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. Author manuscript; available in PMC: 2025 Dec 31.
Published in final edited form as: Epigenet Rep. 2025 Feb 12;3(1):1–13. doi: 10.1080/28361512.2025.2456806

Implication of the Mediator kinase module in CIZ1 recruitment and gene silencing by Xist during the initiation of X inactivation

Jingyi Chen 1, Kevin Halter 1, Timothe Weitzel 1, Anton Wutz 1,✉
PMCID: PMC7618334  EMSID: EMS209667  PMID: 41200585

Abstract

In mammals, dosage compensation between the sexes is mediated by X chromosome inactivation. The long non-coding Xist RNA initiates this process. The molecular mechanism of X-linked gene silencing is beginning to be understood. We have previously reported that the cyclin-dependent kinase CDK8 is required for gene silencing and histone H3 trimethyl-lysine 27 recruitment by Xist. CDK8 functions in cell signalling and transcriptional regulation, in particular, it associates with MED12, MED13, and cyclin C to form a kinase submodule of the Mediator complex. It remains unknown if CDK8 acts as a Mediator kinase in X chromosome inactivation. Here, we analyse a Med12 mutation in an embryonic stem cell model for the initiation of X chromosome inactivation. Loss of Med12 is compatible with self-renewal and survival of mouse embryonic stem cells, but impairs X-linked gene silencing by Xist. In addition, recruitment of CIZ1 and histone H3 trimethyl-lysine 27 by Xist are significantly reduced in the absence of Med12. We further show that CIZ1 recruitment is also modulated by Cdk8. Our study shows that mutations of Med12 and Cdk8 have similar effects on Xist function and provide a cell system for studying the role of the Mediator kinase module in X chromosome inactivation.

Keywords: Med12, Cdk8, Xist, X chromosome inactivation, CIZ1, gene repression

Introduction

In female mouse embryos, one of the two X chromosomes is randomly inactivated through the process of X chromosome inactivation (XCI) for equalizing the expression of X-linked genes between the sexes (Lyon, 1961). XCI is initiated by the long non-coding Xist RNA, which is expressed from the X inactivation centre (Xic) and coats the future inactive X chromosome (Xi). The Xic is a single genetic locus on the X chromosome and regulates XCI. Embryos experience two episodes of XCI, imprinted paternal XCI and random XCI. Imprinted paternal XCI initiates at the 2-cell stage, being completed in the early blastocyst (Huynh & Lee, 2003). Xist expression is observed in most blastomeres at the 4-cell stage leading to inactivation of the paternal X chromosome (Zuccotti et al., 2002). Subsequently, X reactivation is observed in the inner cell mass of early blastocysts (Mak et al., 2004). After implantation, random XCI occurs in the epiblast at embryonic day (E) 5.5 (Rastan, 1982).

Initiation of XCI has been widely studied in mouse embryonic stem cells (ESCs) that retain features of the epiblast in cell culture. Initial gene silencing mediated by Xist is reversible at early stages of differentiation and turns into an irreversible phase during ESC differentiation (Wutz & Jaenisch, 2000). Conversely, Xist is dispensable for the maintenance of XCI in differentiated tissues of mice (Sahakyan et al., 2018). A conserved region at the 5’ end of Xist is required for gene silencing (Wutz et al., 2002). The mammalian homolog of split ends (SPEN) binds to Xist repeat A and is essential for Xist function and the recruitment of chromatin modifications (Asun Monfort et al., 2015; Chu et al., 2015; Keniry & Blewitt, 2023). SPEN directly interacts with Xist recruiting HDAC3 which can deacetylate histones (McHugh et al., 2015). Xist RNA exhibits dynamic localization patterns throughout the cell cycle, tightly associating with the nuclear scaffold of the inactive X chromosome territory during interphase and release during mitosis (Clemson et al., 1996). The release of Xist RNA is regulated by chromatin modifications and the activity of cell division kinase. Aurora B kinase (AURKB) can phosphorylate H3 and chromosomal proteins at prophase, facilitating the release of Xist RNA from the inactive chromosome in the stage of mitosis (Hall et al., 2009).

Xist also recruits chromatin modifying complexes of the Polycomb group of genes. Polycomb-repressive complexes 1 (PRC1) and PRC2 establish histone modifications along the inactive X chromosome (Xi). PRC2 contains the core subunits EED, SUZ12, and EZH1 or EZH2, and catalyses histone H3 trimethyl-lysine 27 (H3K27me3) (Cao et al., 2002; Cao & Zhang, 2004; Sewalt et al., 1998; Shen et al., 2008). Xist initiates PRC1 and PRC2 recruitment through its Polycomb interaction domain (XR-PID) binding hnRNPK, which can recruit Polycomb group RING finger 3/5 (PCGF3/5) (Bousard et al., 2019; Pintacuda et al., 2017). A variant PRC1 complex containing Ring1A/B and one of PCGF3 or PCGF5 proteins catalyses mono-ubiquitylation of histone H2A lysine 119 (H2AK119ub), which in turn mediates PRC2 recruitment (Almeida et al., 2017). JARID is required for PRC2 recruitment and establishment of H3K27me3 (da Rocha et al., 2014). Together PRC1 and PRC2 lead to chromosome-wide modification of histones H3 and H2A on the Xi. Additional factors have been identified that are recruited to the Xi and contribute to DNA cytosine methylation and maintenance of gene repression (Blewitt et al., 2008; Costanzi & Pehrson, 1998; Hernández-Muñoz et al., 2005). Cip1-interacting zinc finger protein (CIZ1) is recruited by Xist during the initiation phase of XCI and accumulates over the Xi (Sofi et al., 2022). CIZ1 becomes essential for Xist localization and maintenance of gene repression in differentiated cells (Ridings-Figueroa et al., 2017). Therefore, CIZ1 is a marker of chromatin of the Xi but its function during initiation of XCI remains to be determined. CIZ1 plays an important role in the regulation of the G1/S transition of the cell cycle by assisting the localization of cyclin A-CDK2 to chromatin, which is crucial for the initiation of DNA replication (Pauzaite et al., 2016). Although, mice with a Ciz1 mutation develop normally, in adult females a lymphoproliferative disorder has been observed that is linked with defects in maintaining Xist localization and gene repression on the Xi (Ridings-Figueroa et al., 2017).

Recently, modulators of XCI have been identified. We have previously shown that a mutation of Cdk8 impairs repression of X-linked genes by Xist and reduces the establishment of H3K27me3 on the Xi (Postlmayr et al., 2020). CDK8 forms a complex with MED12, MED13 and cyclin C. This Mediator kinase module can phosphorylate subunits of core-mediator and RNA polymerase II to suppress the function of the RNA polymerase II carboxy-terminal domain (Hengartner et al., 1998; Lynch et al., 2020; Soutourina, 2018). The tail domain of Mediator forms contacts with enhancer regions including transcription activators, whereas the head and middle domains interact with RNA polymerase II and transcription initiation factor IIA.

Our previous observation of a role of CDK8 in XCI raises the question if MED12 could also function in Xist-mediated repression of X-linked genes. Here, we engineer and analyse a Med12 mutation in ESCs, in which Xist can be induced for studying its effect on X-chromosome-wide chromatin modifications and gene repression. We observe that loss of Med12 impairs gene repression by Xist and causes a defect in the recruitment of H3K27me3 and CIZ1. Notably, we find that mutation of Cdk8 also reduces CIZ1 recruitment by Xist. Our data show that Cdk8 and Med12 mutations have similar effects on Xist function and implicate the Mediator kinase module in the mammalian dosage compensation mechanism.

Results

Establishment of Med12 deficient mouse ESCs

To investigate the function of MED12 in XCI, we used a newly established ESC line from blastocysts of crosses between TX female and Cast/Ei male mice. TX mice carry a tetracycline-inducible promoter at the Xist transcription start site and a reverse tetracycline-controlled transactivator (rtTA) in the ROSA26 locus on chromosome 6 (Savarese et al., 2006) (Figure 1a). In our male ESC line, Xist can therefore be induced by the addition of doxycycline, resulting in the silencing of the X chromosome. We engineered a deletion into the Med12 gene using CRISPR/Cas9 nucleases to remove exons 1 to 7, which code for the N-terminal and phosphoserine domain, including the start codon (Figure 1b). Med12 is an X-linked gene and we obtained three independent male ESC clones with hemizygous Med12 mutations and confirmed the absence of MED12 protein by Western blot, and the deletions using PCR, and Sanger sequencing analysis (Figure 1c, d and e). DNA FISH results showed a single X chromosome in WT and Med12 deficient (ΔMed12) male ESCs (Figure S2f and g). ΔMed12 ESC cultures showed a similar morphology to wild-type control ESCs in 2i medium (Figure 1f). We conclude that Med12 is not required for self-renewal and survival of mouse ESCs, consistent with an earlier report (Rocha, Scholze, et al., 2010).

Figure 1. Engineering a deletion of Med12 in ESCs.

Figure 1

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a) Schematic overview of the inducible Xist system. b) Strategy for engineering a deletion of Med12 exons 1 to 7. PCR primers (horizontal black arrows), product sizes, gRNA positions and deletion size (blue) are indicated. c) PCR using primers indicated in panel b shows wild type and deletion alleles at 846 bp and 417 bp, respectively. Lanes are labelled with clone numbers, whereby ΔMed12 ESC clone F5, H11 and H2 show only the deletion product. d) Sequences of PCR products spanning the Med12 deletion as shown in panel c aligned to the flanking region sequence (top). Deleted bases are indicated by dashes. Blue characters indicate gRNA binding sites. e) Western blot analysis confirming the absence of MED12 protein in ΔMed12 ESC clone F5, H11 and H2. Loading control: HSP90α/β. * unspecific bands. f) The colony morphology of WT and ΔMed12 ESCs. Scale bar: 40 µm.

Xist induction and localization in ΔMed12 ESCs

To further investigate, if Xist could be induced and localize normally in ΔMed12 ESCs, we performed Xist RNA in situ hybridization (RNA FISH) on control TX/Cast (henceforth referred to as WT) and ΔMed12 ESCs after doxycycline treatment. We calculated the percentage of nuclei with clear Xist clusters and observed a comparable number of Xist clusters in ΔMed12 and control ESCs with approximately 50% of nuclei showing Xist clusters after 24h and 48h of doxycycline treatment (Figure 2a and b).

Figure 2. Xist induction and localization in ΔMed12 ESCs.

Figure 2

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a) Bar graph showing a quantification of Xist RNA FISH. The percentage of nuclei with Xist clusters in the presence of doxycycline for 24 hours and 48 hours in WT and ΔMed12 mutant ESCs is plotted. Data are shown as mean ± standard deviation (SD), 24 hours: four replicates for each independent clone. An unpaired two-tailed t-test parametric test assuming the same SD was used to analyse the change between WT and ΔMed12 ESCs. 48 hours: two replicates for each independent clone. Unpaired two-tailed t-test nonparametric test Mann-Whitney test was used to analyse the change between WT and ΔMed12 ESCs. More than 100 nuclei were counted in each sample. ns: not significant. b) Xist RNA FISH (red) in male control WT and ΔMed12 ESCs. DNA is stained with DAPI (blue). Scale bar = 10 μm. c) Cell survival after 96 hours of Xist induction in wild type and ΔMed12 ESCs. The ratio of the cell number of doxycycline-treated relative to untreated ESCs is plotted. Error bars represent SD (three technical replicates). Unpaired two-tailed t-test parametric test assuming the same SD was used to analyse the change between WT and ΔMed12 ESCs. (*: p<0.05, **: p<0.01, *** p<0.001).

Xist induction in our male TX/Cast ESCs results in X-linked gene repression leading to cell death. Therefore, cell survival can be used as a surrogate measure for X-linked gene repression by Xist. To quantify cell survival, we seeded the same number of control and ΔMed12 ESCs in duplicate wells and cultured them for 4 days either in the presence or absence of doxycycline. Cell survival was then calculated as the ratio of the cell number in the presence of doxycycline relative to the cell number in the absence of doxycycline for each ESC line. We determined a low survival rate for our control TX/Cast ESCs as expected (Figure 2c). In contrast, ΔMed12 ESCs showed a statistically significant increase in cell survival after Xist induction (Figure 2c). We conclude that Med12 is not required for Xist localization but contributes to Xist function in ESCs.

Med12 is required for gene repression by Xist

To further investigate the effect of Med12 deletion on Xist-mediated gene silencing, we analysed X-linked gene expression after Xist induction. We observed a strong increase in Xist expression after 24h and 48h of doxycycline treatment in control and ΔMed12 ESCs by qRT-PCR (Figure 3). Autosomal gene expression remained largely unaffected by Xist induction in all ESC lines. Next, we performed RNA-seq transcriptome analysis of two independent control and ΔMed12 ESC clones. Analysis of differential gene expression in ΔMed12 ESCs compared to control ESCs showed that a total of 253 and 182 genes were at least 2-fold up- and down-regulated, respectively (adjusted p-value < 0.05). The downregulation of developmental genes like Gata6, Sox17, Foxd3, Runx1 and Wnt3 suggest that MED12 may be necessary for activating certain differentiation pathways, specifically towards endoderm and mesoderm development (Krivega et al., 2015; Liu et al., 2007; Schrode et al., 2014; Teng et al., 2008) (Figure S1a, Table S1).

Figure 3. Xist and X-linked gene expression in Med12 mutant ESCs.

Figure 3

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qRT-PCR analysis of Xist (red bars) and autosomal genes (blue bars) in WT and ΔMed12 ESCs after 24 and 48 hours of doxycycline induction. Expression is normalised to Gapdh and shown relative to WT uninduced condition. NT: non-treated. +dox: Xist induction. Data represent means ± SD (four technical replicates, relative expression to WT under the no doxycycline treatment condition).

To further investigate X-linked gene silencing in a chromosome-wide manner, we analysed RNA-seq analysis of two independent control and ΔMed12 ESC clones after 48 hours of Xist induction and without Xist induction. The expression of Magix, Itgb1bp2, Col4a5 and Adgrg2 was strongly reduced by Xist induction in control ESCs but remained at a substantially higher level in ΔMed12 ESCs (Figure 4a). Autosomal genes and escape genes were not affected by Xist induction (Figure 4b and c). The expression of other X-linked genes was less affected by Xist expression for 24 hours. qPCR data shows that Usp9x, Uba1, Cdk16, Hprt, Gla and Huwe1 were slightly repressed by Xist induction in control and ΔMed12 ESC clones (Figure S1b). RNA-Seq analysis revealed that the expression of these genes by Xist was slightly impaired by mutation of the Med12 gene (Figure S1c).

Figure 4. Mutation of Med12 reduces X-linked gene silencing by Xist in RNA-seq.

Figure 4

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abc) Fold expression change for a) X-linked genes and Xist b) autosomal genes c) escape genes in WT and ΔMed12 ESCs after 48 hours of Xist induction by doxycycline, calculated as the expression in the dox condition divided by the expression in the sample no dox condition. TPM (transcripts per kilobase million) was used to normalise the abundance value. The positions of genes are indicated by red bars on the X chromosome ideogram (left). d) Heatmap showing z-transformed RNA-seq data representing gene expression in WT and ΔMed12 ESCs on X chromosome (left) and chromosome 1 (right) after 48h of Xist induction. Two WT and ΔMed12 ESC clones F5 and H11 are shown. Red: high expression, blue: low expression.

Although autosomal gene expression appeared unaffected by Xist induction, we had previously made a serendipitous observation that the abundance of NOTCH1 protein was reduced (Postlmayr et al., 2020). To determine whether Xist induction might have subtle effects on autosomal genes we investigated NOTCH1 and HDAC3 (Figure S2a). Immunoblot analysis showed that NOTCH1 and HDAC3 protein were clearly detected in male untreated control and male untreated ΔMed12 ESCs. After Xist induction, both proteins were strongly reduced in male control and male ΔMed12 ESCs (but enriched in female control and ΔCdk8 ESCs (Figure S2a, b and d), see later), which is consistent with our earlier observations (Postlmayr et al., 2020). Next, we determined the transcript levels of Notch1 and Hdac3 in our RNA-Seq datasets from control ESCs and ΔMed12 ESCs with or without Xist induction (Figure S2e). The transcript level of Hdac3 is unaffected with or without Xist induction in control and ΔMed12 ESCs, suggesting that the observed change in Hdac3 is at the protein level rather than the transcript level. The transcript level of Notch1 in ΔMed12 ESCs appears to be higher than control ESCs in the no dox condition, which is consistent with the immunoblot analysis result.

The reason for these expression changes remains to be determined but could be related to the loss of X-linked gene products. Taken together, our results showed that Xist expression leads to repression of X-linked genes but appeared less efficient in the absence of Med12.

We then plotted a heatmap of X-linked gene expression changes between uninduced and induced conditions (Figure 4d). Whereas in control ESCs a strong reduction of gene expression along the X chromosome is observed, X-linked gene expression in ΔMed12 ESCs remains on a higher level after doxycycline treatment. These observations indicate that, although, variability in the strength of silencing between different X-linked genes is observed, genes are less efficiently repressed in the absence of Med12. We conclude that mutation of Med12 had a subtle effect on autosomal gene expression in ESCs but impaired Xist-mediated X-linked gene repression.

Med12 is required for H3K27me3 and CIZ1 recruitment by Xist

To investigate whether Med12 could have a role in chromatin changes, we next quantified the establishment of PRC1-associated H2AK119ub1, PRC2-associated H3K27me3, and the recruitment of the chromatin marker CIZ1 by Xist. For this, we performed combined Xist RNA FISH and immunofluorescence in control and ΔMed12 ESCs. We determined the percentage of Xist clusters that had colocalizing H3K27me3 or CIZ1 foci. After 24 hours of doxycycline treatment, 92.32% of Xist clusters showed colocalizing CIZ1 foci in control ESCs. In ΔMed12 ESCs the percentage was reduced to 62.46% (Figure 5a and b). This indicated that the ability of Xist to recruit CIZ1 was impaired in ΔMed12 ESCs. The transcript level of Ciz1 was slightly increased by Xist induction in ΔMed12 and control ESCs in our RNA-seq dataset (Figure 5e). A similar reduction was observed for the ability of Xist to trigger the establishment of H3K27me3. After 24 hours of doxycycline treatment, 61.78% of Xist clusters had overlapping H3K27me3 foci in control ESCs. In contrast, only 29.94% Xist clusters showed H3K27me3 foci in ΔMed12 ESCs (Figure 5a and c). The expression of Ezh2 was not affected by Xist in control or ΔMed12 ESCs in the RNA-seq dataset (Figure 5e). The percentage of Xist clusters showing H2AK119ub foci was comparable between WT and ΔMed12 ESCs after 24h of doxycycline treatment (Figure 5a and d). We conclude that MED12 contributes to the recruitment of H3K27me3 and CIZ1 by Xist, but is dispensable for establishment of H2AK119ub.

Figure 5. Loss of Med12 reduces CIZ1 and H3K27me3 recruitment.

Figure 5

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a) Quantification of the percentage of Xist clusters co-localizing with CIZ1, H3K27me3, and H2AK119ub in WT and ΔMed12 ESCs in the presence of doxycycline for 24 hours. Four replicates were used and unpaired two-tailed t-test parametric test assuming the same SD were used to analyse the statistical significance of differences in the co-localization of CIZ1/Xist and H3K27me3/Xist between WT and ΔMed12 ESCs. Two replicates were used and unpaired two-tailed t-test nonparametric test Mann-Whitney test was used to analyse the statistical significance of differences in the co-localization of H2AK119ub/Xist between WT and ΔMed12 ESCs. Error bars represent SD. More than 100 nuclei were counted in each sample. ns: not significant. (*: p<0.05, **: p<0.01, *** p<0.001).

bcd) Representative images of combined Xist RNA FISH and CIZ1 b) H3K27me3 c) and H2AK119ub d) immunofluorescence in WT and ΔMed12 ESCs. DAPI stained nuclei. Scale bar = 10 μm. e) Fold expression change for Ciz1 and Ezh2 transcripts in WT and ΔMed12 ESCs after 48 hours of doxycycline induction. RNA-seq data was normalized to TPM (transcripts per kilobase million).

Cdk8 is required for the recruitment of CIZ1 by Xist

Our previous analysis of Cdk8 mutant ESCs did not investigate an effect on CIZ1 recruitment by Xist (Postlmayr et al., 2020). To investigate this aspect further we used a TX/Cast female ESC line that had been established from an independent blastocyst in parallel with our male TX/Cast ESCs. We engineered homozygous deletions in the Cdk8 gene as previously described, confirmed the absence of CDK8 by immunoblot (Halter et al., 2024) and verified Xist induction by RNA FISH (Figure S3a and b). We observed a comparable number of Xist clusters in ΔCdk8 and control ESCs, with approximately 65% of nuclei showing Xist clusters after 24 hours of doxycycline treatment (Figure S3a and b). We next investigated if Xist induction would affect NOTCH1 and HDAC3. Interestingly, female WT and ΔCdk8 ESCs showed higher protein levels after Xist induction, which is in contrast to reduced levels seen in our male TX/Cast ESCs and our previous observation in Cdk8 mutant ESCs with a single TX X chromosome (Figure S2a)(Postlmayr et al., 2020). Although the reason for the differential effect of Xist expression on autosomal Notch1 and Hdac3 expression remains unknown, it might be related to the clonal effects of our ESC lines or potentially reflect the number of X chromosomes. Future work will be needed to determine the biological relevance of our observation. We next performed immunoblot analysis for MED12 in ΔCdk8 ESCs (Figure S2c). MED12 protein was present but appeared reduced in one of our ΔCdk8 ESC clones compared to female TX/Cast ESCs. RNA-seq indicated that the transcript level of Cdk8 in ΔMed12 ESCs was comparable with control ESCs (Figure S2e).

To investigate chromatin changes triggered by Xist in ΔCdk8 ESCs we performed Xist RNA FISH combined with immunofluorescence staining for CIZ1, H3K27me3, and H2AK119ub after Xist induction (Figure 6). After 96 hours of doxycycline treatment, 79.27 % of Xist clusters were positive for CIZ1 in WT #6, while 56.29% of Xist clusters were positive for CIZ1 in ΔCdk8 ESCs. 97.2% of Xist clusters showed H3K27me3 foci in WT female TX/Cast ESCs, while around 70.95% of Xist clusters showed H3K27me3 in ΔCdk8 ESCs. These results suggest that the mutation of Cdk8 significantly affected CIZ1 and reduced H3K27me3 recruitment by Xist (Figure 6a). In conclusion, we observed similar defects in the recruitment of CIZ1 and H3K27me3 in ΔCdk8 and ΔMed12 ESCs.

Figure 6. Reduced recruitment of CIZ1 and H3K27me3 by Xist in the absence of Cdk8.

Figure 6

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a) Quantification of the percentage of CIZ1, H3K27me3, and H2AK119ub foci co-localizing with Xist clusters in WT and ΔCdk8 ESCs after the doxycycline treatment for 24 hours after doxycycline induction. Four replicates were used and unpaired two-tailed t-test parametric test assuming same SD were used to analyse statistical significance of differences in the co-localization of CIZ1/Xist between WT and ΔMed12 ESCs. Two replicates were used and unpaired two-tailed t-test parametric test assuming same SD were used to analyse statistical significance of differences in the co-localization of H2AK119ub/Xist between WT and ΔMed12 ESCs. One replicate in WT and two replicates were used for changes in H3K27me3/Xist between and ΔMed12 ESCs. (*: p<0.05, **: p<0.01, *** p<0.001). Error bars represent SD. More than 100 nuclei were counted in each sample. bcd) Representative images of combined Xist RNA FISH and CIZ1 b) and H3K27me3 c) and H2AK119ub d) immunofluorescence in WT and ΔCdk8 hybrid female mouse ESCs. DAPI stained nuclei. Scale bar = 10 μm.

Discussion

Our study characterizes the role of Med12 during the initiation of XCI. We observe that mutation of Med12 impairs X-linked gene repression by Xist and reduces the establishment of the PRC2-associated histone modification H3K27me3. In contrast, establishment of PRC1-associated H2AK119ub1 appears unaffected by loss of Med12 suggesting a specific requirement of MED12 for PRC2 activity on the Xi. We have previously made similar observations with a mutation of Cdk8 (Postlmayr et al., 2020). In addition, we find that the recruitment of CIZ1 by Xist is similarly dependent on MED12 and CDK8. Taken together these findings suggest that CDK8 functions in the context of the Mediator kinase module. Therefore, our work implicates regulation of the Mediator complex in the initiation of XCI.

Med12 is an X-linked gene in all mammals and translates into a 243 kDa protein in mice. Med12 exon 1 and 2 mutations have been described in human uterine tumours and colorectal cancer that lead to impaired Mediator kinase activity by disrupting the connection between MED12 and CDK8 (Kämpjärvi et al., 2014). The interaction of MED12 with CDK8 proteins also regulates their abundance. We show that MED12 is reduced in ΔCdk8 ESCs, which is consistent with previous results showing reciprocal stabilization of CDK8 and MED12 in colorectal cancer cells (Kuuluvainen et al., 2018) and reduced Med12 expression in cyclin C deficient ESCs (Haarhuis et al., 2022). In mice, a hypomorphic mutation of Med12 causes lethality at E10.5 with severe neural tube closure defects (Rocha, Bleiss, et al., 2010; Rocha, Scholze, et al., 2010). Loss of Med12 is associated with defects in Wnt and planar cell polarity signaling. A role for Med12 in repressing neuronal gene expression has also been reported (Ding et al., 2008). However, it remains to be determined if Med12 mutations would cause sex specific phenotypes in mouse embryos, which might be complicated by the early lethality of a loss of function mutation.

The Mediator complex forms contacts between DNA bound transcription factors at gene regulatory regions and the RNA polymerase II transcription initiation complex (Luyties & Taatjes, 2022). Its composition and activity are regulated by the kinase module, which can have activating as well as repressive functions depending on the cell system and gene locus (Luyties & Taatjes, 2022). Inhibition of CDK8 in human acute myeloid leukaemia cells causes hyperactivation of super-enhancers suggesting a repressive function of the Mediator kinase module (Pelish et al., 2015). Conversely, loss of CDK8 and CDK19 in colorectal cancer cells has been shown to enhanced MED12 and BRD4 binding and to sensitize cancer cells to BET protein inhibitors (Sooraj et al., 2022). This finding raises the possibility that also in Cdk8 deficient ESCs elevated MED12 and BRD4 binding drives transcription, which might not be susceptible to repression by Xist. However, we also observe a defect in X-linked gene repression by Xist in Med12 mutant ESCs, which makes this scenario unlikely. Taken together current data indicate a repressive function of the Mediator kinase module in several biological systems, but details of the molecular mechanisms remain of high interest for future studies.

We show that loss of either Med12 or Cdk8 affects the establishment of Xist-mediated H3K27me3 over the X chromosome. This finding is consistent with our previous analysis of a Cdk8 mutation (Postlmayr et al., 2020) and a report of reduced H3K27me3 in colorectal cancer cells and intestine tissue of mice with a conditional mutation of Cdk8 (McCleland et al., 2015). Our observation that specifically the establishment of PRC2-associated H3K27me3 and not PRC1-associated H2AK119ub1 involves the Mediator kinase module is consistent with the order of Polycomb complex recruitment in XCI. The recruitment of H3K27me3 in female WT was higher than in male WT, likely due to the longer Xist induction in female WT. This finding is consistent with an earlier observation showing continued H3K27me3 accumulation over the course of Xist induction (Figure 5a and 6a) (Tjalsma et al., 2021). PRC2 recruitment depends on PCGF3/5 containing PRC1 complexes and Jarid2 (Almeida et al., 2017; da Rocha et al., 2014). PRC2 recruitment can be genetically separated from gene repression by mutations in Xist (Bousard et al., 2019; Kohlmaier et al., 2004; Plath et al., 2003). The observation that mutations in Cdk8 and Med12 affect both gene repression and establishment of H3K27me3 by Xist place their function at a central position in the silencing mechanism. Also mutations of Hnrnpk (Chu et al., 2015) and Spen (Asun Monfort et al., 2015; McHugh et al., 2015) affect both gene repression and establishment of H3K27me3 by Xist.

Notably, we find that CIZ1 recruitment by Xist is reduced by mutations in Cdk8 and Med12. Both CIZ1 recruitment and the deposition of H3K27me3 were reduced in ΔMed12 ESCs. However, due to only analysing CIZ1 recruitment and the deposition of H3K27me3 after 24 hours of Xist induction, their recruitment and the deposition may be also delayed. CIZ1 is not required for female embryo development or initiation of gene repression by Xist, but becomes essential for Xist localization and maintenance of XCI in somatic tissues (Ridings-Figueroa et al., 2017). CIZ1 recruitment has also been separated from PRC2 recruitment in a recent study investigating specific sequences of human XIST (Navarro-Cobos et al., 2023). Therefore, the Mediator kinase module contributes to regulation of several independent and genetically separable processes in XCI. Mediator is a key component of the transcription units of active genes in mammals. The Mediator kinase module is part of the infrastructure that allows regulators of gene expression to change transcriptional states. We suggest that in XCI its function supports processes that are triggered by factors that are recruited by Xist including SPEN, and PCGF3/5 and ultimately facilitate repression of X-linked genes and chromatin changes. The observation that the ability to repress X-linked genes is reduced but not entirely abolished in Med12 and Cdk8 mutant ESCs suggest, thus far, unknown factors might exist that act in parallel with the Mediator kinase module. It will be of high interest to identify redundant pathways in future studies.

Materials and Methods

ESC culture, genetic engineering, and induction of Xist

ESCs were cultured as previously described (Wutz & Jaenisch, 2000). In brief, ESCs were seeded on gelatine-coated dishes and cultured in DMEM high glucose with L-Glutamine (Life technologies, 41965039) supplemented with 15% fetal bovine serum (Biowest, S1810-500), 1mM sodium pyruvate (Life technologies, 11360039), 100 U/ml penicillin/streptomycin (Life Technologies), 1% Non-Essential Amino Acids (Life Technologies, 11140035), 8 μl/l β-mercaptoethanol (M6250), 1000U/mL leukemia inhibitory factor (LIF), 3 μM Gsk3β inhibitor (Axon Medchem, Chir99021) and 1 μM Mek1/2 inhibitor (Axon Medchem, PD035901). To induce Xist expression, 2 μg/ml doxycycline was used (Sigma-Aldrich, D9891-1G). Cdk8 mutations were generated as previously described (Postlmayr et al., 2020). Med12 mutations were generated using a CRISPR/Cas9 strategy. Two gRNAs were designed using the CCtop program (gRNA1: 5’-CACGGGGACAGCGAGCCGGGCGG-3’; gRNA2: 5’-CTGCTCTTCCTGGAGAATGGGGG-3’) and inserted into PX458 (Addgene: 48138), which contains a green fluorescence protein (EGFP) expression cassette, and used for transfection (Lipofectamine 2000, Life Technologies Europe). Cells were sorted for green fluorescence by Fluorescence-activated cell sorting (FACS). Genomic PCR was performed to detect deletions. (PCR for deletions: F: 5’-GTTTCCGGCAGTAATCGAGAGTTTC-3’, R: 5’-TACATTCAAAGCCGTCAGTTCATCC-3’; PCR for WT bands: F: 5’-GTTTCCGGCAGTAATCGAGAGTTTC-3’, R: 5’-TATCATTCCTGATCCCCATCTTCCT-3’). Xist expression from the TX allele was induced by addition of doxycycline to a final concentration of 2 μg/ml.

Cell survival assay

To determine cell survival after Xist induction, an equal number of cells were plated into gelatine-coated 6 well-plates in medium without GSK3 and MEK1 inhibitors and without penicillin/streptomycin either supplemented with doxycycline or without. The medium was changed every day. The cells were harvested, and the number of cells was counted after 96 hours for doxycycline treated and control wells.

RNA extraction, cDNA synthesis, and qPCR

RNA was extracted using a Qiagen kit (74104) according to the protocol and on-column DNase I digestion (Qiagen: 79254). RNA concentration was measured on a NanoDrop. cDNA synthesis was performed using PrimeScript RT Master Mix (TAKARA: RR036A). RT-PCR performed on a LightCycler 480 using SYBR GREEN (Kapa Biosystems: KK4611). Gapdh was used as a reference gene for normalization. Fold change expression of the target gene was calculated using the ΔΔ ct method and normalized to Gapdh. Primer sequences for gene expression:

Gene Forward primer Reverse primer
Usp9x 5’-ATGCACAACCGAATGCAGTA-3’ 5’-CCCTGGAGGAGGGTTTAAGT-3’
Ubal 5’ -TTTCCTCCTGACCAGCTCAC-3’ 5’-TTTGGGTCCAGACCAGAAAG-3’
Hprt 5’-CAGTCCCAGCGTCGTGATTA-3’ 5’-TGGCCTCCCATCTCCTTCAT-3’
Cdk16 5’-CGACTTGATAGCGATGGAGC-3’ 5’-CAATGGTTGGGTTGACAGGC-3’
Xist 5’ -GCCATCCTCCCTACCTCAGAA-3’ 5’-CCTGACATTGTTTTCCCCCTAA-3’
Gla 5’-CCGCTCCCCTACTCATGTCCCAGGATCT-3’ 5’ -GTCAAAGTCCATTCATAGAAGCC-3’
Huwel 5’-TGACTACCCCCACAACTGCT-3’ 5’ -CACCAACCTTTGCTGGAGAT-3’
Rrm2 5’ -ATGGGAAAGACAACGAAGC-3’ 5’ -CCGAGCTGGAAAGTAAAGC-3 ‘
Eif4a2 5’ -ACACCATCGGGGTCCATTCC-3’ 5’-CCTGTCTTTTCAGTCGGGCG-3’
Sdha 5’-TTCCGTGTGGGGAGTGTATTGC-3’ 5’ -AGGTCTGTGTTCCAAACCATTCC-3’
Actb 5’ -CTAAGGCCAACCGTGAAAAG-3’ 5’ -GGGGTGTTGAAGGTCTCAAA-3’
Tbp 5’ -GGGGAGCTGTGATGTGAAGT-3’ 5’ -CCAGGAAATAATTCTGGCTCA-3’
Gapdh 5’ -CGAAGGTGGAAGAGTGGGAG-3’ 5’- TGAAGCAGGCATCTGAGGG-3’
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RNA FISH analysis

Xist RNA FISH probes were generated by random priming using the Prime-It II Random Primer Labeling Kit (Agilent #300385). Briefly, 1 μg plasmid containing full-length Xist was denatured in water (up to 23 μL) at 95 °C for 5 minutes. Then, 10 μL 9-mer random primers were added to the denatured plasmid and incubated at 95 °C for 5 minutes. Subsequently, the mixture was cooled for 5 minutes. Cy3-dCTP was then added for labelling. The mixture was incubated at 37°C overnight, protected from light. The probe was purified before using. Cells were washed twice with PBS, trypsinized, harvested and diluted to approximately 200’000 cells/mL. Cells were attached to slides using Cytospin 4 by Thermo Scientific. Slides were then washed with PBS, once with CSK buffer for 30 seconds, once with CSK buffer supplemented with 0.5% Triton for 2 minutes and once in CSK buffer for 30 seconds. Cells were fixed in 4% PFA for 10 minutes and subsequently rinsed in 70%, 80%, 95% and 100% EtOH for 2 minutes each. Samples were hybridized with the probe, covered with a coverslip and sealed with rubber cement. The slides were incubated overnight at 37°C. After incubation, slides were washed with 2X SSC/50% formamide for 15 minutes, followed by 2X SSC for 15 minutes in 39 °C, 1X SSC at room temperature for 10 minutes, 4X SSC at room temperature for 5 minutes. Finally, cells were stained with DAPI, covered with a coverslip and sealed with nail polish. Two replicates out of the four for Xist FISH for Figure 2b were then used for CIZ1/H3K27me3 staining for Figure 5 b and c.

Combined RNA FISH and Immunofluorescence analysis

Immunofluorescence staining was performed following Xist RNA FISH. Slides with fixed cells were immersed in 0.2% PBST for 5 minutes. Subsequently, the slides were fixed in 4% PFA for 10 minutes followed by a 3 minute rinse in PBS. Afterward, the cells were incubated in blocking buffer (10% donkey serum, 0.2% Tween, 1X PBS) for 30 minutes. Primary antibodies diluted in antibody buffer (0.5% BSA, 0.2%Tween, 1X PBS) were added and incubated overnight. The following antibodies were used: rabbit anti-Ciz1 (Novus Biologicals, NB100-74624), rabbit anti-H3K27me3 (active motif, 39155), rabbit anti-H2AK119ub (cell signalling). Cells were washed 3 times each 5 minutes with PBST at room temperature (1X PBS, 0.2% Tween). Secondary antibodies were added at a dilution of 1:1000 in the antibody buffer. Cells were washed 3 times each 5 minutes with 0.2% PBST. The slides were mounted with Vectashield containing DAPI (Vector Laboratories, H01200-10), and the coverslip was sealed with nail polish.

DNA FISH chromosome painting

Cells were arrested with 0.1 μg/mL Colcemid (Roche, 10295892001) for 3 hours. Subsequently, the cells were washed with PBS twice and harvested. Cell suspension was centrifuged for 5 minutes at 800 rpm. The supernatant was discarded and the cell pellet was resuspended in hypotonic solution (DMEM: water, 1:1) and incubated for 25 minutes at 37°C in a water bath. Afterwards, samples were centrifuged for 5 minutes at 200g. The supernatant was discarded, and cell pellet was resuspended in 1 mL fixative (methanol: acetic acid, 3:1) and incubated on ice for 20 minutes. Samples were centrifuged for 5 minutes at 800 rpm. Supernatant was discarded and cells resuspended in 1 mL of fresh fixative and incubated on ice for 20 minutes. Samples were again centrifuged for 5 minutes at 800 rpm. The supernatant was discarded and the cells were resuspended in fresh fixative. The volume was adjusted such that the solution appeared slightly cloudy. One drop of cell suspension was dropped on a SuperFrost microscopy slide. After letting the slide air dry, 10 μL of diluted probe (1:3 in Hybrisol) was added. A coverslip was added to the slide and sealed with rubber cement. The slides were heated on a heat block at 75°C for 2 minutes. The slides were incubated overnight at 37°C in a dark and humidified chamber. On the next day, the coverslip and all traces of rubber cement were removed. The slides were first washed in 0.4X SSC (pH=7) at 72°C for 2 minutes, then in 2X SSC 0.05% Tween-20 (pH=7) at RT for 30 seconds. The slides were then briefly dipped in water to avoid crystal formation, and air-dried. The slides were counterstained with 10 μL of DAPI. Coverslips were added and sealed with nail polish.

Western blot

Whole-cell extracts were prepared by suspending cells in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH = 8), 1 mM EDTA, 1% NP-40) supplemented with protease inhibitor (Sigma Aldrich, 505648900). The lysate was rotated on a wheel for 30 minutes at 4°C. The samples were sonicated for DNA degradation at 10% amplitude for 10 seconds. Subsequently, samples were centrifuged at 17’200 g for 20 minutes. The supernatant containing protein was transferred into a new tube. Protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, 5000006). All samples were adjusted to 2 μg/μL. Samples were denatured at 95°C for 5 minutes. The same amount of protein was loaded for each sample in every experiment. The membrane was washed with 1X TBS buffer for 5 minutes with shaking (10X TBS: 24 g/L Tris, 80 g/L NaCl, pH = 7.5) and washed twice with TBST (1X TBS, 0.05% Tween20) for 5 minutes with shaking. The membranes were blocked with 5% milk (2.5 g milk in 50 mL TBST). The primary antibodies were diluted with 5% milk, then the membrane was incubated with the diluted antibodies at 4°C overnight on a shaker. On the next day, the membrane was washed 3 times with TBST for 5 minutes each at room temperature on a shaker. Secondary antibodies (dilution: 1:2000) were added to the membranes and incubated for 1 hour on a shaker. After incubation, the membranes were washed twice with TBST and one time with TBS for 5 minutes each at room temperature on a shaker. Then the membranes were imaged on a ChemiDoc system. Primary antibodies: rabbit anti-MED12 (Novus Biologicals, NB100-2357), rabbit anti-NOTCH1 (Cell Signaling Technology, 3608s), mouse anti-HDAC3 (Santa Cruz Biotechnology, sc-37695), mouse anti-HSP90α/β (Santa Cruz Biotechnology, sc-13119).

Microscopy

Microscopy slides were analyzed using at a Zeiss Axio Observer Z1 microscope, and images were taken with an attached Hamamatsu ORCAFlash 4.0 digital camera. Processing of the images was performed using Zeiss ZEN 2 software.

Transcriptomic and data analysis

RNA was extracted as described above and sent to the Functional Genomics Center Zurich (FGCZ) for sequencing. The library was prepared using the Illumina stranded mRNA prep by FGCZ. Samples were sequenced on an Illumina NovaSeq X plus sequencer, creating 150 bp paired end reads. For the RNA seq analysis, Trimmomatic (Bolger et al., 2014) and HISAT2 (Kim et al., 2019) were used for read trimming and alignment of reads to the mm10 mouse genome reference assembly, respectively. HTSeq were used for counting RNA-seq reads that overlap gene features. For differential gene expression analysis was carried out using DESeq2 (Love et al., 2014). The Script and count data for each sample are available at: https://github.com/HappyJingyi/Med12_project.

GraphPad prism 9 was used for the data analysis panels in Figure 2a and c, Figure 5a and Figure 6a.

Supplementary Material

Supplementary Information
Table S1

Acknowledgements

We thank FGCZ at ETH Zurich for providing the RNA-seq service and technical support. This study was supported by the Swiss National Science Foundation (grant 31003A_175643).

Funding

This study was supported by the Swiss National Science Foundation [grant 31003A_175643].

Footnotes

Competing interests

The authors declare no competing or financial interests.

Author contributions

Study conception and design: JC, TW, AW. Data collection: JC, TW. Analysis and interpretation of results: JC, KH, AW. Funding acquisition: AW. Visualization: JC, KH. Supervision: AW. Writing: JC, AW. Review and editing: JC, AW.

Data availability

The sequencing datasets were uploaded in the NCBI short read archive (SRA) (https://www.ncbi.nlm.nih.gov/sra), and can be accessed in bioproject PRJNA1116844.

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Supplementary Materials

Supplementary Information
EMS209667-supplement-Supplementary_Information.pdf (3.7MB, pdf)
Table S1
EMS209667-supplement-Table_S1.xlsx (185.7KB, xlsx)

Data Availability Statement

The sequencing datasets were uploaded in the NCBI short read archive (SRA) (https://www.ncbi.nlm.nih.gov/sra), and can be accessed in bioproject PRJNA1116844.

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