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

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

NIPP1 maintains EZH2 phosphorylation and promoter occupancy

Polycomb Group (PcG) proteins are repressive chromatin modifiers that regulate stem-cell pluripotency and differentiation, and are involved in cancer development. They function in large multimeric protein collections termed Polycomb Repressive complexes (PRC) and a key step in PcG-mediated gene silencing is the trimethylation of histone H3 at lysine 27 (H3K27me3) by the PRC2-component EZH2. Both the chromatin targeting of EZH2 and its affinity for regulatory proteins is controlled by multisite phosphorylation. NIPP1, a regulator of the Ser/Thr protein phosphatase PP1, is a direct interactor of the core PRC2-components EZH2 and EED. Moreover, NIPP1 represses genes in a PcG-mediated manner and associates with a subset of EZH2-target genes. Additionally, the loss of NIPP1 is embryonically lethal and is correlated with a global reduction of H3K27me3 levels.In the first part of this work, we show that NIPP1 is present in a nuclear complex with both PP1 and PRC2 components. Moreover, depleting either NIPP1 or PP1 reduces the association of EZH2 with a subset of its target genes, whereas overexpressing NIPP1 results in a retargeting of EZH2 in a PP1-dependent manner. Moreover, mapping of the genome-wide NIPP1 chromatin distribution using the DamID technique reveals that NIPP1 is associated with multiple PcG target genes. A PP1-binding mutant of NIPP1 shows a deficient association with these loci but still associates normally with PRC2 components. In the second part of this work, we show that the interaction between NIPP1 and EZH2 depends on the CDK-catalyzed phosphorylation of EZH2 at T416. This creates a docking site for the FHA domain of NIPP1 that subsequently prevents PP1 from dephosphorylating EZH2 at mitotic target motifs. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated and shows a deficient association with proliferation-related target loci, as determined by DamID. In conclusion, we demonstrate that both NIPP1 and PP1 play an essential role in the regulation of EZH2-mediated gene silencing. While PP1 is necessary for dephosphorylating EZH2, NIPP1 functions as a PP1-inhibitor to secure the net phosphorylation of EZH2 by CDKs during mitosis. Possibly, a balanced phosphorylation of EZH2 is necessary to safeguard epigenetic memory during cell division in proliferating cells.nrpages: 127status: publishe

Biochemical characterization of malate synthase G of P. aeruginosa

ABSTRACT: BACKGROUND: Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen. RESULTS: Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5 degrees C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 microM), Km acetyl CoA (12 microM) and Vmax (16.5 micromol/minutes/millig enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source. CONCLUSIONS: The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.status: publishe