894549 cites W2002259190 @default.
• W2021894549 cites W2004271214 @default.
• W2021894549 cites W2006460442 @default.
• W2021894549 cites W2010295908 @default.
• W2021894549 cites W2014143877 @default.
• W2021894549 cites W2016252630 @default.
• W2021894549 cites W2023983390 @default.
• W2021894549 cites W2026180423 @default.
• W2021894549 cites W2029146706 @default.
• W2021894549 cites W2036322758 @default.
• W2021894549 cites W2048791985 @default.
• W2021894549 cites W2052921451 @default.
• W2021894549 cites W2054661742 @default.
• W2021894549 cites W2069865987 @default.
• W2021894549 cites W2082532751 @default.
• W2021894549 cites W2085388793 @default.
• W2021894549 cites W2088727054 @default.
• W2021894549 cites W2091502861 @default.
• W2021894549 cites W2098359404 @default.
• W2021894549 cites W2106420931 @default.
• W2021894549 cites W2107011634 @default.
• W2021894549 cites W2112455395 @default.
• W2021894549 cites W2115709759 @default.
• W2021894549 cites W2118812779 @default.
• W2021894549 cites W2127439151 @default.
• W2021894549 cites W2135302954 @default.
• W2021894549 cites W2137808356 @default.
• W2021894549 cites W2142928043 @default.
• W2021894549 cites W2158240732 @default.
• W2021894549 cites W2169727662 @default.
• W2021894549 cites W2594550962 @default.
• W2021894549 cites W4238656497 @default.
• W2021894549 cites W4378711309 @default.
• W2021894549 doi "https://doi.org/10.1016/j.cell.2011.03.032" @default.
• W2021894549 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3756906" @default.
• W2021894549 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21529716" @default.
• W2021894549 hasPublicationYear "2011" @default.
• W2021894549 type Work @default.
• W2021894549 sameAs 2021894549 @default.
• W2021894549 citedByCount "127" @default.
• W2021894549 countsByYear W20218945492012 @default.
• W2021894549 countsByYear W20218945492013 @default.
• W2021894549 countsByYear W20218945492014 @default.
• W2021894549 countsByYear W20218945492015 @default.
• W2021894549 countsByYear W20218945492016 @default.
• W2021894549 countsByYear W20218945492017 @default.
• W2021894549 countsByYear W20218945492018 @default.
• W2021894549 countsByYear W20218945492019 @default.
• W2021894549 countsByYear W20218945492020 @default.
• W2021894549 countsByYear W20218945492021 @default.
• W2021894549 countsByYear W20218945492022 @default.
• W2021894549 countsByYear W20218945492023 @default.
• W2021894549 crossrefType "journal-article" @default.
• W2021894549 hasAuthorship W2021894549A5007435716 @default.
• W2021894549 hasAuthorship W2021894549A5018287271 @default.
• W2021894549 hasAuthorship W2021894549A5030078016 @default.
• W2021894549 hasAuthorship W2021894549A5032144337 @default.
• W2021894549 hasAuthorship W2021894549A5038201165 @default.
• W2021894549 hasAuthorship W2021894549A5062067186 @default.
• W2021894549 hasAuthorship W2021894549A5062217480 @default.
• W2021894549 hasAuthorship W2021894549A5063035597 @default.
• W2021894549 hasAuthorship W2021894549A5072711950 @default.
• W2021894549 hasAuthorship W2021894549A5087714116 @default.
• W2021894549 hasBestOaLocation W20218945491 @default.
• W2021894549 hasConcept C104317684 @default.
• W2021894549 hasConcept C121332964 @default.
• W2021894549 hasConcept C14103023 @default.
• W2021894549 hasConcept C144237770 @default.
• W2021894549 hasConcept C145912823 @default.
• W2021894549 hasConcept C148220186 @default.
• W2021894549 hasConcept C148738053 @default.
• W2021894549 hasConcept C1491633281 @default.
• W2021894549 hasConcept C24890656 @default.

• next