Sumoylation induced at the H2B C terminus cannot functionally replace H2Bub1 in mediating H3-K4 and -K79 Methylation. (A) Amino acid sequence of the C-terminal region of yeast histone H2B from valine (114) to alanine (130). Two consensus sumoylation sites (bold) separated by a spacer glycine were inserted into H2B to create the chimeric H2B [H2B(2SU)]. To determine the sumoylation site, the two lysines (K1 and K2) were changed to leucine (underlined) to obtain mutants, H2B(2SU)-K1L and H2B(2SU)-K2L. Except for the control (H2B no tag), the wild type, the chimeric H2B and their derivatives contain a single Flag epitope at the N terminus. (B and C) Yeast whole cell extracts of the indicated strains were analyzed for the occurrence of ubiquitination (ub1) and sumoylation (su) by Western blot. Asterisk, a protein cross-reacting with α-Flag. (D) Levels of H2B(2SU)su at the indicated genomic loci were analyzed by sequential ChIP using α-Flag and α-SUMO. Fold enrichment of H2B(2SU)su in the indicated regions is shown relative to the control strain that lacks the sumoylation site [H2B(2SU)-K2L]. Error bars denote standard error of the mean obtained from three independent experiments. (E) Histone methylation levels in whole cell extracts were assessed using the indicated H3-K4 and -K79 methylation-specific antibodies.

Changes in H2Bub1 levels affect the amount of histones on chromatin. Nuclei isolated from the indicated strains were lysed in a hypotonic solution to obtain chromatin. Equal amounts of chromatin were resolved by 15% SDS/PAGE. Histone H2B and H3 levels were assessed by Western blots. Both the initial and normalized amounts of chromatin were measured by quantitative PCR using two-fold serially diluted (triangle) genomic DNA template isolated from the chromatin. Primers for the indicated regions (as in Fig. 1D) were used in multiplex PCR. Graph depicts the fold change in H2B and H3 amounts on chromatin in the mutants relative to wild type. Error bars denote standard error of the mean obtained by measuring the histone amount in the mutant at each dilution normalized to the respective amount in the wild-type chromatin (set as 1).

H2Bub1 levels affect both the sensitivity of chromatin to salt-dependent disruption and cell growth. (A) Equal amounts of nuclei isolated from the indicated strains were resuspended in solutions of increasing NaCl concentration. Following incubation and centrifugation, amount of H3 in the supernatant (soluble fraction) was analyzed by Western blotting. As the soluble H3 amount in the wild type and the mutant remain the same at high NaCl concentrations (≥ 590 mM), the amount of soluble H3 at various salt concentrations (475, 515, or 545 mM) was normalized to the amount obtained after 615 mM NaCl wash (Input). Graph depicts the average soluble H3 amount obtained from two independent experiments. (B) Soluble H3 obtained by resuspension of nuclei in solutions containing low NaCl concentration was analyzed as in (A). The amount of soluble H3 obtained following low salt wash was normalized to the amount obtained from 2.0 M NaCl wash (Input). Fold change in soluble H3 amounts in the mutants were calculated relative to the normalized soluble H3 amount obtained by washing the wild-type nuclei with 200 mM NaCl (set as 1). Graph depicts the average fold change in soluble H3 amounts obtained from two independent experiments. (C) Low salt solubility of H2B was determined as described for H3. Fold change in soluble H2B amounts from one representative experiment is shown. (D) To assess cell growth, the indicated yeast strains were grown on rich media at 30 °C.

H2Bub1 levels can differentially affect the sensitivity of chromatin to micrococcal nuclease digestion. (A) Nuclei isolated from the indicated strains were treated with increasing MNase concentrations. Undigested or partially digested DNA was resolved in a 1.8% agarose gel. (B) Nuclei isolated from the indicated strains were treated with MNase (5 U/mL) for increasing incubation time. Arrowheads indicate the nucleosomal ladder.

Increase in H2Bub1 augments H2B amounts on constitutive and repressed genes. (A) Levels of H2B at the promoter and ORF regions of PMA1 and FUN12 were analyzed by ChIP assay using α-Flag. Fold enrichment of H2B levels on the promoter (P) or ORF regions [proximal (5′), middle (M), distal (3′)] in the mutants are shown relative to the wild type. (B) Ovals with solid line, experimentally-verified positioned nucleosomes; Oval with the dotted line, a speculative positioned nucleosome; rectangle, upstream activating sequence (UAS); Arrow, transcription start site; TATAA, the TATA element; and the thick solid line, region amplified by PCR following ChIP. (C) H2B levels at the promoters were analyzed as in (A). Fold change in H2B levels in the mutants is shown relative to the wild type. (D) H2Bub1 levels were analyzed by sequential ChIP using α-Flag and an antibody that recognizes ubiquitinated proteins. Fold enrichment in H2Bub1 levels in the indicated strains is shown relative to H2B-K123R. Error bars denote standard error of the mean obtained from at least three independent experiments.