Research ArticleCancer

Transcriptional repressor REST drives lineage stage–specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma

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Science Signaling  22 Jan 2019:
Vol. 12, Issue 565, eaan8680
DOI: 10.1126/scisignal.aan8680
  • Fig. 1 Clinical characteristics associated with gene expression profiles.

    (A) Hierarchical clustering analysis of SHH MB patient samples using gene expression. Hierarchical clustering assay identified six distinct clusters based on expression profiles of neuronal differentiation markers (www.ncbi.nlm.nih.gov/geo; dataset GSE85217). The blue to red color scale indicates the expression level (Z score). The key, clinical information [subtype, age, gender, and metastasis (mets) status] regarding patient samples, is provided beneath. NA, not available. (B) Gene expression profiles measured by microarray in six clusters. Each dot corresponds to one individual patient. Data show individual variability and means ± SD. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Overall survival (OS) of six clusters in patients with SHH MB (P value; log-rank Mantel-Cox test). (D) Hierarchical clustering analysis of SHH MB patient samples using gene expression. Hierarchical clustering assay identified five distinct clusters based on expression profiles of REST target genes. The blue to red color scale indicates the expression level based on Z score. The clinical information (subtype, age, gender, and metastasis status) regarding patient samples was shown in the bottom panel. (E) Overall survival of five clusters in patients with SHH MB (P value; log-rank Mantel-Cox test).

  • Fig. 2 Generation and characterization of a novel genetically engineered mouse model with increased REST expression in CGNPs.

    (A) Schema to describe generation of a conditional RESTTG mouse model. Primers targeting the 6× His/3× hemagglutinin (HA) tag and the 5′ end of the hREST complementary DNA (cDNA) were used for genotyping. Agarose gel of PCR product from WT, line 1 (L1), and line 2 (L2) is shown. (B and C) CGNPs harvested from p8 pups that received tamoxifen (TX) injections on p2, p3, and p4 were cultured for up to 15 days. Cells were collected and analyzed for (B) REST Tg mRNA expression by qRT-PCR analyses and (C) REST protein abundance by Western blotting. Data are means ± SD of three individual pups. Representative Westerns are shown. a.u., arbitrary units. (D) Neuronal differentiation in CGNPs was evaluated by qRT-PCR measurement of Syn1 mRNA expression. Data are means ± SD of three individual pups. (E) H&E staining of brain tissue from p8 WT (n = 3) or RESTTG (n = 3) pups injected with tamoxifen. (F and G) Sections were analyzed by IHC for (F) REST and (G) NeuN expression using specific antibodies to assess protein expression in CGNPs in the EGL and granule neurons of the IGL (n = 3). For (B) and (C), bars represent means ± SD of fold changes relative to WT controls. P values for qRT-PCR were calculated by paired two-tailed t test of ΔCp values: Significance is indicated as ns. *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001. Densitometry was obtained using Image Lab software. Scale bars, 50 μm (×10; E) and 20 μm (×40; F and G).

  • Fig. 3 Increased REST abundance alters the kinetics and penetrance of SHH-driven MB development.

    (A) Schema to describe generation of Ptch+/−/RESTTG mice. (B) Survival of WT (n = 45), RESTTG (n = 23), Ptch+/− (n = 31), and Ptch+/−/RESTTG (n = 13) mice after tamoxifen administration to induce REST Tg expression in RESTTG and Ptch+/−/RESTTG mice was assessed using Kaplan-Meier analysis. (C) Representative gross images of brains from p40 WT, RESTTG, Ptch+/−, and Ptch+/−/RESTTG mice are shown (n = 3). The red oval indicates cerebellar tumor in p40 Ptch+/−/RESTTG mice. Scale bar, 2 mm. (D) H&E staining of brain tissue from Ptch+/− and Ptch+/−/RESTTG animals (n = 3) is shown. (E) Immunodeficient mice bearing cerebellar xenografts of human DAOY cells expressing endogenous REST (n = 9) or hREST (DAOY-REST; n = 11) were monitored for tumor growth by bioluminescent imaging (BLI). Images of representative mice and relative flux for the entire cohort are shown before euthanasia on day 47 due to tumor burden. P values were obtained using Student’s t test. H&E staining of brain tissue from (F) DAOY and DAOY-REST xenografts (n = 3) and (G) low-REST and high-REST PDOX (n = 3) are shown. Scale bars, 50 μm (×10; D, F, and G).

  • Fig. 4 REST represses the expression of the gene encoding PTCH1.

    (A to C) Cerebellar sections of (A) tumor-bearing Ptch+/− and Ptch+/−/RESTTG mice (n = 3), (B) DAOY and DAOY-REST xenografts (n = 3), and (C) human SHH subgroup PDOX (n = 3) were analyzed by IHC for REST, PTCH1, and Ki-67 expression using specific antibodies. (D) PTCH1 and GLI1 mRNA expression profiles in SHH-type MB patient samples measured by microarray. Hierarchical clustering based on expression of neuronal differentiation markers divided the SHH-type MB patient samples into six distinct clusters. Each dot corresponds to one individual patient. (E) Ptch1 and Gli1 mRNA expression was measured in CGNPs from WT (white bars) and RESTTG (gray bars) mice after culturing in proliferation (prolif) or differentiation (diff) media. Data are means ± SD from three (WT) or two (RESTTG) pups. Graph shows fold change compared to WT proliferating controls. Scale bars, 20 μm (×40). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns by paired two-tailed t test of ΔCp values.

  • Fig. 5 Transcription factor binding and resulting histone modification changes leads to Ptch1 loss of heterozygosity.

    (A) Schematic representation of mPtch1 promoter with RE1 site and adjacent Gli1 binding site are shown. REST and GLI1 binding to RE1 site on mPtch1 promoter measured by ChIP-qPCR in WT and RESTTG proliferating (prolif) and differentiating CGNPs. Data are represented as fold change over IgG (n = 3 for WT and n = 6 for RESTTG). (B) Enrichment of H3Ac over IgG at mPtch1 promoter in proliferating and differentiating (diff) CGNPs. Bars represent fold change of H3Ac over IgG in the samples (n = 3 for WT and n = 6 for RESTTG). (C) Enrichment of H3K4-me3 evaluated by ChIP-qPCR at the mPtch1 TSS site in WT and RESTTG proliferating and differentiating CGNPs (n = 3). (D) Enrichment of mono-, di-, and trimethylation at histone H3 Lys9 (H3K9-me1, H3K9-me2, and H3K9-me3) evaluated by ChIP-qPCR at the mPtch1 RE1 site in WT and RESTTG proliferating and differentiating CGNPs (n = 3). (E) Arrb1 mRNA expression was measured in WT and RESTTG CGNPs after culturing with proliferation or differentiation media. Data are means ± SD from three (WT) or two (RESTTG) pups. Graph shows fold change compared to WT proliferating controls. (F) ARRB1 mRNA expression profile in human SHH-type MB patient samples measured by microarray. Hierarchical clustering based on expression of neuronal differentiation markers divided the SHH-type MB patient samples into six distinct clusters. Each dot corresponds to one individual patient. (G) Enrichment of H3K4-me3 at hPTCH1 TSS and enrichment of other histone modifications at hPTCH1 RE1 site using ChIP-qPCR from a high-REST PDOX sample. (H) DAOY MB cell line treated with either the HDAC inhibitor MS275 (0.625 to 5 μM) or the G9a inhibitor UNC0638 (0.5 to 5 μM), or a combination of both, and MTT assay was performed at 48 hours after treatment to measure cell viability. Data are means ± SD of independent triplicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns by two-way analysis of variance (ANOVA) in GraphPad and either Dunnett’s method (A to D) or Sidak or Tukey’s test (H) for multiple comparisons or by paired two-tailed t test of ΔCp values (E).

  • Fig. 6 Increased REST expression in the context of constitutive SHH signaling results in increased AKT activation.

    (A to C) IHC was performed with phosphorylated (p)–AKTSer473 or PTEN-specific antibodies in (A) Ptch+/− and Ptch+/−/RESTTG tumors (n = 3), (B) DAOY and DAOY-REST xenografts (n = 3), and (C) human SHH subgroup PDOX (n = 3). Scale bars, 20 μm (×40). (D) PTEN mRNA expression profile was measured by microarray. Hierarchical clustering based on expression levels of neuronal differentiation markers divided the SHH MB patient samples into six distinct clusters. Each dot corresponds to one individual patient. (E) Pten mRNA expression was measured in WT and RESTTG CGNPs after culturing in proliferation or differentiation media. Graph shows fold change compared to WT proliferating controls. Data are means ± SD from three (WT) or two (RESTTG) pups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns by paired two-tailed t test of ΔCp values. (F and G) Western blot analysis of p-AKTSer473 and total AKT abundance in WT and RESTTG CGNPs after culturing with proliferation or differentiation media unperturbed (F) or after 5 hours of treatment with MK2206 at 1 or 5 μM (G). Histone H3 served as loading control. Images are representative of n = 2 independent experiments.

  • Fig. 7 REST-dependent AKT phosphorylation in MB cell lines.

    (A) Western blotting for basal protein abundance of REST, p-AKTSer473, total AKT, and histone H3 (control) in DAOY, UW426, and UW228 cells. Representative blots are shown; long/short indicates exposure times. (B and C) Western blotting for total and p-AKTSer473 protein abundance after either shRNA-mediated REST knockdown in UW228 and DAOY cells (B) or REST overexpression in DAOY cells (C). Blots are representative of three experiments. (D) MTT assay–derived proliferation of UW228 and DAOY cells treated with various doses of MK2206 for 24, 48, or 72 hours. Data are means ± SD of three independent assays. (ns), *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001. (E) Western blotting for abundance of p-AKTSer473, total AKT, cleaved caspase-3, cleaved PARP, and histone H3 (loading control) to assess induction of apoptosis after treatment of UW228 cells with MK2206 (5 μM) for 12 or 24 hours. Blots are representative of three experiments.

  • Fig. 8 Increased REST expression drives progression of SHH-driven MBs.

    (A) Schematic representation of tumor characteristics obtained from CGNPs with perturbed SHH signaling in the presence or absence of increased REST expression. (B) Graphical representation of REST-dependent chromatin remodeling of the Ptch1 gene in WT or RESTTG CGNPs during proliferation and differentiation. (C) Model depicting REST regulation of AKT signaling in SHH-driven MBs.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/565/eaan8680/DC1

    Fig. S1. Clinical characteristics.

    Fig. S2. Cerebellar architecture and protein abundance in age-matched RESTTG and WT mice.

    Fig. S3. Increased REST expression contributes to infiltrative SHH-driven MB development.

    Fig. S4. Differential regulation of PTCH1 expression by REST in MB tumors and CGNPs.

    Fig. S5. REST and SUV39H1 do not coimmunoprecipitate in DAOY cells.

    Fig. S6. Increased REST expression increases AKT phosphorylation at Ser473.

    Fig. S7. REST-dependent AKT phosphorylation in cell lines.

    Table S1. Synergy between MS275 and UNC0638.

    Table S2. IC50 values for UW228 and DAOY cells treated with MK2206.

    Table S3. Antibodies for IHC, qChIP (quantitative ChIP), and Western blotting assays.

    Table S4. Primers for qChIP and qRT-PCR assays.

  • This PDF file includes:

    • Fig. S1. Clinical characteristics.
    • Fig. S2. Cerebellar architecture and protein abundance in age-matched RESTTG and WT mice.
    • Fig. S3. Increased REST expression contributes to infiltrative SHH-driven MB development.
    • Fig. S4. Differential regulation of PTCH1 expression by REST in MB tumors and CGNPs.
    • Fig. S5. REST and SUV39H1 do not coimmunoprecipitate in DAOY cells.
    • Fig. S6. Increased REST expression increases AKT phosphorylation at Ser473.
    • Fig. S7. REST-dependent AKT phosphorylation in cell lines.
    • Table S1. Synergy between MS275 and UNC0638.
    • Table S2. IC50 values for UW228 and DAOY cells treated with MK2206.
    • Table S3. Antibodies for IHC, qChIP (quantitative ChIP), and Western blotting assays.
    • Table S4. Primers for qChIP and qRT-PCR assays.

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