We have previously demonstrated that NIPP1 has distinct binding sites for PP1 (16
) and the PRC2 components EZH2 (18
) and EED (17
). Here, we show that the chromatin-associated fraction of NIPP1 and PP1, unlike its soluble pool, forms a complex with the PRC2 complex, as illustrated by reciprocal co-immunoprecipitation experiments (A and A) and the ability of recombinant NIPP1 to competitively disrupt the chromatin binding of PRC2-type complexes (B). This complex also includes the PRC2 core components SUZ12 and RbAp48, which do not interact directly with NIPP1. Moreover, a microarray- and qPCR-based DamID analysis ( and ) confirmed and extended previous ChIP data (19
), showing that NIPP1 binds to PcG target sites. Among the newly identified NIPP1 targets is the Hox A cluster (A), which belongs to the first and best characterized PcG targets (4
). Unexpectedly, the DamID analysis also identified numerous NIPP1 chromatin-binding sites that were remote from PcG targets, indicating that NIPP1 also has chromatin-associated functions unrelated to PcG signaling. This is in accordance with previous findings showing that NIPP1 binds to RNA (26
), is complexed to the splicing factors SAP155 (28
) and CDC5L (29
), and has a role in (alternative) pre-mRNA splicing (21
). It indeed seems likely that Dam-NIPP1, as a component of the spliceosomes or splicing enhancer/silencing complexes, also leaves methylation marks on neighboring DNA during co-transcriptional (alternative) splicing. However, it cannot be ruled out that the transcriptional and splicing functions of NIPP1 are somehow connected. For example, the NIPP1 ligand and splicing factor SAP155 also functions as a linker between the PRC2 and PRC1 complex (31
). Moreover, NIPP1 binds with high affinity to RNA (26
) and noncoding RNAs have been implicated in the recruitment of PRC complexes (2
). It will therefore be interesting to investigate whether NIPP1 regulates the targeting of PRC2-type complexes by an RNA-guided mechanism.
Our observation that NIPP1 interacts with PcG target genes as well as with the PRC2 complex makes it a prime candidate-regulator of PRC2 recruitment. Consistent with this notion, we found that the knockdown of NIPP1 resulted in a loss of EZH2 from PcG target genes and a decreased trimethylation of H3K27 ( and Supplementary Figure S3
). Conversely, the stable overexpression of NIPP1 caused a redistribution of EZH2 among PcG target genes, with corresponding changes in H3K27 trimethylation (). Intriguingly, overexpressed NIPP1 redistributed EZH2 from maximally repressed PcG targets to genes that were still partially active (D). Yet, both sets of PcG genes were direct NIPP1 targets, as suggested by DamID analysis () and their increased expression following the knockdown of NIPP1 (Supplementary Figure S2
). Collectively, these data suggest that maximally repressed PcG target genes are no longer accessible to overexpressed NIPP1 or are already saturated with NIPP1. In either case, the redistribution of EZH2 can be explained by the competitive disruption of the PRC2-type complexes from fully repressed genes and their re-targeting to genes that bind the overexpressed NIPP1. At present, we do not know whether NIPP1 remains associated with PcG target genes once the PRC2 complex is recruited. It is possible that NIPP1 is only transiently associated with PcG targets and dissociates again once a chromatin structure is established that stabilizes the binding of PRC2. In this respect, it is important to note that the PRC2 complex and EED (13
) have recently been shown to bind directly to trimethylated H3K27, which could represent a mechanism to keep PcG targets silenced once the triggers of their initial inactivation are gone. We also want to point out that the recruitment of PRC complexes in Drosophila
is complexly regulated by multiple proteins (3
). It seems likely that vertebrates also express multiple PRC recruiters or recruiter regulating proteins, as has been suggested recently (5
). Additional PcG recruiter (regulating) proteins may act independently or in concert with NIPP1.
Intriguingly, the PP1-binding mutant NIPP1m still fractionated with chromatin (D) and interacted with the PRC2 complex (A). Yet, NIPP1m only had minor effects on transcription () and on the distribution and function of EZH2 (). This is good evidence that NIPP1-associated PP1 plays a key role in NIPP1-regulated PRC2 signaling. Strikingly, we mapped numerous chromatin-binding sites for both NIPP1 and NIPP1m (Supplementary Table 4
), but NIPP1m was conspicuously less associated with PcG targets ( and ). This suggests that NIPP1-associated PP1 is specifically needed for the targeting of NIPP1 to a subset of PcG loci. Interestingly, phosphorylation of PcG proteins is generally associated with their dissociation from chromatin (32
). For example, the phosphorylation of EZH2 on Ser21 precludes its association with chromatin (33
). However, the phosphorylation of this site was not different between Wt and NIPP1−/–
blastocyst outgrowths (20
). PP1-interacting proteins often act themselves as substrates or substrate targeting subunits (34
). However, metabolic labeling experiments of the HTO cell lines with 32
did not disclose a different extent of phosphorylation of NIPP1 and NIPP1m, or their co-immunoprecipitating proteins (our unpublished data). Therefore, the substrate(s) of NIPP1-associated PP1 that enable the binding of NIPP1 to PcG targets remain elusive and it can currently even not be ruled out entirely that the role of NIPP1-associated PP1 is structural rather than catalytic.
In conclusion, we have found that NIPP1 modulates the binding of the PRC2 complex to at least a subset of its target genes. The binding of NIPP1 to these genes depends on associated PP1, disclosing a novel interaction between PcG signaling and PP1.