1. Volume 8, Issue 2, Pages 473-479 (August 2001)
Highly Specific Antibodies Determine Histone Acetylation Site Usage in Yeast Heterochromatin and Euchromatin 
Noriyuki Suka, Yuko Suka, Andrew A Carmen, Jiansheng Wu, Michael Grunstein 
Molecular Cell 
Volume 8, Issue 2, Pages (August 2001)
DOI: /S (01)00301-X

2. Figure 1
Antibodies Specific to a Site of Acetylation in the ELISA Are Not Necessarily Specific in Chr-IP
α172 was generated using tetracetylated histone H4 N-terminal peptide. (A) Competition is demonstrated for singly acetylated peptides against fully acetylated peptide, bound to the microtiter plate in the ELISA. Peptide sequences used for competition are described in Experimental Procedures. As shown, α172 is specific against the singly acetylated peptide (K5-Ac) in the ELISA since only that peptide strongly inhibited antiserum binding to the fully acetylated peptide.
(B) Chr-IP assays were performed using chromatin from rpd3Δ strains that contained either wild-type (WT, NSY104) or a substitution mutant of K5 to R in H4 (K5R, NSY105). Multiplex PCR was done with primer pairs directed against INO1, SPS2, and IME2 promoter regions and Chr VI-R telomeric (TEL) regions. Separated band intensity was quantitated at INO1 relative to TEL (average of two independent experiments) as measured by the PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA)
Molecular Cell 2001 8, DOI: ( /S (01)00301-X)

3. Figure 2
Histone Substitution Mutations Are Used to Determine the Specificity of Antibodies to Individual Acetylation Sites
Shown are Chr-IP/multiplex PCR using strains containing a disruption of RPD3 containing the WT histone or histone substitution designated and its quantitation at INO1 (average of two Chr-IP). The sera found to be specific in ELISA and against the appropriate histone mutation were tested as shown here.
(A) Histone H4-specific antibodies and Chr-IP tests. The strains used are NSY104 (WT), NSY105 (K5R), NSY106 (K8R), NSY107 (K12R) and NSY108 (K16R).
(B) H3-specific antibodies and Chr-IP. The strains used are NSY115 (WT), NSY119 (K9R), NSY120 (K14R), NSY130 (K18R), NSY131 (K23R), and NSY132 (K27R).
(C) H2B-specific antibodies and Chr-IP. The strains used are NSY185 (WT), NSY188 (K11R), and NSY189 (K16R).
(D) H2A-specific antibody and Chr-IP. The strains used are NSY184 (WT), NSY186 (K4R), and NSY187 (K7R). In each Chr-IP, a mutation in the site of interest reduced to near background level the amount of immunoprecipitated INO1 and IME2 promoter DNA as assayed by PCR
Molecular Cell 2001 8, DOI: ( /S (01)00301-X)

4. Figure 3
All Histone Sites of Acetylation Are Hypoacetylated at Heterochromatin
sir3Δ causes increased acetylation of all sites examined in all four core histones at HML, HMR, and TEL (telomeric) heterochromatin. Chr-IP was performed using antibodies against acetylated lysines of histones as shown above each lane (A, H4; B, H3; C, H2B; D, H2A lysine sites). Chr-IP were performed on strains that were either AYH2.8 wild-type (+) or AYH2.45 mutant (−) for SIR3 (Hecht et al., 1996). Multiplex PCR was done with primer pairs directed against HMLα, SPS2, MATa, HMRa, and TEL. Below each multiplex PCR, quantitation (average of four Chr-IP) shows the increase in acetylation level at the HMLα (L), HMRa (R), and telomeric regions (T) relative to MATa. Black bar, wild-type; gray bar, sir3Δ
Molecular Cell 2001 8, DOI: ( /S (01)00301-X)

5. Figure 4
Histone Sites Modified by RPD3, GCN5, and ESA1 at the INO1 Promoter
(A) RPD3 is responsible for strongly deacetylating all core histone sites with the exception of H4 K16 at the INO1 promoter. GCN5 is responsible for strongly acetylating H2B and H3 sites with the exception of H3 K14. Chr-IP was performed using wild-type (YDS21U [WT]), SRYR39 (rpd3Δ), NSY223 (gcn5Δ), and NSY227 (rpd3Δgcn5Δ) strains. Illustrated are the quantitations of multiplex PCR (data not shown) for the INO1 promoter relative to TEL (average of three Chr-IP experiments) as described in the Figure 1 legend.
(B) ESA1 is responsible for acetylating H2A, H2B, and H4 sites with the exception of H4 K16. Chr-IP was performed from wild-type (LPY3431 [WT]), NSY164 (rpd3Δ), LPY3430 (esa1ts), and NSY165 (rpd3Δesa1ts) strains grown at 37°C for 4 hr. Shown are the quantitations of INO1 relative to TEL (average of three independent Chr-IP experiments) obtained from multiplex PCR
Molecular Cell 2001 8, DOI: ( /S (01)00301-X)