Dynamic Lysine Methylation on Histone H3 Defines the Regulatory Phase of Gene Transcription  Antonin Morillon, Nickoletta Karabetsou, Anitha Nair, Jane Mellor  Molecular Cell  Volume 18, Issue 6, Pages 723-734 (June 2005) DOI: 10.1016/j.molcel.2005.05.009 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 A Regulatory Phase Precedes Transcription Elongation at MET16 (A) Northern blot of total MET16 and 18S rRNA isolated from wild-type (wt) (BY4741) minutes after activation by transfer to Hc-Met from YPD (Y). (B) Indirect end-label analysis of chromatin at MET16 in wt incubated in YPD (Y) or Hc-Met for the times indicated. Chromatin was processed as in Morillon et al. (2003a) by using 150 U/ml micrococcal nuclease (MNase) and a secondary digest with EcoRI. The two Isw1-positioned nucleosomes (−1 and +1) are separated by a hypersensitive site (large asterisk), and the changes in position during induction are shown schematically. Triangle (down) represents the TATA region. (C and D) ChIP at MET16 using anti-HA antibodies to detect Rbp3-HA, anti-Ser5-P CTD (H14, Covance), anti-Ser2-P CTD (H5, Covance), or anti-CTD (8WG16, Covance) in a wt strain incubated in Y or Hc-Met for the times indicated. Real-time PCR was used to amplify regions corresponding to those shown at MET16. Each value is derived from three PCR reactions on an immunoprecipitation (IP) in duplicate (n = 3) × 2. Signals are expressed as a percentage of input. Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). The vertical broken line separates the regulatory and elongation phases. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Isw1 Controls RNAPII during the Regulatory Phase (A) Indirect end-label analysis of chromatin at MET16 in wt and isw1Δ strains incubated in Y or Hc-Met for the times indicated. See Figure 1 for details. (B) Northern blot of total MET16 and 18S rRNA isolated from wt and isw1Δ strains minutes after activation by transfer to Hc-Met from Y. (C and D) ChIP at MET16 using anti-myc antibodies (Sigma) to detect Isw1-myc or anti-Rpb1 antibodies (H224; Santa Cruz) to detect RNAPII in a wt strain ([C] and [D], top) or an isw1Δ strain ([D], bottom) incubated in Y or Hc-Met for the times indicated. Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). See Figure 1 for details. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Dynamic H3 Methylation Defines the Regulatory Phase ChIP profiles using antibodies with the specificities shown (all antibodies from Abcam) across MET16 in YPD and minutes after transfer to medium lacking methionine (−Met). Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). See Figure 1 for details. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Functionally Distinct States of K4 Methylation (A and B) ChIP using antibodies specific to (A) K4me1 (Abcam/Upstate), K4me2 (Abcam), K4me3 (Abcam), or K36me2 ([B]; Abcam) detected at MET16 in the yeast strains indicated cultured in Y or Hc-Met for 120 min in the strains indicated. For (A), n = 4 experiments, although for clarity error bars are not shown. For (B), error bars reflect the SD of the average obtained between different experiments (n = 2). See Figure 1 for details. (C) Western blots of total yeast protein isolated from cells grown in YPD by using antibody specific to K4me2 (Abcam), K4me3 (Abcam), or histone H3 (N20; Santa Cruz). (D) 6-azauracil (6AU) sensitivity drop test for strains with genotypes indicated transformed to uracil prototrophy with pRS316 grown on Hc-Uracil plates (3 days, 30°C) supplemented with 100 μg/ml of 6AU (Sigma) or 100 μg/ml 6AU and 100 μg/ml of guanine (control plate). (E and F) ChIP using anti-HA (Roche) in yeast expressing Rbp3-HA (E) or anti- Ser5P CTD (H14; Covance; [F]) at MET16 in the yeast strains indicated cultured in Y or Hc-Met for 30 min. Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). See Figure 1 for details. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Transient Isw1-myc Recruitment during the Regulatory Phase ChIP profiles of Isw1-myc at MET16 in wt or strain backgrounds indicated cultured in Y (A) after 120 min in Hc-Met (B) or minutes after activation at the promoter and 3′ region of MET16 (C). Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). See Figure 1 for details. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Transient HA-Esa1 Recruitment during the Regulatory Phase ChIP profiles of HA-Esa1 (A) and H4K8ac (C) at the MET16 promoter for strains indicated incubated in Y (0) or for the time indicated in Hc-Met. Error bars reflect the SD of the average obtained between different experiments (n = 2). See Figure 1 for details. (B) Western blot probed with anti-HA and antitubulin in strains of the genotypes indicated expressing HA-Esa1. (D) Sequential ChIP (double ChIP) at the MET16 promoter on chromatin prepared from the strains indicated after induction of transcription for the times indicated. The antibodies used for the first and second IPs are shown. See Supplemental Data for experimental details. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Esa1 Antagonizes the Negative Function of Isw1 Strains with genotypes indicated, cultured in YPD, were treated at 39°C for 60 min before transfer to fresh Y or Hc-Met for the times indicated, total RNA isolated and analyzed by northern blot (A), or the cells fixed and subject to ChIP to detect RNAPII (Y80; Santa Cruz) (B) or RNAPII phosphorylated on CTD at Ser5 (Covance; H14) (C). Error bars reflect the SD of the average signal obtained between different experiments (n = 2–4). See Figure 1 for details. Note that the timing of induction is altered at high temperature. Molecular Cell 2005 18, 723-734DOI: (10.1016/j.molcel.2005.05.009) Copyright © 2005 Elsevier Inc. Terms and Conditions