The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets

Abstract

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex.

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

“…The cell lines expressed the fusions in a doxycycline (Dox)-dependent manner and to a similar extent (Figs S1A and S2A). As expected (Tanuma et al, 2008;Van Dessel et al, 2010), EGFP traps of the NIPP1-Wt and NIPP1-Fm fusions showed an association with endogenous PP1, which was not seen with the NIPP1-Pm and NIPP1-Fm+Pm fusions (Figs S1B and S2B). The associated PP1 was inactive with glycogen phosphorylase as substrate but the phosphatase activity could be revealed by trypsinolysis, which destroys NIPP1 but not PP1 (Beullens et al, 1998), confirming that NIPP1-Wt and NIPP1-Fm inhibit PP1 (Fig.…”

“…Anti-GAPDH and anti-cleaved caspase 3 antibodies were purchased from Cell Signaling Technology, and anti-BubR1 and antiKif18A antibodies from Bethyl laboratories. The anti-NIPP1 and anti-PP1 antibodies were made in-house, as previously described (Van Dessel et al, 2010). Anti-Sap155, anti-cyclin B1, anti-γ-H2Ax, anti-Aurora B, anti-Aca and anti-Flag antibodies were purchased from MBL International, BD Pharmingen, Millipore, BD Transduction Laboratories, ImmunoVision (Springdale, AR) and Stratagene, respectively.…”

“…7A) (Tanuma et al, 2008;Van Dessel et al, 2010). This enabled us to study the effect of the selective inhibition of PP1 on colony formation and tumor growth in the absence of an inducing agent.…”

Roles of enhancer of zeste homolog 2: From skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis

“…73 It has been demonstrated that several factors are involved in PRC2 recruitment to target genes. The serine/threonine protein phosphatase-1 (PP1), along with its regulatory partner NPP1, is able to modulate the DNA occupancy of EZH2 and its activity, enabling or reducing EZH2 phosphorylation; 74,75 Twist-1, in mesenchymal stem cells (MSCs), recruits EZH2 at the ARFINK4a locus, inducing transcriptional repression of both p14 and p16; 76 PHF1, another PcG protein, influences the recruitment and enzymatic activity of PRC2. 77,78 Additionally, recent studies demonstrated that the protooncogene n-Myc is able, in neuroblastoma, to recruit EZH2 at clusterin promoter, inducing the silencing of this tumor suppressor gene and promoting tumorigenesis.…”

Tautomycetin Induces Apoptosis by Inactivating Akt Through a PP1-Independent Signaling Pathway in Human Breast Cancer Cells

“…siRNA mediated suppression of PP1 was achieved as described previously (27). The following siRNA sense olgonucleotide sequences were used: Control, 5′-UAAGGCUAUGAAGAGAUACdTdT-3′; PP1α, 5′-CCGCAUCUAUGGUUUCUACdTdT-3′; PP1λ, 5′-GC AUGAUUUGGAUCUUAUAdTdT-3′.…”