Research
Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation
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* Corresponding author: Susanna F Greer sgreer@gsu.edu
1 Division of Molecular Genetics and Biochemistry, Georgia State University, Atlanta, Georgia, USA
2 Division of Cellular and Molecular Biology and Physiology, Georgia State University, Atlanta, Georgia, USA
3 Division of Biotechnology, Department of Biology, Georgia State University, Atlanta, Georgia, USA
Epigenetics & Chromatin 2010, 3:5 doi:10.1186/1756-8935-3-5
Published: 4 February 2010Additional files
Additional file 1:
Supplemental Figure 1. IFN-γ inducible CIITA promoter IV (pIV) transcription drives expression of MHC-II. Before IFN-γ stimulation, both MHC-II and CIITA pIV exhibit low to moderate acetylation of histone H3 and H4 and are occupied by ubiquitiously expressed factors. MHC-II is bound by an enhanceosome complex of nuclear factor Y (NFY), regulatory factor X (RFX) and CREB, and pIV is bound in a highly conserved E-box by upstream stimulating factor (USF)-1 and c-Myc. Upon stimulation with the pro-inflammatory cytokine IFN-γ, the JAK/STAT1 pathway is triggered, leading to enhanced pIV acetylation and methylation, to rapid recruitment of the STAT1 homodimer to the pIV GAS element and to IFN response factors 1 and 2 (IRF1/2) binding to the pIV IRE. Once expressed, CIITA binds each component of the enhanceosome complex and recruits basal transcriptional components to initiate the switch to an elongation complex.
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Additional file 2:
Supplemental Figure 2. Common MLL/COMPASS subunits associate with the GAPDH promoter. (a-c) ChIP assays were carried out in HeLa cells stimulated with IFN-γ for 0 to 18 hours. Lysates were immunoprecipitated with control or endogenous (a) WDR5, (b) Ash2L or (c) RbBP5 antibody. Associated DNA was isolated and analyzed via real-time PCR using primers and probe spanning the GAPDH promoter. Data are presented as percentage input. Values represent mean ± SEM of (n = 3) independent experiments. IgG isotype control values were 0.001 ± 0.0005
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Additional file 3:
Supplemental Figure 3. Knockdown of a common MLL/COMPASS subunit decreases H3K4me3 but not H3K18ac at the GAPDH promoter. (a, b) HeLa cells transfected with scrambled control or WDR5-specific siRNA were stimulated with IFN-γ and subjected to ChIP assay. Lysates were immunoprecipitated with control, (a) endogenous H3K4me3 or (b) endogenous H3K18ac antibody. Associated DNA was isolated and analyzed via real-time PCR as described in Figure 1, using primers and probes specific for the GAPDH promoter. Data are presented as percentage input. IgG Isotype control values were 0.1 ± 0.05. Values represent mean ± SEM of (n = 2-3) independent experiments.
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Additional file 4:
Supplemental Figure 4. Neither IFN-γ stimulation nor siRNA transfection affect levels of histone H3. (a, b) HeLa cells stimulated with (a) IFN-γ or (b) transfected with scrambled control or WDR5-specific siRNA were subjected to ChIP assay. Lysates were immunoprecipitated with control or endogenous H3 antibody. Associated DNA was isolated and analyzed via real-time PCR as described in Figure 2 using primers and probes specific for CIITA pIV. Data are presented as percentage input. Values represent mean ± SEM of (n = 2-3) independent experiments.
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