Nodal signaling recruits the histone demethylase Jmjd3 to counteract polycomb-mediated repression at target genes

Abstract

Both intercellular signaling and epigenetic mechanisms regulate embryonic development, but it is unclear how they are integrated to establish and maintain lineage-specific gene expression programs. Here, we show that a key function of the developmentally essential Nodal-Smads2/3 (Smad2 and Smad3) signaling pathway is to recruit the histone demethylase Jmjd3 to target genes, thereby counteracting repression by Polycomb. Smads2/3 bound to Jmjd3 and recruited it to chromatin in a manner that was dependent on active Nodal signaling. Knockdown of Jmjd3 alone substantially reduced Nodal target gene expression, whereas in the absence of Polycomb, target loci were expressed independently of Nodal signaling. These data establish a role for Polycomb in imposing a dependency on Nodal signaling for the expression of target genes and reveal how developmental signaling integrates with epigenetic processes to control gene expression.

Fig. 1.

Polycomb represses Nodal upon inhibition of Smads2/3 signaling. (A) Diagram of the Nodal locus, indicating the positions of PCR primers used for ChIP analysis (red arrows) and RT-PCR (black arrows). (B and C) ChIP analysis of H3K27me3 (B) and Suz12 occupancy (C) at the Nodal locus in untreated ES cells (black bars), in ES cells 96 hours after withdrawal of LIF (gray bars), in ES cells treated with SB431542 (5 μM) for 96 hours in the presence or absence of LIF (red filled bars or red striped bars, respectively), and in B16 melanoma cells (blue bars). ChIP assays were performed on both wild-type (WT) ES cells (left panels) and Suz12^−/− ES cells (right panels). Error bars represent the SEM; n = 3 experiments. (D) Quantification of the abundances of Nodal, Nanog, and Oct4 mRNAs in WT ES cells (left panels) and Suz12^−/− ES cells (right panels) as percentages of the total amount of actin mRNA. Cells were treated as in (B) and (C). Error bars represent the SEM; n = 3 experiments. *P ≤ 0.01. ns, not significant.

Fig. 2.

Smads2/3 do not recruit core Trithorax components. (A to C) ChIP analysis of H3K4me3 (A) and the Trithorax component proteins Rbbp5 (B) and Ash1 (C) at the Nodal locus in untreated WT ES cells (black bars), in ES cells 96 hours after withdrawal of LIF (gray bars), in ES cells after treatment with SB431542 (5 μM) for 96 hours in the presence or absence of LIF (red filled bars and red striped bars, respectively), and in B16 melanoma cells (blue bars). Error bars represent the SEM; n = 3 experiments.

Fig. 3.

Nodal activates Smads2/3 to recruit Jmjd3 to the Nodal locus. (A) ChIP analysis of Jmjd3 at the Nodal locus in WT ES cells treated as described for Fig. 1. Error bars represent the SEM; n = 3 experiments. (B) Coimmunoprecipitation of activated Smads2/3 and Jmjd3 in lysates from untreated, WT ES cells (left set of panels). No coimmunoprecipitation was found in lysates from SB431542-treated cells (right set of panels). Samples immunoprecipitated (IP) with nonspecific IgG as a control or with antibodies against Jmjd3 were analyzed by Western blotting (WB) with antibodies against Smads2/3 (top panel) or Jmjd3 (lower panel). Data are representative of four independent experiments with similar results. (C) shRNA-mediated knockdown of Jmjd3. The abundances of Jmjd3 and Nodal mRNAs after treatment with control shRNA (gray bars) or Jmjd3-specific shRNA (white bars) are shown. The data represent the average from six clonally distinct colonies from three independent infections with lentiviral vectors, demonstrating a ~90% reduction in the abundance of Jmjd3. Error bars represent the SEM; n = 3 experiments.

Fig. 4.

Brachyury is a direct target of Smads2/3 signaling. (A) The Brachyury locus contains elements responsive to Wnt signaling in the 5′ URR. Positions of the primers used for ChIP analysis (red arrows) and RT-PCR (black arrows) are indicated. 3′UTR, 3′ untranslated region. (B) Abundance of Brachyury mRNA in WT ES cells (left panel) and Suz12^−/− ES cells (right panel). Cells were left untreated (black bars), treated with BIO (2 μM) for 48 hours (green bars), treated with SB431542 (5 μM) for 48 hours (green stippled bars), or treated with both BIO (2 μM) and SB431542 (5 μM) for 48 hours (green striped bars). Active (+) or inactive (−) Nodal and Wnt signaling as a consequence of these treatments is indicated under the graph. Error bars represent the SEM; n = 3 experiments. *P ≤0.01. (C and D) ChIP analysis of the binding of β-catenin (C) and Smads2/3 (D) at the Brachyury URR. WT ES cells were left untreated (black bars), treated with BIO alone (green bars), or treated with BIO and SB431542 (green striped bars). Cells were analyzed after 48 hours of treatment. Error bars represent the SEM; n = 3 experiments. (E) Coimmunoprecipitation of β-catenin and Smads2/3. WT ES cells were left untreated, treated with BIO alone, or treated with BIO and SB431542 for 48 hours. Samples immunoprecipitated with nonspecific IgG as a control or with antibodies against Smads2/3 were analyzed by Western blotting with antibodies against β-catenin (top panel) or Smads2/3 (lower panel). Data are representative of three independent experiments with similar results.

Fig. 5.

Nodal-Smads2/3 signaling counteracts Polycomb-mediated repression at Brachyury by recruiting Jmjd3. (A to C) ChIP analyses of Suz12 (A), H3K27me3 (B), and Jmjd3 (C) at the Brachyury locus. WT ES cells were left untreated (black bars), treated with BIO alone (green bars), or treated with BIO and SB431542 (green striped bars). Cells were analyzed after 48 hours. Error bars represent the SEM; n = 3 experiments. *P ≤ 0.01.

Fig. 6.

Model for the Jmjd3-dependent activation of Polycomb-repressed Smads2/3 target genes. Nodal signaling activates Smads2/3, which are recruited to target loci or are stabilized at target loci, or both, either by intrinsic transcription factors, such as Foxh1 for Nodal, or by signal-induced factors, such as Wnt-activated β-catenin for Brachyury. Smads2/3 then recruit Jmjd3 to chromatin, resulting in demethylation of H3K27me3. This initiates derepression, which involves the subsequent recruitment of Trithorax group complexes (TrxG) and the general transcriptional machinery (GTM), ultimately resulting in gene activation.