Jarid2 and PRC2, partners in regulating gene expression

Abstract

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.

Figure 1.

Jarid2 interacts with the PRC2 complex. (A) Flag tag purification of Ezh2 from 293F cells. After immunoprecipitation (IP), Ezh2 and associated polypeptides were eluted with Flag peptide, loaded onto SDS-PAGE, and stained with Coomassie blue. The proteins identified by mass spectrometric analyses are indicated on the right. (B) Flag tag purification of Ezh2 from mouse thymus. (Left) Purification scheme. (Center) Silver-stained SDS-PAGE of Flag-IP eluate. Control wild-type and knock-in samples were subjected to mass spectrometry. Results are presented on the right, after subtraction of peptides that were present in both control and knock-in samples. (C) Immunoprecipitation from ES cell nuclear extract using antibodies against Ezh2, Jarid2, or MTF2 and probed with the antibodies indicated on the left. IgG served as control. (D) Nuclear extracts of ES cells were fractionated on a DE52 column followed by a Superose 6 gel filtration column. The fractions were analyzed by Western blot. Molecular weight standards are shown on top. (E) Western analysis using the antibodies shown on the left of immunoprecipitations performed with antibodies shown on the right after recombinant Jarid2 was mixed with recombinant Ezh2, Suz12, or Eed protein.

Figure 2.

Jarid2 regulates PRC2-Ezh2 enzymatic activity. (A) HKMT assay with PRC2-Ezh2 alone or in the presence of increasing amounts of Jarid2 or Rbp2. Recombinant oligonucleosomes (top) or octamers (bottom) were used as substrate. (B, top) Schematic representing the known Jarid2 domains. Also shown are the regions of Jarid2 protein that were expressed in bacteria. (Bottom) Band shift assay in the presence of increasing amounts of each of the Jarid2 fragments using the radiolabeled DNA probes selected by SELEX. The asterisks indicate nonspecific signals present in the free probes. The arrow indicates the specific band shift. (C) Coimmunoprecipitations of purified Ezh2 protein and Jarid2 fragments, as indicated. Immunoprecipitation was performed using the Ezh2 antibody. (D) HKMT assay with PRC2-Ezh2 alone or in the presence of increasing amounts of fragment 2 or fragment 4 of Jarid2 using oligonucleosomes (left) or octamers (right) as substrate.

Figure 3.

Jarid2 recruits Ezh2 to an artificial promoter. (A) Schematic of the luciferase reporter under the control of the TK promoter, with Gal4 DNA-binding sites upstream. (B) Luciferase activity was measured 24 h after doxycyclin induction of the stably integrated transgene encoding Jarid2 fused to the Gal4 DNA-binding domain (Gal4-Jarid2). The values were normalized to the amount of protein. Error bars represent SD of three independent experiments. (Inset) Gal4-Jarid2 expression was checked by Western blot. Actin was used as a loading control. (C) ChIP experiments were performed before (−Dox) and after (+Dox) Gal4-Jarid2 induction. Chromatin was immunoprecipitated with antibodies against the proteins indicated at the bottom. DNA enrichment was analyzed at the luciferase transgene. Results are presented as fold of enrichment over control. Error bars represent SD of three independent experiments. (D) ChIP experiments were performed with or without induction of the stably integrated transgene encoding Gal4-Ezh2. DNA enrichment was analyzed at the luciferase transgene. (E,F) Same as C, but DNA enrichments were analyzed at the endogenous Otx1 (E) and Irx4 (F) promoters. Insets show the levels of Otx1 and Irx4 mRNAs, normalized to those of GADPH.

Figure 4.

Jarid2 binds to PRC2 target cenes in ES cells. (A) ChIP-seq tracks showing H3K4me3, H3K27me3, Ezh2, Mtf2, and Jarid2 at three representative genes in mouse ES cells. (B) Venn diagram showing overlap among the top 1000 gene promoters occupied by Ezh2, Mtf2, or Jarid2, respectively. (C) Box plot comparing H3K27me3 ChIP-seq signals for all genes and for the top 1000 Jarid2 targets in mouse ES cells. (*) P < 0.001. (D) Box plot showing H3K4me3 ChIP-seq signals for the top 1000 Jarid2 targets, for genes enriched for H3K4me3 but not H3K27me3, for “bivalent” genes (enriched for both H3K4me3 and H3K27me3), and for those genes that do not show a significant signal for either H3K4me3 or H3K27me3 in mouse ES cells. (*) P < 0.001. Significance was scored using the Student t-test.

Figure 5.

Jarid2 contributes to PRC2 recruitment to its target genes. (A,B) Two clones in which Jarid2 was stably knocked down were compared with the parental cell line with respect to the levels of mRNA (A) and protein (B) of PRC2 components. mRNA levels were normalized to those of GADPH (A) and GADPH served as a loading control for the Western blot (B). (C–F) ChIP experiments comparing the control parental ES cells versus ES cells in which Jarid2 was stably knocked down. Chromatin was immunoprecipitated with either Jarid2 (C), Ezh2 (D), H3 (E), or H3K27me3 and H3K4me3 (F) antibodies. (C–E) Results are represented as percent of input. Error bars represent SD of triplicates. (F) Results are represented as fold change over control. Error bars represent SD of triplicates.

Figure 6.

PRC2 is required for Jarid2 targeting. (A,B) The levels of protein (A) and mRNA (B) with respect to PRC2 components present in wild-type versus Eed^−/− ES cells are shown. (A) Actin served as a loading control. (B) mRNA levels are presented relative to the wild type, and were normalized to those of GADPH. (C–E) ChIP experiments comparing wild-type with Eed^−/− ES cells. Chromatin was immunoprecipitated with Ezh2 (C), Jarid2 (D), or MTF2 (E) antibodies. Results are represented as percent of input. Error bars represent SD of triplicates.

Figure 7.

Jarid2 is required for proper ES cell differentiation. (A) Western blot of PRC components present in ES cells as a function of time of RA-induced differentiation, as shown at the top. The antibodies used are indicated on the left. (B) Control parental ES or Jarid2 knockdown stable clones were induced for differentiation under conditions conducive to the formation of EBs. Bright-field images of representative EBs at day 8 after induction of differentiation are shown. (C) EBs at day 7 after induction of differentiation were plated on regular cell culture dishes to promote cardiomyocyte differentiation. After 4 d, spontaneous contractions of the EBs were quantified for control versus Jarid2 knockdown ES cells. (D) mRNA levels of a representative set of genes (noted at the bottom) that are targeted by PRC2 and Jarid2, in the case of undifferentiated ES cells stably knocked down for Jarid2, shown relative to the control parental cell line and normalized against those of GADPH. Error bars represent SD of triplicates. (*) P < 0.05 indicates statistically significant changes (Student's t-test). (E) mRNA levels of three markers of ES cell differentiation (Pou5f1, Nanog, and Sox17) as a function of time of induction of ES cell differentiation into EBs. mRNA levels are represented relative to undifferentiated ES cells (left panel), or relative to the control parental cell line (right panel) at each time point. Error bars represent SD of triplicates. (F) Enrichment of H3K27me3 (top panel) or H3K4me3 (bottom panel) was analyzed by ChIP at different times during differentiation, as indicated, in the case of the Pou5f1, Nanog, and Sox17 promoters. Results are expressed as percent of input. Error bars represent SD of triplicates.