PRC2 complexes with JARID2, MTF2, and esPRC2p48 in ES cells to modulate ES cell pluripotency and somatic cell reprogramming

Abstract

Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprogramming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2's activity and gene expression both in ES cells and during somatic cell reprogramming.

Figure 1

Purification and identification of PRC2 from mouse ES cells. (A): Silver staining (top panel) and Western Blot assay (bottom panel) of an SDS-PAGE containing an aliquot of fractions derived from the Superose six column. Polypeptides co-purified with SUZ12 and EZH2 are indicated by asterisks. The elution profile of the protein markers is indicated on top of the panel. (B): Western blot analysis of an aliquot of Input (In), Flowthrough (Ft), and Bound (B) of samples derived from anti-SUZ12 and control IgG immunoprecipitation. (C): Western blot analysis of an aliquot of Superose six fractions. (D–F): Western blot analysis of an aliquot of Input (In), Flowthrough (Ft), and Bound (B) of samples derived from anti-JARID2 (D), anti-MTF2 (E), and anti-esPRC2p48 (F) and control IgG immunoprecipitation.

Figure 2

JARID2, MTF2, and esPRC2p48 are highly expressed in ES cells. Western blot analysis (A, C) and semi-quantitative Reverse Transcription Polymerase Chain Reaction assay (B, D) of selected PRC2 subunits in mouse ES cells and MEFs (A, B) and during ES cell differentiation (C, D). GAPDH serves as a loading control. Parallel experiments, during which the Reverse Transcription reaction was omitted, did not give any specific signal and thus were not shown.

Figure 3

JARID2, MTF2, and esPRC2p48 regulate H3K27 methylation and gene expression in ES cells. (A): Western blot analysis of selected subunits of PRC2 in ES cells treated with shRNA as indicated on the top of the panels. GAPDH is used as a loading control. The numbers below the panels represent the relative proteins levels as compared to control (cells transduced with virus encoding non-targeting shRNAs CFP). (B): Western blot analysis of histone H3K27 methylation in ES cells treated with shRNA as indicated on the top of the panels. Histone H3 was used as a loading control. The numbers below the top panel represent the relative methylation levels. Quantification of top panel is shown under the image of western blot of H3K27me3. (C): Semi-quantitative Reverse Transcription Polymerase Chain Reaction assay of differentiation-associated genes in mouse ES cells treated with interference RNA as indicated on the top of the panels. GAPDH was used as a control. Parallel experiments, during which the Reverse Transcription reaction was omitted, did not give any specific signal and thus were not shown. (D): Chromatin immunoprecipitation (ChIP) assay of histone H3K27 methylation, JARID2, MTF2, esPRC2p48, and SUZ12 on TGFβR2 gene in ES cells treated with shRNAs as indicated on the top of the panels. Antibodies used are indicated in the top of the panels. A diagram of the TGFβR2 gene is illustrated in Figure 6C.

Figure 4

JARID2, MTF2, and esPRC2p48 facilitate somatic cell reprograming. (A): MEFs were infected with the polycistronic OSK vector (left) or in combination with JARID2/MTF2/esPRC2p48 lentiviral vectors (right). Emerging colonies of iPS cells were visualized by alkaline phosphatase (AP) staining on day 14 post infection. Representative plates from three independent experiments are shown. Scale bars: 200 μm. (B): Quantification of iPS cell colony number shown in A. Reprograming efficiency was shown as relative fold changes. Data correspond to the average and SD of three independent experiments. (C): Morphology (top rows) and SSEA1 staining (bottom rows) of selected iPS colonies derived from MEFs infected with the polycistronic OSK vector plus JARID2, MTF2, and esPRC2p48 lentiviral vectors. Original magnification is ×100. (D, E): Morphology (top rows) and Nanog staining (bottom rows) of selected iPS colonies derived from MEFs infected with the polycistronic OSK vector plus JARID2, MTF2, and esPRC2p48 lentiviral vectors. Staining omitted primary antibody was shown as a negative control. (F): RT-PCR analysis of MEFs, mouse ES cells, and iPS colonies derived from MEFs infected with the polycistronic OSK vector in combination with JARID2, MTF2, and esPRC2p48 lentiviral vectors. Nat1 was used as a control. Parallel experiments, during which the Reverse Transcription reaction was omitted, did not give any specific signal and thus were not shown.

Figure 5

Role of JARID2, MTF2, and esPRC2p48 in somatic cell reprograming. (A, B): Effects of individual JARID2, MTF2, and esPRC2p48 on somatic cell reprograming. iPS cell colonies were visualized by alkaline phosphatase (AP) staining. Quantification (average and SD) of two independent experiments is shown. (C, D): Effects of knockdown of JARID2, MTF2, esPRC2p48, and SUZ12 on somatic cell reprograming. iPS cell colonies were visualized by AP staining at day 18. Quantification (average and SD) of iPS cell colonies from two independent experiments at day 12 after transduction of MEFs is shown. (E, F): Effects of JARID2 knockout on somatic cell reprograming. iPS cell colonies were visualized by AP staining at day 32. Quantification (average and SD) of iPS cell colonies from two independent experiments at day 32 is shown.

Figure 6

JARID2, MTF2, and esPRC2p48 facilitate lineage-specific gene repression during somatic cell reprograming. (A): Semi-quantitative Reverse Transcription Polymerase Chain Reaction assay of fibroblast-associated genes in MEFs infected with lentiviral vectors as indicated on the top of the panels. GAPDH was used as a loading control. Parallel experiments, during which the Reverse Transcription reaction was omitted, did not give any specific signal and thus were not shown. (B): Western blot analysis of H3K27 methylation in MEFs infected with lentiviral vectors as indicated on the top of the panels. Histone H3 was used as a loading control. (C): Chromatin immunoprecipitation (ChIP) assay of histone H3K27 methylation on TGFβR2 gene in MEFs infected with the indicated lentiviral vectors. Antibodies used for ChIP are indicated on the top of the images. A diagram of the TGFβR2 gene is illustrated on the top of the panels.

Figure 7

JARID2, MTF2, and esPRC2p48 regulate the function of PRC2. (A): JARID2, MTF2, and esPRC2p48 modulate the histone methyl-transferase activity of PRC2 core complex (EZH2, SUZ12, EED, and RbAP46/68). PRC2 was incubated with recombinant proteins as indicated on the top of the panels on ice for 40 min before histone methyltransferase reaction was started. The top panel is an autoradiograph of the bottom panel. Quantification of two independent experiments is shown in the middle panel. (B): Western blot assay of the histone methyltransferase reaction products with antibodies as indicated at the left side of the panels. Histone methyltransferase assay were performed as above except cold SAM was used in the assay. (C): Peptide array binding assay of JARID2. Histone peptides containing indicated modifications (Table S3) were incubated with recombinant JARID2 and the binding was revealed by anti-His antibody. (D): JARID2 inhibits histone demethylation. Histone demethylation reactions were performed in the presence or absence of JARID2 and reaction product was analyzed by western blot assay with antibodies as indicated at the left side of the panels. (E): Model for JARID2, MTF2, and esPRC2p48 in the regulation of PRC2 function and gene expression. JARID2 recruits PRC2 to target loci. JARID2, MTF2, and esPRC2p48 then synergistically stimulate the histone methyltransferase activity of PRC2. JARID2 could also protect nucleosomes from demethylation by binding to methylated histone tails. All these mechanisms contribute to the high levels of H3K27 methylation and gene repression.