Volume 19, Issue 3, Pages 381-391 (August 2005) Histone H3 Lysine 9 Methylation and HP1γ Are Associated with Transcription Elongation through Mammalian Chromatin  Christopher R. Vakoc, Sean A. Mandat, Benjamin A. Olenchock, Gerd A. Blobel  Molecular Cell  Volume 19, Issue 3, Pages 381-391 (August 2005) DOI: 10.1016/j.molcel.2005.06.011 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Levels of H3K9 Trimethylation Correlate with Transcriptional Activity in Mammalian Chromatin (A) ChIP assay with three anti-H3K9-trimethyl antibodies and control IgG in G1E cells before or after differentiation induction with tamoxifen (OHT) for 21 hr. PCR primers were specific for promoters and transcribed regions in genes that are activated (β-major, band3), repressed (GATA-2, c-myc), or unchanged (GAPDH). Major satellite repeats served as positive controls. Results are the average of two or three independent experiments. Error bars denote standard deviation. IgG signals that appear absent are less than 0.001. (B) ChIP with anti-dimethyl H3K9 at the β-major gene, the GATA-2 gene, and major satellite repeats. (C and D) ChIP with anti-trimethyl H3K9 (ab8898) and anti-pol II in primary erythroid cells and primary T cells, respectively. Molecular Cell 2005 19, 381-391DOI: (10.1016/j.molcel.2005.06.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 The Distribution of H3K9 Methylation in Mammalian Chromatin Is Distinct from Other Histone Trimethyl-Lysine Marks (A) ChIP analysis in G1E cells with anti-trimethyl H3K9 (ab8898), anti-trimethyl H3K27 (07-449), and anti-trimethyl H4K20 (07-463) at the β-major gene and, as controls, major satellite repeats and MYT1. (B) ChIP analysis of the IL-2 gene with anti-trimethyl H3K4 (07-473) and anti-trimethyl H3K9 (ab8898) before and after T cell activation. Position of primer pairs are indicated below. (C) Trimethyl HK9-specific antibodies (ab8898, ab1186, and 07-442) fail to react with transcriptionally active chromatin in S. cerevisiae. ChIP analysis with chromatin from cells grown in glucose- or galactose-containing media. A trimethyl H3K4 antibody served as a positive control. Primers were specific for the GAL10 5′-transcribed region. ChIP analysis of the active β-major gene in G1E cells was performed in parallel for comparison. (D) Double ChIP of the β-major globin gene in differentiated G1E cells with anti-trimethyl H3K9 (ab1186) antibody for the first immunoprecipitation (1st IP) followed by elution and reimmunoprecipitation with the indicated second antibody (IgG or anti-trimethyl H3K4 07-473). The graph shown is the average of two independent experiments. Error bars represent standard deviation. Molecular Cell 2005 19, 381-391DOI: (10.1016/j.molcel.2005.06.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 HP1γ Is Associated with Active Genes ChIP Analysis with Anti-HP1γ and Anti-trimethyl H3K9: (A), at the c-kit gene in G1E cells (07-442) (B), at the IL-2 gene in primary T cells (ab8898) (C), at the β-major gene in primary proerythroblasts (ab8898). Position of primer pairs is indicated below. The graphs shown are the averages of three (A) or two (B and C) independent experiments. Error bars represent standard deviation. (D) HP1γ is physically associated with RNA polymerase II. Immunoprecipitation with anti-HP1γ antibodies or nonimmune IgG (IgG) followed by Western blot with antibodies raised against unphosphorylated RNA polymerase II (8wg16), RNA polymerase II phosphorylated on serine 5 (H14), or on serine 2 (H5). 10% of unprecipitated material was analyzed in parallel (input). Of note, the H5 antibody may recognize serine 5 CTD phosphorylation in addition to serine 2 phosphorylation (Jones et al., 2004). Molecular Cell 2005 19, 381-391DOI: (10.1016/j.molcel.2005.06.011) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 H3K9 Trimethylation and HP1γ Association Is Dynamic and Depends on Elongating RNA Polymerase II (A) Time-course ChIP of the transcribed region of the GATA-2 gene with anti-trimethyl H3K9 (ab8898) and HP1γ at indicated time points after tamoxifen treatment of G1E cells. Result shown is a representative experiment. (B) ChIP with anti-trimethyl H3K9 (ab1186 and ab8898), anti-HP1γ, and anti-RNA polymerase II at the induced β-major gene after DRB treatment in MEL cells. The graph shown is the average of two independent experiments. Error bars represent standard deviation. Molecular Cell 2005 19, 381-391DOI: (10.1016/j.molcel.2005.06.011) Copyright © 2005 Elsevier Inc. Terms and Conditions