RNF12 activates Xist and is essential for X chromosome inactivation

Abstract

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12⁻/⁻ knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

Figure 1. RNF12 activates X chromosome inactivation in trans.

A) Allele specific RT-PCR analysis of Rnf12 expression with RNA isolated from day 3 differentiated female Rnf12 ^+/− ES cells (Cas/129, 129 Rnf12 targeted), and rescued cell lines obtained after stable integration of an 129 Rnf12 transgene. NheI digested 129 products were separated from undigested Cas products. B) Overview of RNA-FISH experiments detecting Xist expression in female wild type, Rnf12 ^+/− and Rnf12^+/− rescued cell lines. qPCR copynumber analysis was performed on genomic DNA. RNA-FISH analysis was performed on day 3 differentiated ES cells, and the percentage of cells harbouring one Xist coated X chromosome (Xist cloud ( = Xi), 1x Xist) or two Xist coated X chromosomes (2x Xist) was determined. C) Representative pictures of RNA-FISH analysis, detecting Xist (FITC) in day 3 differentiated female wild type and Rnf12^+/− rescued ES cells (line 20, Rnf12 overexpression). DNA is counterstained with DAPI in all RNA-FISH slides. D) Allele specific RT-PCR analysis of day 3 differentiated wild type, Rnf12^+/− and Rnf12^+/− rescued cell lines, detecting an Xist length polymorphism that discriminates 129 and Cas Xist.

Figure 2. Counteracting roles for RNF12 and NANOG in XCI.

A) Immunocytochemistry detecting RNF12 (Alexa 546) and SUZ12 (Alexa 488) in day 3 differentiated female ES cells. Cells showing accumulation of SUZ12 on the X chromosome (Xi) show low levels of nuclear RNF12, suggesting that RNF12 is downregulated upon XCI. RNF12 does not accumulate on the SUZ12 coated Xi. B) Quantification of RNF12 staining intensities in female and male ES cells at different timepoints of differentiation. Red and blue box plots show results for female and male cells, respectively. Mean, interquartile range and standard deviation are indicated. N>100 cells per timepoint. Female cells show higher staining intensities and more fluctuation of RNF12 expression compared to male cells. C) Immunocytochemistry detecting RNF12 (rhodamine) and NANOG (FITC) in undifferentiated and day 2 and 3 differentiated male and female ES cells. D) FACS analysis of NANOG-GFP (right panel) and OCT4-GFP (left panel) ES cells transgenic for an Rnf12-mCherry promoter construct. FACS plots show results of undifferentiated ES cells. Cells are gated for GFP+, Cherry+, GFP+Cherry+ or negative. Results of a representative experiment are shown. E) Quantification of FACS analysis of NANOG-GFP (right panel) and OCT4-GFP (left panel) ES cells transgenic for an Rnf12 mCherry promoter construct. Cells were differentiated for up to 7 days, and the percentage of positive cells was determined (Cherry+, red line; GFP+, green line; Cherry+GFP+, yellow line).

Figure 3. RNF12 initiates XCI independent of pluripotency factor binding to Xist intron 1.

A) Schematic representation of part of the X chromosome and the strategy to target the Xist intron 1 pluripotency factor binding sites. A BAC targeting construct replacing Xist intron 1 by a floxed neomycin resistance cassette (Neo) was used to target specifically the 129 allele in Cas/129 female ES cells. The Neo cassette was looped out after transient expression of Cre recombinase. B) RNA-FISH analysis detecting Xist (FITC) in undifferentiated female wild type and Xist ^{intron 1+/−} ES cells. In both wild type and Xist intron 1 deleted cells, only pinpoint signals are visible, representing basal Xist and Tsix expression. C) Bar graph showing the percentage of wild type and Xist ^{intron 1+/−} ES cells that initiated XCI, detected by Xist RNA-FISH, at different time points of EB differentiation. No statistical significant differences were noticed between the wild type control and the cell lines harbouring a deletion of Xist intron 1 (95% confidence interval, N>100 cells per time point ‡ p>0.05). D) Allele specific RT-PCR analysis detecting Xist expression in female wild type and Xist ^{intron 1+/−} cell lines (clone 3, 8 and 10) during differentiation. E) qPCR analysis to determine the Rnf12 copy number in Xist ^{intron 1+/−} ES cells transgenic ES cell lines (transgenic, grey, and endogenous, black, copy number), and percentage of cells with two Xist clouds at day 3 of differentiation. F) RNA-FISH analysis detecting Xist (FITC) in day 3 differentiated Xist ^{intron 1+/−} ES cells, without (left panels) and with (right panels) an Rnf12 transgene. The Xist clusters in one cell with two Xist clusters are indicated with arrowheads. G) RFLP RT-PCR amplifying a NheI RFLP present on the endogenous129 Rnf12 allele, and the Rnf12 transgene. Relative expression analysis was performed with RNA isolated from undifferentiated and day 3 differentiated ES cell lines. H) RT-PCR amplifying a length polymorphism distinguishing Xist emanating from the mutated 129 allele and the wild type Cas allele, with RNA isolated from undifferentiated and day 3 differentiated ES cell lines. I) Xist expression in undifferentiated Rnf12 transgenic Xist ^{intron 1+/−} ES cells, and an Xist ^{intron 1+/−} control cell line was quantified qPCR. J) Table summarizing the results obtained with female wild type, Rnf12 transgenic, Xist ^{intron 1+/−} and Xist ^{intron 1+/−} Rnf12 transgenic ES cell lines.

Figure 4. RNF12 activates Xist directly, but does not inhibit Tsix.

A) Map showing part of the mouse X chromosome, the location of the BAC sequences used, and the position of ms2 repeats within Xist. RNA-FISH probes are indicated in green and red, and non-annotated genes in grey. B) RNA-FISH analysis detecting endogenous Xist (ms2, rhodamine and FITC positive) and exogenous Xist (FITC) from the autosomally integrated Xist-only BAC RP24-180B23 in day 3 differentiated male ES cells transgenic for Rnf12 (BAC RP24-240J16). C) Table summarizing RNA-FISH results from B). Copy number of the Rnf12 transgene was determined by gDNA qPCR. Shown are the percentage of autosomal and endogenous Xist clouds; N, number of cells analyzed. D) RNA-FISH analysis detecting endogenous and transgenic Tsix (FITC, pinpoint signals) and endogenous Xist (FITC, clouds) in day 3 differentiated Tsix transgenic male cells (left panels) and day 4 differentiated Tsix transgenic male cells with additional copies of an Rnf12 transgene (right panels). E) Table summarizing results obtained with single copy Tsix transgenic male ES cell lines with a Rnf12 transgene, 4 days after differentiation. Shown are copy number of the Rnf12 transgene, percentage of cells with two Tsix signals, cells with an Xist cloud, and the percentage of cells with an Xist cloud and Tsix pinpoint signal (n is number of cells analyzed). F) Allele specific RT-PCR detecting transgenic (129) and endogenous (Cas) Tsix in undifferentiated and day 4 differentiated Tsix/Rnf12 double transgenic ES cells. G) qPCR analysis to quantify Xist and Tsix expression in day 4 differentiated Tsix/Rnf12 double transgenic ES cells, and a control cell line without an Rnf12 transgene.

Figure 5. RNF12 is essential for XCI.

A) Targeting strategy to generate Rnf12^−/− ES cells. The Cas Rnf12 allele of the previously generated heterozygous Rnf12^+/− ES cells (Cas/129) was targeted with a BAC construct containing a puromycin selection cassette disrupting the open reading frame of Rnf12. B) PCR RFLP analysis with primers spanning a NheI RFLP descriminating the Cas (no NheI site) and the 129 (NheI site present) alleles, which was used to insert the targeting cassette. C) PCR RFLP analysis confirming the presence of two X chromosomes in Rnf12^−/− ES cells. PCR primers span a BsrgI RFLP located in Xist. D) Western analysis of RNF12 protein and ACTIN in wild type and Rnf12^−/− ES cells. E) qRT-PCR analysis detecting Tsix expression in female wild type and Rnf12^−/− ES cells differentiated for up to 10 days. Results were normalized to Actin. F) qRT-PCR analysis as in (H), but now detecting Nanog (left graph) and Klf4 (right graph) expression. G) RNA-FISH analysis detecting Xist (FITC) in day 3 differentiated female wild type and Rnf12^−/− ES cells. H) Bar graph showing the percentage of female wild type, Rnf12^+/− and Rnf12^−/− ES cells that initiated XCI, as determined by Xist RNA-FISH, at different time points of differentiation. *** p<0,001; ** p<0,01; * p<0,05, Student's T-test. I) qRT-PCR detecting Xist in female wild type and Rnf12^−/− ES cells differentiated for up to 7 days. Results were normalized to Actin. J) Genome wide expression analysis comparing day 3 differentiated Rnf12^−/− and wild type ES cells. Shown are the Log fold expression change and the adjusted P value. K) Luciferase assay detecting expression of an Xist-promoter-luciferase construct in female wild type and Rnf12^−/− ES cells differentiated for 3 days. For transient experiments, cells were co-transfected at day 0 with the Xist-promoter-luciferase or control vector (empty luciferase vector) and a Renilla plasmid. Results were normalized to Renilla expression. For stable pooled clones, the promoter constructs were transfected, clones were pooled after selection and differentiated 3 days prior to analysis.

Figure 6. Rnf12 and its role in the XCI regulatory network.

A) In wild type and Rnf12^+/− cells the XCI activator concentration is above the threshold required to generate a probability to initiate XCI. In contrast, in most Rnf12^−/− cells the XCI activator concentration is not sufficient to reach the threshold required to initiate XCI. B) The regulatory network of XCI. Xist is repressed in Tsix dependent and independent pathways (NANOG binding in intron 1). Activation of XCI is accomplished by RNF12 through activation of the Xist promoter, and possibly Xist mediated silencing of Tsix. Finally, Rnf12 is repressed by Xist and possibly NANOG.