Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells

<div><p>X-chromosome inactivation (XCI) in female lymphocytes is uniquely regulated, as the inactive X (Xi) chromosome lacks localized Xist RNA and heterochromatin modifications. Epigenetic profiling reveals that Xist RNA is lost from the Xi at the pro-B cell stage and that additional heterochromatic modifications are gradually lost during B cell development. Activation of mature B cells restores Xist RNA and heterochromatin to the Xi in a dynamic two-step process that differs in timing and pattern, depending on the method of B cell stimulation. Finally, we find that DNA binding domain of YY1 is necessary for XCI in activated B cells, as <i>ex-vivo</i> YY1 deletion results in loss of Xi heterochromatin marks and up-regulation of X-linked genes. Ectopic expression of the YY1 zinc finger domain is sufficient to restore Xist RNA localization during B cell activation. Together, our results indicate that Xist RNA localization is critical for maintaining XCI in female lymphocytes, and that chromatin changes on the Xi during B cell development and the dynamic nature of YY1-dependent XCI maintenance in mature B cells predisposes X-linked immunity genes to reactivation.</p></div

YY1 is required for Xi heterochromatin and X-linked gene dosage compensation in activated splenic B cells.

<p>(A) Sequential Xist RNA FISH (red) then immunofluorescence detection for H3K27me3 or H2a-Ub (green) in wild type (left) and <i>ex vivo</i> YY1 deleted (-/-; right) splenic B cells stimulated with CpG for 72 hrs. White arrows denote heterochromatic foci that overlap with an Xist RNA cloud. Representative results from one experiment are shown (n = 3). (B) Quantification of the number of nuclei in (A) containing a focus of heterochromatin. The averages from 3 experiments are shown, and statistical significance was determined using two-tailed t-test. Error bars denote standard deviations from the mean for biological replicates between independent experiments. (C) Sex-specific differentially expressed genes (DEGs) in YY1^-/- B cells. Venn diagram shows the comparison of RNA-seq results for unique male (blue) versus unique female (pink) YY1^-/- activated B cells. Shared DEGs shown in purple. Heat map shows hierarchical clustering of expression for the 68 female-specific X-linked transcripts for female wildtype and female YY1^-/- activated B cells. Cells were harvested two days after Cre-mediated deletion, corresponding to 48 hrs of stimulation. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s012" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s013" target="_blank">S2</a> Tables for complete gene lists. Three independent mice for each condition (wildtype and YY1^-/-) and sex were used for RNA isolation. (D) List of top 15 female-specific up-regulated X-linked genes in YY1^-/- B cells. Complete lists are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s012" target="_blank">S1 Table</a>. (E) Gene Ontology analysis for the complete list of X-linked genes upregulated in YY1^-/- B cells compared to female wildtype cells. Enrichment Score (ES) > 1, p < 0.05. The complete gene lists are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s013" target="_blank">S2 Table</a>.</p

The C-terminal zinc finger domain of YY1 localizes Xist RNA to the Xi.

<p>(A) YY1 protein domains (left). A representative Xist RNA FISH image for wildtype LPS activated splenic B cells (right). (B) Rescue experiments in YY1^-/- B cells using various YY1 mutant proteins. YY1 was deleted using TAT-CRE protein, then cells were stimulated with LPS overnight and then infected with YY1 protein constructs co-expressing GFP. GFP positive and negative cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s011" target="_blank">S11 Fig</a>) were sorted 48 hrs after transduction and then used for Xist RNA FISH. Clockwise from top left: empty vector; full length YY1 protein; N-terminal domain of YY1 (amino acids 1–200); C-terminal domain of YY1 (amino acids 201–414); full length YY1 lacking the REPO domain (deletion of amino acids 201–226); YY1 zinc finger domain (amino acids 290–414). Representative Xist RNA FISH (red) fields from one experiment are shown (n = 2). (C) Quantification of nuclei containing Type I and II Xist RNA localization patterns for a representative YY1 rescue experiment. The total number of nuclei counted is shown above each column. Statistical significance was determined using Chi-squared tests comparing wildtype to each rescue mutant for both experiments; p values are shown above total nuclei; n.s. not significant. One representative experiment is shown; similar <i>p</i> values for Chi-squared tests were obtained within each experiment and chi-squared tests between experiments 1 & 2 were not significantly different (p = 0.68) reflecting high reproducibility between experiments.</p

Model: Two phases for Xist RNA localization to the Xi during B cell stimulation.

<p>Naïve B cells lack detectible Xist RNA signals despite continuous <i>Xist</i> transcription. Xist RNA transcripts (pink) diffuse away from the Xi; YY1 protein is present, but at lower levels. During Phase 1 of Xist RNA localization (4–8 hrs post-stimulation), Xist RNA pinpoints are visible across the nucleus and RNA binding proteins cluster transcripts together to produce a visible signal by RNA FISH. During Phase 2 of localization (12–16 hrs post-stimulation), YY1 recruits Xist RNA transcripts, either directly binding RNA or indirectly interacting with Xist binding proteins (Xist BP), back to the Xi through the zinc-finger domain. Nuclei with robust Type I Xist RNA clouds have Xist RNA localized across the Xi.</p

Timing of Xist RNA localization to the Xi during B cell stimulation.

<p>(A) Time course analysis, using RNA FISH, to monitor Xist RNA (red) localization changes immediately following B cell stimulation using CpG. Representative field images from one experiment are shown; similar results were obtained across 4 independent replicate experiments. (B) Representative results from one experiment quantifying the Xist RNA localization patterns for each time point after B cell stimulation using CpG. The total number of nuclei counted is shown above each column. Results from independent replicate experiments are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s006" target="_blank">S6A Fig</a>, along with statistical significance testing across all three experiments. (C) Representative Xist RNA FISH images of naïve splenic B cells and <i>in vitro</i> stimulated B cells (for 24 hrs) using three different stimuli. (D) Quantification of Xist RNA localization patterns for splenic B cells stimulated for 24 hrs using LPS, CpG, or anti-mu (Mu). Each column shows the average localization patterns for splenocytes from one female mouse. The total number of nuclei counted is shown above each column. (E) Time course analysis of splenic B cells stimulated with anti-mu. Representative results from one experiment quantifying Xist RNA localization patterns. The total number of nuclei counted is shown above each column.</p

Simultaneous return of Xist RNA and H3K27me3 enrichment at the Xi during B cell activation.

<p>(A) Sequential Xist RNA FISH (red) then immunofluorescence detection (green) for H3K27me3 at 5, 12, 24 hrs post-stimulation. Representative results from one experiment (#1) are shown; results from experiments #2, 3 are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s008" target="_blank">S8 Fig</a>. One-way ANOVA analyses comparing all three experiments were not statistically different. (B) Examples of individual nuclei displaying different co-localization patterns of Xist RNA (red) and Xi heterochromatin (H3K27me3, green); taken 5 hrs post-stimulation. White arrows denote heterochromatic foci. (C) Quantification of co-localization patterns for Xist RNA and H3K27me3 (blue bars), Xist RNA signals alone (orange), H3K27me3 foci alone (green), or nuclei without either signals (purple) during B cell stimulation. Xist RNA signals corresponded to Type I, II, or III patterns. Results from one experiment are shown; three independent experiments were performed and these results are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007050#pgen.1007050.s008" target="_blank">S8 Fig</a> together with statistical tests across all three independent replicates. The total number of nuclei counted is shown above each column.</p