1. Volume 20, Issue 4, Pages 589-599 (November 2005)
The Bur1/Bur2 Complex Is Required for Histone H2B Monoubiquitination by Rad6/Bre1 and Histone Methylation by COMPASS 
Adam Wood, Jessica Schneider, Jim Dover, Mark Johnston, Ali Shilatifard 
Molecular Cell 
Volume 20, Issue 4, Pages (November 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Deletion of BUR2 Results in Reduction of the Levels of Histone H3 Lysine 4 and 79 Methylation
(A) Extracts prepared from plates containing single deletion mutants from the nonessential deletion consortium (lanes A–D) were subjected to SDS-PAGE and Western analysis by using antibodies directed against dimethylated lysine 4 and lysine 79 of histone H3. Blue arrows indicate the lane containing the BUR2 deletion mutant. Several other sites that are null for histone methylation represent slow-growing strains or plate markers.
(B) Deletion of BUR2 results in a decrease in di- and trimethylation of H3K4 (lanes 3 and 4). After a CEN-URA3 plasmid containing HA-tagged Bur2 was introduced into the BUR2 deletion mutant, histone H3K4 methylation was restored to levels comparable to wild-type (wt) (lanes 9 and 10).
(C) The loss of BUR2 results in reduction of the level of histone H3K79 methylation by Dot1p. A titration study was performed by the analysis of bur2 null strain extracts. Each extract was analyzed by its application to SDS-PAGE and tested for the presence of modified H3K79 by using appropriate antibodies. As load controls, antibodies against either unmodified or modified histone H3 and actin were also used.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The Loss of Bur1/Bur2 Results in the Reduction in the Levels of Histone H2B Monoubiquitination
(A) A strain carrying a plasmid bearing a single copy of histone H2B FLAG tagged at its N terminus was mated to a bur2 null strain, and the resulting viable tetrads were dissected. Because the KanMX cassette was used to disrupt the BUR2 open reading frame, strains carrying the bur2 deletion will be resistant to G418. The top panel displays the growth analysis of the mating cross between a bur2 null and wt strain. Growth phenotypes associated with Bur2 mutations migrate with kanamycin resistance (lanes A1 and A2, top). Strains produced from the cross that are not resistant to G418 are wt progeny (lanes A3 and A4). Protein extracts were also prepared from the parental strains and each of the four strains resulting from the tetrad dissection. After SDS-PAGE, the blots were probed with antisera against the FLAG epitope, dimethylated H3K4, trimethylated H3K4, and acetylated histone H3 (anti-FLAG antibody was used to visualize the amount of monoubiquitinated histone H2B).
(B) Comparative analysis between the bur2 mutant and wt progeny of a separate mating cross. Two spores, one bearing the KanMX::bur2 deletion (B1) and one containing the wt BUR2 gene (B2), were also analyzed for defects in histone H2B monoubiquitination and histone H3K4 methylation.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The Bur1/Bur2 Complex Can Phosphorylate Rad6
(A) The mRNA levels of the several known genes involved in histone H2B monoubiquitination were measured by using RT-PCR in wt and bur2 null strains with primers generated against the indicated genes (top). When BUR2 is deleted, Rad6 protein expression is not affected (bottom). These data indicate that the expression or stability of Rad6 is not altered in a bur2 null strain.
(B) Rad6 exists as a phosphoprotein in vivo. Lysates from a wt strain containing TAP-tagged Rad6 were subjected to 2D gel analysis and probed with antibody directed against Rad6-TAP. Addition of phosphate groups causes Rad6 to migrate toward the anode (positive electrode). Treatment of the extracts with potato acid phosphatase (Sigma) results in a loss of the phosphorylated forms of Rad6 (bottom). A red dot denotes the free form of Rad6, and the asterisks indicate crossreacting proteins from the phosphatase mixture.
(C) In order to ensure the purity of the purified Rad6 substrate used in the in vitro kinase assays and that no other contaminating proteins migrate with Rad6 in the Bur1 or Bur2 purification, a fraction of the TAP-purified proteins was resolved by SDS-PAGE and was silver stained.
(D) TAP-tag-purified Bur1, Bur2, and Rad6 from (C) were used to reconstitute an in vitro kinase assay. Increasing amounts of either Rad6 (lanes 3–5) or Bur1/Bur2 (lanes 8–10) were titrated into the reaction as described in the Experimental Procedures. Individual reactions were resolved by SDS-PAGE, and kinase activity was determined by using a phosphoimager plate (Molecular Dynamics).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Mutation of Rad6 on Serine 120 Reduces Histone H2B Ubiquitination In Vivo
(A) Serine 120 of Rad6 is an in vitro substrate for Bur1/Bur2 kinase. The top panel demonstrates the sequence alignment of Rad6 and its homologs ranging from yeast to humans. Residues colored with red are completely conserved in all species. The asterisk denotes the position of serine 120. TAP-purified Bur1/Bur2 was used to reconstitute an in vitro kinase assay. Rad6-TAP or Rad6-TAP carrying the S120A mutation (3C) was purified and used as substrates for the assay. Kinase and substrates were either incubated alone (lanes 1–3) or in combination (lanes 4 and 5). The individual assays were resolved by SDS-PAGE, and kinase activity was determined by using a phosphoimager screen after 6 hr of exposure.
(B) An additional kinase assay was run by using the identical conditions, and one reaction containing wt Rad6 was then incubated with phosphatase (middle lane).
(C) Strains carrying plasmids (URA3 CEN) with either the TAP-tagged Rad6 or Rad6 (S120A) (pRAD6-TAP) or (pS120A-TAP) were assayed for their ability to compliment histone H2B monoubiquitination (lanes 3 and 4). The parental strain contains a sole copy of FLAG-tagged histone H2B (lane 1). When RAD6 gene is deleted, the level of the slow-migrating band (ubH2B) is significantly reduced. Wt Rad6 can compliment the monoubiquitination phenotype, but the S120A substitution results in a substantial decrease in histone H2B monoubiquitination when compared to wt (compare lanes 3 and 4).
(D) Two-dimensional gel analysis of Rad6-TAP, Rad6-TAP in a bur2 null strain, and Rad6-TAP mutated at serine residue 120 (S120A) in a wt background. When either the BUR2 gene is deleted or serine 120 of Rad6 is mutated to alanine, the abundance of phosphorylated Rad6 shifts drastically toward the unmodified form.
(E and F) ChIP demonstrates that the S120A substitution or the deletion of BUR2 does not significantly alter the localization of Rad6 to chromatin on the PMA1 gene. To monitor the presence of Rad6 on chromatin at the PMA1 gene, chromatin was immunoprecipitated with rabbit IgG-agarose from a strain containing either wt Rad6-TAP or Rad6-TAP(S120A). (F) ChIP analyses for Rad6 were also performed in wt or bur2 null backgrounds. PCR amplifications were carried out by using primer pairs recognizing the early transcribed region of the PMA1 gene. Each PCR reaction contained a second primer pair that amplified a region of chromosome V devoid of ORFs, thus providing an internal control for background. The ratio of the experimental to the control signal for the precipitated DNA was divided by the ratio of the experimental to the control signal for the input DNA. Error bars represent the standard deviation from the mean.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Localization of the Paf1 Complex Is Partially Reduced When BUR2 Is Deleted
(A) ChIPs were performed to determine if the localization of Bur2 to chromatin on constitutive genes such as ADH1 and PYK1 is altered when the Paf1 complex is dissociated (Paf1 null).
(B) The localization of Paf1 to the same regions of chromatin was determined in a bur2 deletion background following the ChIP method as described in Figure 4.
In (A) and (B), error bars represent the standard deviation from the mean.
(C) The transcription of BUR2, ADH1, PYK1, and actin genes were measured by using RT-PCR with primers generated against the indicated genes in wt and BUR2 strains.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Serine 120 of Rad6 Is Required for Several Functions In Vivo
(A) To determine if Rad6 serine 120 is required for several known functions of Rad6, we tested the Rad6 (S120A) mutant alongside other strains deleted for known components of the histone H2B monoubiquitination machinery to assay phenotypic similarities. Cells were tested for sensitivity to HU, formamide, and heat shock at 37°C.
(B) Serine residue 120 of Rad6 is also required for telomeric silencing. Components of the machinery required for proper histone H2B monoubiquitination were knocked out in a strain with the URA3 reporter inserted into the telomere of chromosome 7. Either wt or Rad6 (S120A) was tested for its ability to grow on 5FOA.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
The Bur1/Bur2 Kinase Complex Is Required for Partial Recruitment of the Paf1 Complex and the Activation of Rad6 Activity via Its Phosphorylation
These activities of the Bur1/Bur2 complex are required for proper monoubiquitination of histone H2B and methylation of histone H3 by COMPASS and Dot1p linking Bur1/Bur2 complex to transcriptional elongation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions