Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed

Anumber of mammalian genes are expressed from only one of the two homologous chromosomes, selected at random in each cell. These include genes subject to X-inactivation, olfactory receptor (OR) genes, and several classes of immune system genes. The means by which monoallelic expression is established are only beginning to be understood. Using a cytological assay, we show that the two homologous alleles of autosomal random monoallelic loci differ from each other in embryonic stem (ES) cells, before establishment of monoallelic expression. The Polycomb Group gene Eed is required to establish this distinctive behavior. In addition, we found that when Eed mutant ES cells are differentiated, they fail to establish asynchronous replication timing at OR loci. These results suggest a common mechanism for random monoallelic expression on autosomes and the X chromosome, and implicate Eed in establishing differences between homologous OR loci before and after differentiation

X Chromosomes Alternate between Two States prior to Random X-Inactivation

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells

Investigation into the Molecular Mechanisms that Control Random X Chromosome Inactivation

In eutherian mammals, dosage compensation between XX females and XY males is achieved by the transcriptional silencing of one X-chromosome in each female cell. The choice of which chromosome will be silenced is random in that both X chromosomes have an equal probability of being silenced. Despite nearly fifty years of research, the mechanisms that enable a cell to designate precisely one active and one inactive X chromosome remain elusive. What follows is an investigation into how X chromosomal fate is assigned. I present evidence that the two X chromosomes in a female cell adopt distinct states and that these states correlate with fate. I also explore the role of Xist's A-repeat in this process. The A-repeat is a highly conserved Xist element that is necessary for Xist-dependent silencing. The work within this thesis shows that the A-repeat is also required for random choice. I demonstrate that the A-repeat is important for post-transcriptional processing of Xist RNA and that the A-repeat binds the essential splicing factor ASF/SF2. In combination, these findings provide the foundation for a model in which regulation of Xist RNA splicing in ES cells is part of the stochastic process that determines which X will be inactivated in wild-type cells

SIAR Is Specific to Pluripotent Cells In Vitro and In Vivo

<div><p>(A) Percent SD signals observed by FISH for the <i>Xic</i> in cell types that are poised for X-inactivation (ES and ICM), and in trophectoderm (TE) cells and MEFs, which have completed X-inactivation. </p> <p>(B) Allele-specific FISH for the <i>Xic</i> (red) in Δ <i>Xist</i>/+ ICM cells. An <i>Xist</i> probe (green) identifies the wild-type allele; white arrowhead indicates the Δ <i>Xist</i> allele. In 12 of 15 SD cells ( <i>p</i> = 0.02), the Δ <i>Xist</i> allele exhibited the doublet signal. </p> <p>(C) Percent SD signals observed for an autosomal biallelic locus <i>(Hba1)</i> and two X chromosomal loci <i>(Mecp2</i> and the <i>Xic)</i> in MEFs fixed with PFA (dark gray) or MeOH (light gray). Average data from two experiments ( <i>n</i> ≥ 150) are presented; error bars indicate one standard deviation. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#sg004" target="_blank">Figure S4</a>B for complete scoring of SS, SD, and DD signals. </p></div

X-Chromosomal Loci Display SD FISH Signals Independent of Asynchronous DNA Replication

<div><p>(A) FISH for <i>Xic</i> genomic sequences (red) demonstrates the three classes of signals in PFA-fixed female ES cells. DNA was stained with DAPI (blue). </p> <p>(B) ES cells display an elevated proportion of SD signals at X-chromosomal loci. Average data from two to four experiments ( <i>n</i> > 150), scored by two independent scorers, are presented. Error bars indicate one standard deviation. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#sg001" target="_blank">Figure S1</a>A for complete scoring of SS, SD, and DD signals in these cells. </p> <p>(C) The <i>Xic</i> and <i>Pgk1</i> show a single peak of replication in female ES cells. DNA was isolated from cells that were arrested in G1, released into S phase, and pulsed with BrdU at hourly intervals. Progression through S phase was monitored by FACS ( <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#sg001" target="_blank">Figure S1</a>B). An equal amount of BrdU-labeled human DNA was added to each time point. BrdU-labeled DNA was immunopurified, and sequences present in each fraction were assessed by PCR. Standard shows amplification of a human sequence, as a control for variability in immunoprecipitations. IgG represents PCR analysis from labeled DNA purified with mouse IgG instead of anti-BrdU antiserum. –BrdU indicates analysis of an anti-BrdU immunoprecipitation from unlabeled DNA. Pre-IP depicts PCR analysis of the input DNA. </p> <p>(D) Live female ES cells, pulse-labeled with BrdU, were sorted into six fractions by Hoechst staining for DNA content (upper panel). FISH for the <i>Xic</i> (middle panel), and <i>Pgk1</i> (lower panel) in these fractions shows constant, high proportions of SD signals (red triangles) throughout S-phase. Proportions of SS (black circles) and DD (gray squares) are also shown. The high proportion of BrdU-positive cells in all fractions (bold black line) shows that a substantial proportion of cells in all six fractions are in S phase. Over 80% of cycling ES cells are in S phase and fewer than 10% are in G1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#pbio-0040159-b040" target="_blank">40</a>], inevitably leading to the inclusion of early S phase cells in fraction 1. Data are representative of two or three independent experiments. </p> <p>(E) In ES cells, singlet and doublet FISH signals for X-chromosomal loci exhibit equivalent fluorescence intensity. Plots show the ratios of S/D (solid symbols) or D/D (open symbols) FISH signal intensities in individual MEF or ES cell nuclei displaying an SD or DD pattern for <i>Pgk1</i> or the <i>Xic</i> as indicated. The intensity of both pinpoints in each doublet was summed to calculate the total intensity of doublet signals. When calculating the D/D intensity ratios, the two doublets in a cell were randomly assigned to the numerator or denominator. Mean ratio values and 95% confidence intervals for the means are indicated. </p> <p>(F) Comparison of the proportions of cells displaying SS (white), SD (black), and DD (gray) signals for an autosomal locus <i>(Fn1)</i> and three X-chromosomal loci <i>(Mecp2, Xic, and Pgk1)</i> in S-phase ES cells upon PFA or MeOH fixation. </p></div

The Future Xa and Future Xi Exhibit Distinct Frequencies of Singlet FISH Signals

<div><p>(A) Allele-specific FISH for the <i>Xic</i> (red) in Δ <i>Xist</i>/+ ES cells. An <i>Xist</i> probe (green) identifies the wild-type allele. White arrowhead indicates the Δ <i>Xist</i> allele. </p> <p>(B) Allele-specific FISH for the <i>Xic</i> (red) in <i>Tsix</i>–pA/+ ES cells. <i>Tsix</i> RNA (green) identifies the wild-type allele. Grey arrowhead indicates the <i>Tsix</i>–pA allele. </p> <p>(C) Table summarizing scoring of allele-specific FISH in Δ <i>Xist</i>/+ and <i>Tsix</i>–pA/+ ES cells. For three X-chromosomal loci, SD cells were scored for identity of the allele displaying the singlet signal. The X chromosome indicated in black always becomes the Xa, and that in gray and marked with an asterisk always becomes the Xi. The allele indicated in green will be the expressed allele after X-inactivation and the allele indicated in red will be the silent allele. <i>p</i>-Values reflect the probability that the observed distributions are random. </p></div

X Chromosomes Differ from One Another in ES Cells

<div><p>(A) Map of the X chromosome showing positions (Mb) of loci assayed for SIAR.</p> <p>(B) Concordant <i>Mecp2</i> (red) and <i>Hprt</i> (green) (left) and discordant <i>Xic</i> (red) and <i>Mecp2</i> (green) (right) FISH signals. </p> <p>(C) Frequencies of concordance and discordance for specified locus pairs in ES cells. <i>p</i>-Values, determined using a χ² test, reflect the probability that the observed distributions are random. </p></div

Models for Achieving Randomness in X-Inactivation

<div><p>(A) The data in the present study suggest a model in which the X chromosomes in pluripotent cells (m indicates maternal X chromosome, p indicates paternal X chromosome) coordinately switch between future Xa (light blue with dark blue <i>Xic</i>) and future Xi (dark blue with light blue <i>Xic</i>) states in cycling cells. The fates of the X chromosomes as the Xa (green with red <i>Xic</i>) or the Xi (red with green <i>Xic</i>) are determined by their states at the time that the cell receives the cue to initiate X-inactivation. </p> <p>(B) The prevailing model holds that the two X chromosomes in pluripotent cells (black, <i>Xic</i> indicated in white) are equivalent until the cue that initiates X-inactivation causes differential marking of the two X chromosomes (gray cross), thus designating the Xa and the Xi. </p></div

X Chromosomes Appear to Switch between States

<div><p>(A) The two SD signal configurations observed for <i>Ccnb3</i> (red) by allele-specific FISH in X.2/X^wt ES cells, in which one X chromosome is fused to Chromosome 2. The marked allele (asterisk) is scored by its proximity to a <i>CEN-2</i> probe (green). The <i>CEN-2</i> signal on wild-type Chromosome 2 is indicated by parentheses. </p> <p>(B) Allele-specific scoring of <i>Ccnb3</i> in X.2/X^wt ES and four single cell-derived clones. Nonsignificant <i>p</i>-values indicate a random distribution. </p> <p>(C) Scoring of allele-specific FISH for the <i>Xic</i> ( <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040159#sg002" target="_blank">Figure S2</a>) in two independently derived 129-tet/ <i>cas</i> ES cell lines <b>.</b><i>p</i>-Values indicate that the <i>Xic</i> on the 129 chromosome exhibits a singlet signal at a higher frequency than would be expected by random chance. </p></div