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PRDM14 Enters the Pluripotency Network



By Stuart P. Atkinson

Gene by gene, we are beginning to unravel and understand those members of the pluripotency network which give embryonic stem cells (ESCs) their identity. Other than giving us an understanding of the molecular networks, signalling pathways and regulatory mechanisms which exist in ESCs, such knowledge is also allowing us to make more and more critical studies of induced pluripotent stem cells (iPSCs) to establish whether these are bona fide “replacements” for embryo derived pluripotent cells. To this end, the group of Huck-Hui Ng utilising genome wide RNAi screens have attempted to further identify genes and pathways present in ESCs, with more than 21,000 genes targeted. This study, published in Nature, initially used an OCT4-GFP reporter to establish a list of potentially important genes for ESC pluripotency, which showed enrichment for transcription and translation factors. Further protein-protein interaction analysis found components of the INO80 chromatin remodelling complex, the mediator complex, the COP9 signalosome, the TAF complex, the eukaryotic initiation factor complex and the spliceosome complex which had not been associated with important functions in hESC.

200 of the 566 genes were studied in greater depth and in multiple hESC lines and led to identification of PRDM14 (a transcriptional regulator and potential methyltransferase previously identified as an inhibitor of ESC differentiation) and NFRKB (a component of the INO80 chromatin remodelling complex associated with DNA damage repair) as potentially important genes in hESCs. Both genes were shown to lead to OCT4 loss in hESC upon knockdown, increased OCT4/SOX2/KLF4/MYC-mediated iPSC generation efficiency and could replace MYC and KLF4 in the reprogramming process, thus demonstrating the importance of these genes to the pluripotent nature of hESCs.

PRDM14 was further studied as it ranked higher in the primary screen of important genes. Its knockdown led to the reduction of hESC-associated and an increase in differentiation-associated gene expression in three hESC lines, with these effects demonstrated to be specific to PRDM14 by rescue following PRDM14 cDNA expression. Chromatin immunoprecipitation (ChIP) assays further showed that PRDM14 co-localised with OCT4, NANOG and p300 and functional assays showed that the DNA-binding domain and amino-terminal region required for transcriptional activation are required for pluripotency using the reprogramming assay. Analysis of PRDM14-bound genes show that upon PRDM14 depletion, 13.5% of these genes were down-regulated while 24.1% were up-regulated, suggesting that PRDM14-mediated regulation is complex.

These results merit further studies, as relatively little is known of PRDM14 function. It has been suggested to have methyltransferase activity, although this has not yet been demonstrated, to be involved in germ cell specification in mice and dysregulation of PRDM14 expression has been linked to cancer. NFRKB perhaps also deserves further study; chromatin remodelling is of obvious importance to ESCs while a potential link between DNA damage repair mechanisms and active DNA methylation makes NFRKB a possibly exciting research topic.


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