Ubiquitin E3 ligase Ring1b/Rnf2 of polycomb repressive complex 1 contributes to stable maintenance of mouse embryonic stem cells

Abstract

Background: Polycomb repressive complex 1 (PRC1) core member Ring1b/Rnf2, with ubiquitin E3 ligase activity towards histone H2A at lysine 119, is essential for early embryogenesis. To obtain more insight into the role of Ring1b in early development, we studied its function in mouse embryonic stem (ES) cells.

Methodology/principal findings: We investigated the effects of Ring1b ablation on transcriptional regulation using Ring1b conditional knockout ES cells and large-scale gene expression analysis. The absence of Ring1b results in aberrant expression of key developmental genes and deregulation of specific differentiation-related pathways, including TGFbeta signaling, cell cycle regulation and cellular communication. Moreover, ES cell markers, including Zfp42/Rex-1 and Sox2, are downregulated. Importantly, retained expression of ES cell regulators Oct4, Nanog and alkaline phosphatase indicates that Ring1b-deficient ES cells retain important ES cell specific characteristics. Comparative analysis of our expression profiling data with previously published global binding studies shows that the genes that are bound by Ring1b in ES cells have bivalent histone marks, i.e. both active H3K4me3 and repressive H3K27me3, or the active H3K4me3 histone mark alone and are associated with CpG-'rich' promoters. However, deletion of Ring1b results in deregulation, mainly derepression, of only a subset of these genes, suggesting that additional silencing mechanisms are involved in repression of the other Ring1b bound genes in ES cells.

Conclusions: Ring1b is essential to stably maintain an undifferentiated state of mouse ES cells by repressing genes with important roles during differentiation and development. These genes are characterized by high CpG content promoters and bivalent histone marks or the active H3K4me3 histone mark alone.

Figure 1. Generating Ring1b conditional knockout ES cells.

(A) Schematic overview of the Ring1b locus targeting strategy in mouse Ring1b^+/− ES cells. The wild type Ring1b allele is shown with main restrictions sites, introns and exons, in the targeted genomic region. The Ring1b^{LoxHy g} allele was generated by targeting a floxed exon3+4/Hygromycin replacement vector to the wild type Ring1b locus of the Ring1b^+/− ES cells. The hygromycin cassette allowed for selection after the initial targeting and was removed by transiently expressing adenoviral Cre to generate the conditional Ring1b^Lox allele. Acute loss of Ring1b from Ring1b^Lox ES cells was induced by adding 4-OHT to the medium to activate CreER^T2 (Cre-recombinase fused to a mutated ligand binding domain of the human estrogen receptor (ER), targeted to the ROSA locus, not shown) resulting in deletion of exons 3 and 4, represented as the Ring1b^Del allele. Exons are indicated by white boxes with the corresponding exon number, loxP sequences by black triangles, and HYG indicates the hygromycin cassette. Main restriction sites are indicated (H = HindIII, E = EcoRI, X = XhoI) as well as the location of the 5′end probe used for Southern blot analysis. (B) Southern blot analysis of HindIII digested genomic DNA of Ring1b^−/Lox;CreER^T2 ES cells showing recombination of the Ring1b^Lox allele following treatment with 4-OHT for the indicated time in days. The 5′end probe visualizes the conventional knockout allele (5 kb), the conditional Lox allele (7 kb) and the recombined Del allele (4 kb). (C) Western blot analysis showing loss of Ring1b protein and uH2A in Ring1b^−/Lox;CreER^T2 ES cells following treatment with 4-OHT for the indicated time in days. Tubulin serves as a loading control.

Figure 2. Deletion of Ring1b results in loss of uH2A but not H3K27me3.

Examples of immunofluorescence stainings in Ring1b^−/Lox;CreER^T2 ES cells showing loss of uH2A (A), but not H3K27me3 (B) in cells that have lost Ring1b four days following 4-OHT treatment.

Figure 3. Downregulation of protein levels of PRC1, but not PRC2, following Ring1b deletion in ES cells.

Western blot analysis of total cell protein extracts (A) or nuclear extracts (B) of Ring1b^−/Lox;CreER^T2 ES cells shows that deletion of Ring1b results in downregulation of Phc1/Mph1 and Bmi1 protein levels, but not of Ezh2, Eed or H3K27me3 levels following treatment with 4-OHT for the indicated number of days.

Figure 4. Gene expression profiling in Ring1b-deficient ES cells.

(A) Expression profiling data clustered by the software program Genesis shows regulation of gene expression (p<0.05) after deletion of Ring1b in mouse ES cells over time. (B) Graph representing the number of all upregulated (red) and downregulated (green) genes (p<0.05). (C) Graph representing the number of up- (red) or downregulated (green) Ring1b bound genes specifically . (D) QPCR validation of microarray data for a selection of genes. Time indicates number of days of 4-OHT treatment. (E) Heatmap of the statistically overrepresented GO categories, as identified by BiNGO analysis. The color scale bar represents the relation between the color intensity and the level of significance (–log10(p-value)). (F) Table showing the significantly enriched GO categories and the percentage (numbers between brackets) of up- or downregulated genes and the number of Ring1b bound genes per GO category (the ones most-downstream in the GO-tree, see also Figure S2).

Figure 5. Ring1b-deficient ES cells retain expression of stem cell specific regulators.

(A,D) Examples of immunofluorescence stainings in Ring1b^−/Lox;CreER^T2 ES cells of (A) Oct4 (red) and DAPI (blue), and (D) Nestin (green) and DAPI (blue), either untreated (upper panels) or treated for four days with 4-OHT (middle panels) or three days with RA (lower panels). (B,C) Histograms represent the distribution of the mean immunofluorescence levels per cell retrieved after quantification of immunofluorescence stainings of Oct4 (B) or Ring1b (C) in Ring1b^−/Lox;CreER^T2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA. (E) Graph representing the number of Ring1b^−/Lox;CreER^T2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA that show ubiquitous (black bars) or filamentous (grey bars) Nestin staining. (F) Alkaline phosphatase activity stainings in Ring1b^−/Lox;CreER^T2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA.

Figure 6. Deregulation of genes bivalent or H3K4me3 marked genes with HCPs in Ring1b-deficient ES cells.

(A–D) Circle graphs show that Ring1b primarily occupies (C) and regulates (D) transcription of genes with high CpG content promoters (HCPs) rather than intermediate (ICP) or ‘low’ (LCP) CpG content promoters, compared to the distribution of HCPs in all genes (A) or only the deregulated genes (B) at the microarray (‘genome-wide’). (E) Bar graph showing the distribution (percentages) of the chromatin state of HCPs for all genes on the whole microarray (‘genome-wide’); only the genes deregulated in absence of Ring1b; Ring1b bound genes; only deregulated Ring1b bound genes in Ring1b-deficient ES cells. Graph shows that Ring1b binds and regulates transcription of bivalent, and to a lesser extend H3K4me3 marked genes (final two columns), in contrast to the genome-wide distribution of these marks on promoters (first two columns). (F) as (E), but for genes with ICPs. Graph shows that bivalent Ring1b bound genes with ICPs are not deregulated in Ring1b-deficient ES cells. (G) Blotplot representing Affimetrix array data of RNA expression levels in wild type ES cells , for the genes represented in (E). The blotplot shows that the median expression levels of H3K4me3 marked genes on the whole microarray (‘genome-wide’ ) are higher compared to the median RNA expression levels of H3K4me3+H3K27me3 marked genes (first two columns). This is similar for genes that are bound by Ring1b (middle two columns), and for Ring1b bound genes that are deregulated in Ring1b-deficient ES cells (last two columns). Black diamond represents median RNA expression level. (H–K) Line graph showing changes (log2 ratio) in gene expression of bivalently (H–I) or H3K4me3 (J–K) marked genes bound by Ring1b following deletion of Ring1b in Ring1b^−/Lox;CreER^T2 ES cells. Time is indicated in number of days of 4-OHT treatment.