1. Bromodomains Mediate an Acetyl-Histone Encoded Antisilencing Function at Heterochromatin Boundaries 
Andreas G. Ladurner, Carla Inouye, Rajan Jain, Robert Tjian 
Molecular Cell 
Volume 11, Issue 2, Pages (February 2003)
DOI: /S (03)

2. Figure 1
Recognition of Acetylated Histones by the Double Bromodomains of Yeast Bdf1
(A) ITC profile for the binding of tetraacetylated histone H4 peptide (1-22) to the Bdf1 double bromodomain (DBD) module.
(B) GST-pull-down assays of endogenous hyperacetylated core histones. Lane 1, input (hyperacetylated core histones); lane 2, histones bound to GST-Bdf1 DBD at 50 mM KCl; lane 3, unbound fraction; lane 4, histones bound after six washes at 500 mM KCl.
(C) Histone H4 peptide competition assays. Hyperacetylated H3/H4 tetramers (lane 1), immobilized on GST-Bdf1 DBD beads (lane 2), in the presence of 0.5 mM tetraacetylated (lane 3) or unacetylated (lane 4) H4 peptide.
(D) Schematic representation of the acetyl-lysine binding pocket of the Bdf1 bromodomains, highlighting the mutated Bdf1 tyrosine residue(s).
(E) Binding of histones to the Mut-Bdf1 mutant. Unbound fraction (Sup) and bound fraction (B) from the interaction of GST-Bdf1 (YF186/YF353) DBD with hyperacetylated histones (cf. lanes 2 and 4 in [B]).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Changes in Gene Expression upon Deletion of BDF1 and upon the Mutation of Bdf1 Residues YF186/YF353
(A) DNA microarray measurements of RNA levels in BDF mutants. Select downregulated (red arrow) and upregulated (green arrow) promoters.
(B) RNase protection assays for select yeast promoters in BDF mutants confirm the overall changes measured by DNA microarray experiments.
(C) Correlation between fold change in the ΔBDF1 deletion strain and in the Mut-BDF1 double point mutant. Except for one open reading frame, 252 genes are either upregulated or downregulated consistently in both ΔBDF1 and Mut-BDF1. The numbers in brackets indicate the proportion of affected genes relative to the total theoretical number of yeast genes.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 2
Changes in Gene Expression upon Deletion of BDF1 and upon the Mutation of Bdf1 Residues YF186/YF353
(A) DNA microarray measurements of RNA levels in BDF mutants. Select downregulated (red arrow) and upregulated (green arrow) promoters.
(B) RNase protection assays for select yeast promoters in BDF mutants confirm the overall changes measured by DNA microarray experiments.
(C) Correlation between fold change in the ΔBDF1 deletion strain and in the Mut-BDF1 double point mutant. Except for one open reading frame, 252 genes are either upregulated or downregulated consistently in both ΔBDF1 and Mut-BDF1. The numbers in brackets indicate the proportion of affected genes relative to the total theoretical number of yeast genes.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 3
Association of Wild-Type and Mutant Bdf1 Protein with Chromatin
(A) Western blot and coimmunoprecipitations of wild-type (WT) and mutant YF186/YF353 Bdf1 (Mut-Bdf1) protein expressed in a chromosomal ΔBDF1/ΔBDF2 knockout yeast strain. Bdf1 protein immunoprecipitated and probed with polyclonal α-Bdf1 antibody.
(B) Association of wild-type and acetyl-histone binding deficient mutant Bdf1 protein with select euchromatic promoters, detected by chromatin immunopurification (ChIP) experiments.
(C) Association of Bdf1 and TFIID-subunit Tsm1 (TAFX/TAF2) with chromatin surrounding the ADR1 gene in a wild-type yeast strain (WT). Gene regions probed by ChIP correspond to nucleotides −400 to 0, +200 to +600, and to relative to the translation start sites of the ADR1 and RMA1 genes.
(D) Association of Bdf1 and TFIID-subunit Tsm1 (TAFX/TAF2) with chromatin surrounding the RMA1 gene in a wild-type yeast strain (WT), in a histone H4-tail deletion mutant (ΔH4-tail), and in ΔBDF1. Gene regions were probed as in (C).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 4
Telomere-Proximal Bias of Gene Downregulation in ΔBDF1 and in the Acetyl-Histone Binding Defective Bdf1 Mutant
Open reading frames (ORFs) are ordered according to their positions along each chromosome, beginning at the centromere (gray circle, left) and ending at the telomere (yellow circle, right). Downregulated ORFs are shown in red, upregulated ORFs in green.
(A) Chromosomal display of changes in gene expression for chromosomes with telomeres not containing Y′ elements.
(B) Chromosomal changes in gene expression at telomeres containing Y′ elements are slightly different, consistent with their altered chromatin structure (Pryde and Louis, 1999).
(C) Correspondence between ΔBDF1 and YF186/YF353 Bdf1 mutant strains in the telomere-proximal downregulation of ORFs on the right arm of chromosome I. Average fold changes were used in the red color-coding for each ORF to highlight the average downregulation caused by each mutation. Shown in blue is the corresponding downregulation for PHO11 as determined by RNase protection assays.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 5
Bias of Gene Downregulation in BDF1 Deletion Mutants at Telomeric Heterochromatin-Euchromatin Boundaries
(A) Telomere-proximal genes affected by ΔBDF1. ORFs that were consistently downregulated or upregulated more than 2-fold were counted at 10 kb intervals for all the 36 S. cerevisiae telomeres (starting from the telomere). χ2 values for downregulated genes versus expected genes for each 10 kb interval are indicated and reflect the telomere-proximal bias of gene downregulation.
(B) ChIP of Sir3 at subtelomeric PHO11 promoter and actin control in rescued wild-type BDF1 (WT) and Mut-BDF1 yeast strains. Lanes 1 and 2, input; lanes 3 and 4, α-Sir3 immunoprecipitates; lanes 1 and 3, WT; lanes 2 and 4, Mut.
(C) Sir3 and Bdf1 association in the telomere-proximal region of chromosome V R. DNA from WT BDF1 and Mut-BDF1 rescue experiments was probed at 2.5 kb intervals starting from the telomere. For Bdf1 ChIPs only the data from wild-type rescue experiments is shown. Lanes 1, 2, and 5, input; lanes 3 and 4, α-Sir3 ChIP; lane 6, α-Bdf1 ChIP; lanes 1, 3, 5, and 6, WT; lanes 2 and 4, Mut.
(D) Quantification of α-Sir3 ChIP signals from independent experiments. The amount of signal from the immunoprecipitations is expressed as a percentage relative to the diluted input material. α-Sir3 ChIP signal in WT-BDF1 and Mut-BDF1 rescue strains relative to the distance from the telomere (n = 4).
(E) Quantification of α-Bdf1 ChIP signals in WT-BDF1 rescue strain, relative to the distance from the telomere (n = 4).
Molecular Cell  , DOI: ( /S (03) )

8. Figure 6
Histone H4 Tetraacetylation at Heterochromatin Boundaries, Sir3/Bdf1 Association at the HML Mating Locus, and Spreading of Bdf1 toward the Telomere in a SIR3-Deletion Strain
(A) Determination of histone H4 acetylation close to the telomere in wild-type rescued WT-BDF1 and Mut-BDF1 rescue strains (n = 4).
(B) Bdf1 chromatin association at subtelomeric sites in a SIR3-deletion strain relative to its isogenic wild-type strain, relative to distance from the telomere.
(C) Sir3 presence at the MATα1 gene of the HML mating locus in wild-type (WT), ΔBDF1, and ΔSIR3 strains.
(D) Chromatin association of Sir3 and Bdf1 proteins at the CHA1 promoter in a ΔBDF1 and ΔSIR3 strain.
Molecular Cell  , DOI: ( /S (03) )

9. Figure 7
Bdf1 Bromodomain Provides a Barrier Function at Heterochromatin-Euchromatin Boundaries by Competing with Sir2 for Binding to Acetylated H4
(A) Protection from Sir2 deacetylase activity of acetylated histone H4 in the presence of GST-Bdf1 double bromodomains. Fifty nanograms Sir2 (light gray) and 400 ng (dark gray) were incubated for 2 or 12 hr with hyperacetylated core histones (HCH). Following incubation, Bdf1-bound (B) and unbound material (Sup) was loaded on TAU gels. Gel sections encompassing histone H4 and H2B are shown. Significant amounts of diacetylated H4 (denoted by an asterisk) are retained by the Bdf1 bromodomains, at a time point when Sir2 has fully deacetylated both unbound H2B and H4.
(B) Binding of hyperacetylated core histones to GST-Sir3. Interaction was performed as in (A) and Figure 1A, except washes were performed in the presence of 150 mM NaCl.
(C) Sir2-mediated deacetylation of hyperacetylated core histones in the presence of Bdf1 bromodomains. Following incubation of the HCH, Sir2, Bdf1, and cofactor mixture, the reaction was terminated and, for the purposes of improved clarity, one-third of the sample was loaded (right), relative to unmodified HCH (left). Bdf1 bromodomains preferentially protect acetylated H4 from Sir2 deacetylation.
(D) Mutations of critical acetyl-histone binding residues in Bdf1 lead to increased spreading of Sir3 protein at the telomere of chromosome V R. This correlates with a decreased transcriptional activity of the underlying promoters in the spread domain from 7.5 to 12.5 kb.
(E) The acetyl-histone binding function of Bdf1 is necessary for chromatin association and for the efficient transcription of a select set of euchromatic genes. Following acetyl-histone recognition, the Bdf1 protein may help TFIID's function by associating with yTAFII67. In subtelomeric compartments, and close to mating loci, the Bdf1 protein may be additionally required in order to antagonistically compete with the repressive function of SIR silencing complexes (Sir2 histone deacetylation and Sir3/Sir4 unacetylated histone binding). This function of Bdf1 at heterochromatin-euchromatin boundaries may be TFIID independent.
Molecular Cell  , DOI: ( /S (03) )