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PRC2 Complexes with JARID2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprogramming

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From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

Recently, the Polycomb repressor complex PRC2 and its known constituents (notably Jarid2 and Pcl2 (or Mtf2)) have received an appraisal of their roles in the pluripotent nature and differentiation of embryonic stem cells (ESC) (Reviewed in Margueron and Reinberg). PRC2 is known to have methyltransferase activity targeted towards lysine 27 of histone H3 (K27 H3) catalysed by Ezh2 which initiates epigenetic silencing of genes, maintained by the subsequent binding of the methylated K27 H3 by the CBX subunits in the PRC1 multi-subunit complex. This mechanism of regulation is vitally important in the silencing of non-specific lineage associated gene expression in development, and as demonstrated recently, in ESCs. A new study (Zhang et al.) from the laboratories of Tim M. Townes and Hengbin Wang at the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham in the February edition of Stem Cells, reports similar findings to previous studies (see further reading below) by identifying Jarid2 and Mtf2 (Pcl2) as members of the PRC2 complex in mouse ESCs (mESCs). However they also go on to describe a novel mESC specific subunit, esPRC2p48 (Hypothetical protein E130012A19Rik) which is demonstrated to be as critical to proper PRC2 function as the other subunits.

Initial experiments involved the purification of the PRC2 complex from mESCs and subsequent liquid chromatography-mass spectrometry (LC-MS) to analyse PRC2 complex composition. Among the known subunits (Suz12, Ezh2, Eed, Rbbp4, Rbbp7) were other unreported immunoprecipitates; Jarid2, Ezh1, Mtf2, Hspa9 and esPRC2p48. Several further stringent immunoprecipitation assays suggested that Jarid2, Mtf2 and esPRC2p48 were indeed additional PRC2 subunits, while it was suggested that Ezh1 may form a different non-canonical PRC2 complex, taking the place of Ezh2. Encouragingly, Jarid2, Ezh1, Mtf2 and esPRC2p48 were highly expressed in mESCs at the RNA and protein level, whereas only Ezh1 was expressed at high levels in mouse embryonic fibroblasts (MEFs). Correspondingly, embryoid body (EB) mediated differentiation of mESCs showed that Jarid2, Mtf2 and esPRC2p48 protein and RNA levels fell upon differentiation suggesting transcriptional regulation of these subunits during differentiation and a possible link to the pluripotent nature of mESCs.

Further work showed that shRNA-mediated knockdown of Jarid2, Mtf2 or esPRC2p48 led to a reciprocal decrease in the other subunits, suggesting that each of these subunits relies on one or more of the others for protein stability as part of the PRC2 complex. While no morphological changes were observed with the knockdowns, a specific downregulation of tri-methylation on K27 H3 (TriMeK27 H3) was observed, in line with what is known of the functions of the PRC2 complex. This was also correlated to an increase in differentiation associated gene expression (including Tgfbr2, Gata1, 3 and 4 and Hox genes). Again, this suggests a link between these novel subunits and pluripotency, which was further enhanced upon the finding that the expression of Jarid2, Mtf2 and esPRC2p48 could accelerate the reprogramming of MEFs to induced pluripotent stem cells (iPSCs) with an Oct4/Sox2/Klf4 polycistronic vector. Interestingly, these effects were only apparent when all three subunits were included; the addition of Jarid2, Mtf2 and esPRC2p48 in isolation alongside Oct47Sox2/Klf4 did not affect reprogramming efficiency. The importance of these factors to reprogramming was further shown by the reduction of reprogramming efficiency upon the shRNA-mediated downregulation of the endogenous PRC2 subunits in MEFs during iPSC reprogramming. This could be attributed to the ability of Jarid2, Mtf2 and esPRC2p48 to increase TriMeK27 at the promoters of differentiation associated genes and, therefore, silence gene expression allowing progression to pluripotency. Without the repression of such genes, a pluripotent network of expression may not be achieved. Further mechanistic analyses demonstrated that Jarid2 and esPRC2p48 aid in the potentiation of Mtf2´s ability to stimulate the K27 histone methyltransferase activity of the PRC2 complex, while in depth studies of Jarid2 also suggest that it may act to recruit PRC2 to target loci, through interactions with di-methylated lysine 4 and 9 of histone H3 and also to protect the de-methylation of K27 H3 by Kdm6b (Jmjd3). These data taken together suggest interlinked and elaborate interactions between PRC2 subunits, based on the principal of binding of non-repressed lineage associated genes by PRC2, the subsequent methylation and of lysine 27 and finally the repression of gene expression.

Overall, this paper reveals the results from an in depth analysis of the roles of both old and new PRC2 subunits and the links they have to pluripotency. As this paper highlights in its introduction, many multi-subunit complexes are thought to be homogenous and perhaps the reason we have not found new subunits for other complexes is that we have not been actively seeking them. Therefore, this suggests that in many cellular contexts we may need to seek out novel components rather than just novel roles for some protein complexes.

 

References

The Polycomb complex PRC2 and its mark in life.
Margueron R, Reinberg D.
Nature. 2011 Jan 20;469(7330):343-9.

PRC2 Complexes with JARID2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprogramming.
Zhang Z, Jones A, Sun CW, Li C, Chang CW, Joo HY, Dai Q, Mysliwiec MR, Wu LC, Guo Y, Yang W, Liu K, Pawlik KM, Erdjument-Bromage H, Tempst P, Lee Y, Min J, Townes TM, Wang H.
Stem Cells. 2010

 

Further Reading

The JARID2-PRC2 duality.
Herz HM, Shilatifard A.
Genes Dev. 2010 May;24(9):857-61.

Jarid2 and PRC2, partners in regulating gene expression.
Li G, Margueron R, Ku M, Chambon P, Bernstein BE, Reinberg D.
Genes Dev. 2010 Feb 15;24(4):368-80.

Also see Pasini et al., Landeira et al., Pereira et al, Walker et al., Shen et al, Peng et al, and Li et al for recent papers on PRC2 members in ESC biology.