Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun;12(6):618-24.
doi: 10.1038/ncb2065. Epub 2010 May 16.

Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators

David Landeira  1 , Stephan Sauer, Raymond Poot, Maria Dvorkina, Luca Mazzarella, Helle F Jørgensen, C Filipe Pereira, Marion Leleu, Francesco M Piccolo, Mikhail Spivakov, Emily Brookes, Ana Pombo, Cynthia Fisher, William C Skarnes, Tim Snoek, Karel Bezstarosti, Jeroen Demmers, Robert J Klose, Miguel Casanova, Ligia Tavares, Neil Brockdorff, Matthias Merkenschlager, Amanda G Fisher
Affiliations

Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators

David Landeira et al. Nat Cell Biol. 2010 Jun.

Abstract

Polycomb Repressor Complexes (PRCs) are important regulators of embryogenesis. In embryonic stem (ES) cells many genes that regulate subsequent stages in development are enriched at their promoters for PRC1, PRC2 and Ser 5-phosphorylated RNA Polymerase II (RNAP), and contain domains of 'bivalent' chromatin (enriched for H3K4me3; histone H3 di- or trimethylated at Lys 4 and H3K27me3; histone H3 trimethylated at Lys 27). Loss of individual PRC components in ES cells can lead to gene de-repression and to unscheduled differentiation. Here we show that Jarid2 is a novel subunit of PRC2 that is required for the co-recruitment of PRC1 and RNAP to genes that regulate development in ES cells. Jarid2-deficient ES cells showed reduced H3K4me2/me3 and H3K27me3 marking and PRC1/PRC2 recruitment, and did not efficiently establish Ser 5-phosporylated RNAP at target genes. ES cells lacking Jarid2, in contrast to previously characterized PRC1 and PRC2 mutants, did not inappropriately express PRC2 target genes. Instead, they show a severely compromised capacity for successful differentiation towards neural or mesodermal fates and failed to correctly initiate lineage-specific gene expression in vitro. Collectively, these data indicate that transcriptional priming of bivalent genes in pluripotent ES cells is Jarid2-dependent, and suggests that priming is critical for subsequent multi-lineage differentiation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Jarid2 selectively associates with PRC2 proteins in mouse ES cells and localizes to PRC2-bound promoters. (a, b) Mass spectrometric analysis of Flag-immunoprecipitates from 2×Flag–Eed- or 2×Flag–Jarid2-expressing ES cells. Silver staining of 2×Flag–Eed immunoprecipitation (a) and Coomassie staining of 2×Flag–Jarid2 immunoprecipitation (b) is shown. Insets show bands present only in the Flag-transfected samples that correspond to PRC2 subunits, assigned according to molecular size. Unique peptides detected in two independent mass spectrometric experiments and their mascot scores are indicated. (c) Western blot analysis of nuclear extracts of E14 ES cells immunoprecipitated with a mouse anti-Eed (in the presence or absence of ethidium bromide; EtBr) and blotted with antiserum to Jarid2, Eed, Ezh2 and Suz12. Mouse immunoglobulin-G (IgG) was used as a negative control. Anti-Eed detected Eed isoforms 1, 3 and 4 as indicated (i1, i3 and i4). (d) Western blot analysis using antibodies described in (c) of anti-Flag immunoprecipitated material from parental (E14) and 2×Flag–Jarid2-transfected ES cells. (e) Overlay of data from 2×Flag–Jarid2 ChIP-chip genomic tiling array (grey, two replicates; as designed in ref. 19) with published ChIP-chip promoter array analysis of polycomb group proteins (green) and H3K27 trimethylation (blue). (f) Comparison of 2×Flag–Jarid2 (black) with ChIP-chip data for H3K4me2 (K4+) and H3K27me3 (K27+; ref. 20) shows significant (P = 2.31×10−62, chi-squared test) preference of Jarid2 for bivalent (K4+K27+) promoters.
Figure 2
Figure 2
Jarid2-null ES cells express pluripotency-associated factors and do not show global de-repression of PRC2-target genes. (a) Western blot, using anti-Jarid2 and anti-Oct4 antibodies, of whole cell extracts of wild-type (JM8+/+) ES cells, a Jarid2 heterozygous (A08+/−) line and two Jarid2 knockout cell lines (E4−/− and E8−/−). Lamin was used as a loading control. Uncropped versions of the western blots are shown in Supplementary Information, Figure S7. (b) Percentage of alkaline phosphatase-positive colonies showing a compact, flat or spread morphology (mean ± s.d.). (c) Growth curve of JM8 (black line), A08 (grey line), E4 (dashed line) and E8 (dashed line) ES cells over 4 days. (d) Percentage of methylene blue-stained colonies (mean ± s.d.) detected 8 days after plating 100 cells per well; representative images of methylene blue-stained colonies plated at 500 cells per well. (e) Expression of PRC2-regulated genes in Jarid2-deficient cells (A08; grey, E4 and E8; white) relative to wild-type control (JM8). Analysis of an Eed deficient ES cell line (clone B1.3), in which PRC2 genes are de-repressed (blue), was included as a control. Mean ± s.d. of three experiments is shown.
Figure 3
Figure 3
Jarid2 is required to recruit PRC2 to target genes in ES cells. (a, b) Binding of Ezh2 and Suz12 to the promoter regions of genes, or across the Nkx2-2 locus, was assessed by ChIP. Results show mean ± s.d. of three experiments where values are expressed relative to input. Asterisks indicate statistical significant differences between JM8 wild-type and Jarid2 knockout cells (p < 0.05; Student’s t-test). (c, d) The abundance of modified histones was assessed by ChIP using specific antibodies. Results show mean of two experiments and values are expressed relative to H3 abundance. In (b) and (d) the abundance (mean) along Nkx2-2, using primer pairs detailed in Supplementary Information, Figure S3, is plotted as a function of distance to TSS (error bars are shown in Supplementary Information, Fig. S3). Experiments were performed using wild-type (JM8, black), heterozygous (A08, grey) and Jarid2-knockout ES cells (E4, E8, white bars or E4; red and E8; blue lines). A scheme of the Nkx2-2 locus displaying TSS (arrow) and three exons (black boxes) is shown in b. Ccnd1, a putative Jarid2 target gene in cardiac myocytes, and Oct4, Hprt and Myf5 were included as controls.
Figure 4
Figure 4
Efficient recruitment of PRC1 and Ser5-phosphorylated RNAP to target genes in ES cells requires Jarid2. (a) Ring1B and Mel18 binding at gene promoter regions (left panel), or across the Nkx2-2 locus (right panel), was assessed by ChIP. Values and error bars are shown in Supplementary Information, Figure S3, and the distance from the Nkx2-2 TSS is indicated. (b) RNAP binding (Ser 5 phosphorylated or detected by 8WG16) was assessed by ChIP. Right panel shows a schematic representation of the Nkx2-2 locus where the TSS, a conserved region at −2.5 kb (light grey box), the coding region (8.7 kb containing three exons, black boxes), untranslated regions (light grey boxes) and the position of primer pairs are indicated. Results show mean ± s.d. of three experiments, where values are expressed relative to input chromatin. Experiments were performed using wild-type (JM8, black), heterozygous (A08, grey) and Jarid2-knockout ES cells (E4, E8, white bars or E4; red and E8; blue lines). Hprt, Oct4 and Ccnd1 (active genes), and Gata1 and Myf5 (silent genes) provide controls. Asterisks indicate statistical significant differences between JM8 wild-type and Jarid2 knockout cells (P < 0.05; Student’s t-test).
Figure 5
Figure 5
Jarid2 is required for successful ES cell differentiation. Brightfield images of parental (JM8+/+), heterozygous (A08+/−) and Jarid2-null (E8−/−) ES cells induced by (a) withdrawal of LIF (upper panel, day 9), culture in neural-inducing N2B27 media (lower panel, day 5) and (b) culture on Collagen IV-coated plates under mesoderm-inducing conditions (upper panel, day 4). Flk1-expressing hemangioblasts were induced from embryoid bodies using standard approaches and quantified by FACS (lower panel, day 3.75) where bar shows gates used to identify Flk1+ cells. (c) Upper panel shows brightfield images where the absence of Jarid2 results in fewer and smaller embryoid bodies induced upon LIF withdrawal, compared with wild-type (day 5). Time course analysis of Oct4, Nanog and Nestin expression in embryoid bodies by western blot (lower panel). (d) Kinetics of gene expression during embryoid body formation (relative to undifferentiated ES cell controls). Values for parental (black) and Jarid2-null (E8, white) ES cells are shown. Scale bars, 100 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bernstein BE, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125:315–326. - PubMed
    1. Boyer LA, et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature. 2006;441:349–353. - PubMed
    1. Lee TI, et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell. 2006;125:301–313. - PMC - PubMed
    1. Azuara V, et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 2006;8:532–538. - PubMed
    1. Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell. 2008;132:567–582. - PMC - PubMed

Publication types