Volume 41, Issue 6, Pages 704-719 (March 2011) Chromatin-Associated Protein Kinase C-θ Regulates an Inducible Gene Expression Program and MicroRNAs in Human T Lymphocytes  Elissa L. Sutcliffe, Karen L. Bunting, Yi Qing He, Jasmine Li, Chansavath Phetsouphanh, Nabila Seddiki, Anjum Zafar, Elizabeth J. Hindmarsh, Christopher R. Parish, Anthony D. Kelleher, Russell L. McInnes, Toshiki Taya, Peter J. Milburn, Sudha Rao  Molecular Cell  Volume 41, Issue 6, Pages 704-719 (March 2011) DOI: 10.1016/j.molcel.2011.02.030 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 PKC-θ Associates with Active Chromatin in the Nucleus of Human T Cells (A) Immunoblot with anti-PKC-θ (top) and anti-Sp1 (bottom) antibodies on nuclear extracts prepared from either nonstimulated (NS) or stimulated (ST with P/I for 4 hr) Jurkat T cells. (B) Fluorescence microscopy on fixed whole cells and nuclei stained with DAPI and anti-integrin β1 antibody. (C) Fixed nuclei from Jurkat T cells ST stained with DAPI and anti-PKC-θ antibody with either anti-Pol II, anti-HP1α or anti-H3K9me2 antibody. (D) PKC ELISA-based kinase assays of nuclear and whole-cell extracts of NS and ST-treated Jurkat T cells. Recombinant active PKC (postitive control) and secondary alone (negative control). Data represent the mean ± SE of two independent experiments performed in duplicate. (E and F) Halfway ChIP of either NS or 4 hr P/I-treated Jurkat T cells with the anti-PKC-θ antibody. (–Ab) is in the absence of antibody. Immunoblot with anti-PKC-θ, anti-histone H3 and anti-histone H4 antibodies (E) or anti-Pol II, anti-Pol IIp and anti-histone H3K9ac antibodies (F). Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 PKC-θ Forms an Active Transcription Complex with Pol II and MSK-1 across Proximal Regulatory Regions of Inducible Genes (A) Sequential ChIP of NS or ST Jurkat T cells with anti-histone H3 antibody, then anti-PKC-θ, anti-PKC-θp, and anti-MSK-1 antibodies. Real-time PCR of CD69 (−0.15 Kb). (B) Sequential ChIP as for (A) with anti-PKC-θ followed by anti-MSK-1 antibody. (C and D) PKC-θ (C) and MSK-1 (D) ChIPs with Jurkat T cells stimulated for the times indicated. (E) ChIPs as above with anti-PKC-θp and primers for CD69 (−0.15 and +0.5 Kb). (F) Sequential ChIP as for (A) with anti-Pol II then anti-PKC-θ and anti-MSK-1. (G and H) ChIPs as above on ex vivo human CD4+ T cells with anti-PKC-θ (G) or anti-H3K9ac antibody on IL-2 (H) (−0.15 Kb). (I) Total RNA from CD4+ T cell subsets and TaqMan real-time PCR for IL-2. Data are expressed as arbitrary copy number normalized to GAPDH and representative of the mean ± standard error (SE) of two independent experiments. (J) Fixed nuclei from CD4+ T cell subsets stained as in Figure 1C. All ChIP data were calculated as n-fold enrichment ratio of immunoprecipitated DNA relative to no antibody control, normalized against total input DNA. NS ChIP enrichment was set to 1 for sequential ChIP. ChIP data with error bars are shown as the mean ± SE of two independent experiments, otherwise representative of at least two independent experiments performed in duplicate. See also Figure S1. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 PKC-θ Recruitment Is Dependent on Active Transcription Jurkat T cells NS, ST, and SW. Depicted schematically (A, top). TaqMan real-time PCR for CD69, TNF-α, and IFN-γ (A, bottom). Fold change (FC) relative to NS. Data show the mean ± SE of two independent experiments performed in duplicate. ChIPs on NS, ST and SW with anti-PKC-θ (B), anti-MSK-1 (C), anti-histone H2A.Z (D), and anti-Pol IIp (E) antibodies across CD69, TNF-α, and IFN-γ (−0.15 Kb). ChIP data are shown as the mean ± SE of two independent experiments performed in duplicate. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Recruitment of PKC-θ to Inducible Gene Promoters Is Required for 14-3-3ζ Occupancy (A and B) Halfway ChIP with anti-histone H3 (A) or anti-PKC-θ (B) antibody with NS or 4 hr P/I-treated Jurkat T cells and immunoblot with anti-14-3-3ζ. (–Ab) is in absence of antibody. (C) ChIPs on Jurkat T cells NS or stimulated with P/I for 4 or 8 hr across CD69 and TNF-α (−0.15 Kb). (D) Sequential ChIP as in Figure 2A, but with anti-PKC-θp and anti-14-3-3ζ antibody. (E) ChIPs on Jurkat T cells transfected with V, WT, or KR. Real-time PCR on DNA recovered with anti-PKC-θ, anti-14-3-3ζ, or anti-Pol II across CD69 and heparanase (−0.15 Kb). ChIP data are shown as the mean ± SE of two independent experiments performed in duplicate. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 PKC-θ Is a Prerequisite for Chromatin Accessibility on Inducible Immune Response Genes in T Cells, but Is Not Required for H3S10p on the CD69 Gene (A) H3S10p, PKC-θ, or MSK-1 ChIP on Jurkat T cells transfected with V, WT, or KR across CD69 (−0.15 Kb). (B and C) H3S10p (B) and MSK-1 (C) ChIPs on Jurkat T cells untreated or pretreated with H89 across CD69 (−0.15 Kb). Average percentage inhibition values shown above graphs. (D and E) H3S10p (D) and PKC-θ (E) ChIPs repeated as above but with Rottlerin. (F) CHART on Jurkat nuclei from NS or 4 hr P/I left untreated or pretreated with Rottlerin (ROTT) or H89 across CD69 and TNF-α (−0.15 Kb). Data are graphed as the percentage change in accessibility and plotted as the mean ± SE of two independent experiments performed in duplicate. (G) Jurkat T cells transfected with scrambled RNAi (mock) or PKC-θ RNAi and subsequently left untreated (NS) or stimulated for 2 hr with P/I (ST). ChIP with PKC-θ, MSK-1, Pol II, 14-3-3ζ, and LSD1 antibodies across IL-2 (−0.15 Kb). (H) MSK-1 RNAi as in (G). ChIP data with error bars are shown as the mean ± SE of two independent experiments, otherwise representative of at least two individual experiments performed in duplicate. (I and J) IL-2 TaqMan real-time PCR for PKC-θ RNAi-treated cells (I) and MSK-1 RNAi-treated cells (J) or mock samples. Data expressed as arbitrary copy number normalized to GAPDH and representative of the mean ± SE of two independent experiments performed in duplicate. See also Figure S2, S3, and S4. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Direct Targets of PKC-θ Identified by Genome-wide ChIP-on-chip (A) Pie chart of PKC-θp bound genes (ratio > 5-fold, p < 0.05, n = 2). (B) The fifty most significant (p < 0.01, n = 2) bound PKC-θ targeted genes within transcribed (top) and promoter (bottom) regions. (C) Table of most significant biological and molecular functions of PKC-θ targeted genes from (A). (D) Correlation between PKC-θ with Pol II-bound genes from duplicate microarray experiments. (E) PKC-θ targeted microRNAs (p < 0.05, n = 2) either PKC-θ bound or PKC-θ & Pol II bound. (F) Phase-contrast microscopy of MCF-7 cells transfected with mock or WT and left untreated or treated for 24 hr with TNF-α. (G) MCF-7 cells transfected with mock or WT and analyzed by the wound-healing assay. Images were taken at zero hr (red line) and after 60 hr (green line). See also Tables S1 and S2 and Figure S5. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Functional Analysis of a Subset of MicroRNAs Targeted by PKC-θ in T Cells (A) TaqMan microRNA complementary DNA (miR-9, miR-200c, and miR-183) from resting and activated Jurkat T cells for time points indicated. (B) As in (A), but in the presence or absence of Rottlerin (ROTT) or H89. (C) As above from Jurkat T cells transfected with V or WT. Data are expressed as arbitrary copy number normalized to RNU6B and are representative of the mean ± SE of three independent experiments. (D) Synthetic miR-200c or scrambled Pre-miR (mock) transfected into Jurkat T cells and left untreated (NS) or stimulated for 2 hr (ST) with P/I. TaqMan real-time PCR for CD69, IL-2, TNF-α (TNF), IFN-γ (IFN), and heparanase (HPSE). (E) Zeb1 real-time PCR on samples as in (D). (F) Repeated as for (D) with synthetic miR-9. Data are expressed as arbitrary copy number normalized to GAPDH and are representative of the mean ± SE of two independent experiments performed in duplicate. (G) Zeb1, PKC-θ, and H3K9me2 ChIP on Jurkat T cells transfected with miR-200c or mock and stimulated 2 hr P/I. ChIP data are shown as the mean ± SE of two independent experiments performed in duplicate. See also Figure S6. Molecular Cell 2011 41, 704-719DOI: (10.1016/j.molcel.2011.02.030) Copyright © 2011 Elsevier Inc. Terms and Conditions