The Groucho-associated phosphatase PPM1B displaces Pax transactivation domain interacting protein (PTIP) to switch the transcription factor Pax2 from a transcriptional activator to a repressor

Abstract

Pax genes encode developmental regulatory proteins that specify cell lineages and tissues in metazoans. Upon binding to DNA through the conserved paired domain, Pax proteins can recruit both activating and repressing complexes that imprint distinct patterns of histone methylation associated with either gene activation or silencing. How the switch from Pax-mediated activation to repression is regulated remains poorly understood. In this report, we identify the phosphatase PPM1B as an essential component of the Groucho4 repressor complex that is recruited by Pax2 to chromatin. PPM1B can dephosphorylate the Pax2 activation domain and displace the adaptor protein PTIP, thus inhibiting H3K4 methylation and gene activation. Loss of PPM1B prevents Groucho-mediated gene repression. Thus, PPM1B helps switch Pax2 from a transcriptional activator to a repressor protein. This can have profound implications for developmental regulation by Pax proteins and suggests a model for imprinting specific epigenetic marks depending on the availability of co-factors.

Keywords: Epigenetics; Groucho; PPM1B; Pax; Phosphatase; Protein Phosphatase 2C (PP2C); Transcription; Transcription Corepressor; Transcription Factor.

FIGURE 1.

Grg4 and PPM1B co-IP. Rabbit anti-PPM1B or goat anti-Grg4 were used to IP proteins from nuclear extracts directly in the presence of EtBr and Western blotted (WB) as indicated. Controls are rabbit IgG for PPM1B and goat IgG for Grg4.

FIGURE 2.

Grg4 displaces PTIP to inhibit Pax2-dependent reporter gene expression. A, a schematic of the Pax-responsive reporter gene integrated into HEK293 cells. TK, minimal HSV-TK promoter; EGFP, green fluorescent protein coding; arrow T, forward primer; arrow B, reverse primer used for real time PCR. B, HEK293 cells containing the integrated PRS-GFP reporter were transfected with control vectors (lane 1), Pax2 alone (lane 2), Pax2 and Grg4 (lane 3), or Grg4 alone (lane 4). Whole cell lysates were blotted for the proteins indicated. The lower panel shows GFP expression quantitated by scanning densitometry. GFP expression in the CMV empty vector alone (lane 1) was normalized to 1, and expression in other lysates are relative to this control. Note that the significant increase in GFP expression with Pax2 alone (lane 2) is suppressed by co-expression of Pax2 and Grg4 (lane 3). Grg4 alone has no effect on GFP expression (lane 4). Note comparisons are made for each transfection, and significant comparisons are shown. *, p < 0.01. C–I, ChIP assays from HEK293 cells transfected as in B. The antibodies used for each ChIP are indicated above the graph. Primer pairs flank the PRS. D and E, note that Pax2 binds to PRS independent of Grg4 (D) and Grg4 localizes to PRS in a Pax2-dependent manner (E). F, PTIP binds to PRS in a Pax2-dependent manner but is displaced upon Grg4 co-expression. G, PPM1B is localized to PRS in a Pax2/Grg4-dependent manner. H, Pax2/Grg4 expression suppresses H3K4me3 at the Pax2 binding element. I, DNA polymerase II (Pol II) does not accumulate at PRS4 with Pax2/Grg4 co-expression. Averages from triplicates are shown with error bars representing 1 S.E.

FIGURE 3.

Recombinant PPM1B dephosphorylates Pax2. A, expression of PPM1B in the control shRNA cell line (Neg) and six shRNA stable reporter cell lines. Note PPM1B expression is markedly attenuated in cells transduced with shRNA 1, 2, and 4. B, PPM1B expression is markedly reduced in 20 μg of nuclear extracts from cells transduced with PPM1B sh4 versus sh negative. Histone H3 is used to control for protein loading. C, recombinant purified His-Pax2-(197–415) phosphorylated with recombinant JNK, and ATP was incubated with 20 μg of nuclear extract prepared from negative sh cells or from cells transduced with PPM1B sh4 in the presence or absence of magnesium and various inhibitors of phosphatases. The upper panel is for Pax2 in reactions from negative sh cells, and the lower panel represents reactions from PPM1B sh4 cells. Lane 1, unphosphorylated Pax2. Lane 2, phosphorylated Pax2 (P-Pax2), note the mobility shift. Lane 3, positive control, Pax2 incubated with calf intestinal alkaline phosphatase. Lane 4, no inhibitors. Lane 5, 5 mm tetramisole and 5 μm microcystin-LR. Lane 6, sodium orthovanadate 1 mm. Lane 7, 10 mm β-glycerophosphate. Lane 8, 6 mm EDTA. Lane 9, 20 mm sodium fluoride. Lane 10, 5 mm calcium chloride instead of magnesium. Lane 11, NE without inhibitors or Mg. Note dephosphorylation in lanes 2–7, with mobility shift down to that of unphosphorylated Pax2 in the reactions with negative sh extract. There is no dephosphorylation of Pax2 in the presence or absence of Mg and any inhibitors in the reactions with PPM1B sh4 extract. NE required magnesium for dephosphorylation activity. D, recombinant purified His-Pax2-(197–415) phosphorylated with recombinant JNK and ATP was incubated with 30 ng of recombinant His-PPM1B. Concentrations of inhibitors are as in C. Sigma phosphatase inhibitor sets 1 and 2 were used at the recommended concentrations. Note that PPM1B dephosphorylation activity requires magnesium (compare lane 1 with lane 5). Fluoride and EDTA inhibit PPM1B activity (lanes 6 and 7). β-Glycerophosphate (lane 8), orthovanadate (lane 9), and sigma inhibitors (lanes 10 and 11) had little effect on PPM1B activity. Western blots (WB) for PPM1B and Pax2 are shown. E, recombinant purified His-Pax2-(197–415) phosphorylated with recombinant JNK and ATP was incubated with 10 or 30 ng of recombinant wild-type His-PPM1B (WT) or phosphatase-deficient PPM1B (Mut) in the presence of Mg. Western blot for Pax2 shows an increase in mobility to a lower molecular weight in the incubations with wild-type enzyme denoting loss of phosphorylation. Western blot for PPM1B is shown in the upper panel.

FIGURE 4.

PPM1B is required for Pax2- and Grg4-mediated gene repression. A, control negative sh GFP reporter cells or GFP reporter cells with shRNA 1, 2, or 4 were transfected with Grg4, Pax2, wild-type, or phosphatase-deficient PPM1B as indicated. Whole cell lysates were Western blotted (WB) for the indicated proteins with β-actin used as a loading control. Note that GFP expression increased upon Pax2 transfection (lanes 2, 7, 12, and 17) in all cell lines; this activation was inhibited by Grg4 (lane 3) in control cells but not in PPM1B knockdown cells (lanes 8, 13, and 18). The Grg4-mediated inhibition was rescued with wild-type PPM1B in the knockdown cells (lanes 9, 14, and 19) but not mutant enzyme (lanes 10, 15, and 20). B, scanning densitometry for GFP expression quantitated by ImageJ is shown. GFP expression in the CMV empty vector alone (lanes 1, 6, 11, and 16) are normalized to 1, and expression in other lysates is relative to the respective control. Note that GFP expression is completely suppressed in the control sh cells transfected with Pax2/Grg4 in the presence or absence of PPM1B or mutant PPM1B (compare lane 2 with lanes 3–5). In the knock-out cells, co-expression of Pax2/Grg4 did not repress GFP expression (compare lanes 7 and 8; lanes 12 and 13; and lanes 17 and 18). Wild-type (lanes 9, 14, and 19) but not mutant PPM1B (lanes 10, 15, and 20) restores repression of GFP. All bars are averages of duplicates with error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p < 0.01.

FIGURE 5.

Pax2/Grg4-mediated PTIP displacement is dependent on PPM1B. A–E, ChIP assays were performed with the indicated antibodies from negative sh cells and PPM1Bsh1 cells. The lanes correspond to transfections shown in Fig. 4. A, note that Pax2 binds to the PRS element in all cells transfected with Pax2 (lanes 2–5 and 7–10). B, Grg4 localizes to PRS only when co-transfected with Pax2 (lanes 3–5 and 8–10). C, PPM1B localizes to PRS4 when Pax2 and Grg4 are co-expressed (lanes 3–5, 9, and 10). D, PTIP localizes to the PRS when Pax2 is expressed (lanes 2 and 7) and when wild-type PPM1B expression is attenuated in the presence of Pax2 and Grg4 (lane 8) or when mutant PPM1B is used in the rescue (lane 10). Wild-type PPM1B rescue restores Pax2- and Grg4-mediated PTIP displacement (lane 9). E, H3K4 is methylated when PTIP is localized to PRS. All bars are averages of triplicates with error bars indicating S.E.

FIGURE 6.

Pax2/Grg4-mediated chromatin remodeling at the Rap1A locus is PPM1B-dependent. A, GFP reporter cells with the control sh or cells with PPM1B shRNA 1 and shRNA 4 were transfected as noted and Western blotted (WB) for Grg4, PPM1B, and Pax2. Loading was normalized for β-galactosidase activity. B, RAP1A mRNA expression in the transfected cells from A. Rap1A expression in the CMV empty vector alone (lanes 1, 6, and 11) are normalized to 1 and expression in other lanes are relative to the respective control. Note that in all cell lines, Pax2 stimulates Rap1a expression (lanes 2, 7, and 12). In control cells, Pax2-Grg4 co-expression represses Rap1A expression (lane 3) but not in the absence of PPM1B (lanes 8,13). Rescue with wild-type PPM1B (lanes 9 and 14) but not phosphatase-deficient PPM1B (lanes 10 and 15) restores repression of Rap1A expression in the knock-out cells. All bars are averages of two experiments with PCR done in triplicate and error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p < 0.01. C–G, ChIP assays were performed with the indicated antibodies. Lanes 1–5, control cells; lanes 6–10, PPM1B sh1 knockdown cells. C, note that Pax2 binds to the Rap1a promoter in all cells transfected with Pax2 (lanes 2–5 and 7–10). D, Grg4 localizes to the Rap1A promoter when co-transfected with Pax2 (lanes 3–5 and 8–10). E, PPM1B localizes to this site when Pax2 and Grg4 are co-expressed (lanes 3–5, 9, and 10). F, PTIP localizes to the Rap1A promoter when Pax2 is expressed (lanes 2 and 7) and when wild-type PPM1B expression is attenuated in the presence of Pax2 and Grg4 (lane 8) or when mutant PPM1B is used in the rescue (lane 10). Wild-type PPM1B rescue restores Pax2- and Grg4-mediated PTIP displacement (lane 9). G, H3K4 is methylated when PTIP is localized to this Rap1A promoter site. All bars are averages of triplicates with error bars being S.E.