Volume 31, Issue 1, Pages 57-66 (July 2008) H2B Ubiquitylation Plays a Role in Nucleosome Dynamics during Transcription Elongation  Alastair B. Fleming, Cheng-Fu Kao, Cory Hillyer, Michael Pikaart, Mary Ann Osley  Molecular Cell  Volume 31, Issue 1, Pages 57-66 (July 2008) DOI: 10.1016/j.molcel.2008.04.025 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 H2B Ubiquitylation and H3 Lysine 4 Methylation Differentially Affect GAL-Regulated Transcription Wild-type, htb-K123R, or hht-K4A cells containing a GAL-YLR454w gene were grown at 30° in YP + 2% raffinose and then shifted to YP + 2% galactose. (A) GAL-YLR454w transcript levels were measured by RT-qPCR and normalized to ACT1 mRNA levels. (B) Two hours after galactose induction, Pol II occupancy was measured by ChIP at the promoter and 2 kb intervals across the GAL-YLR454w ORF. The results represent the means from two to three independent experiments, with bars depicting SEM. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 H2B Ubiquitylation and Spt16 Functionally Interact to Control Histone and Pol II Occupancy at GAL1 Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown at 30° in YP + 2% raffinose for 2 hr, shifted to YP + 2% galactose for 2 hr, followed by an additional 1 hr at 37°. (A) Histone occupancy at the GAL1 promoter and 5′ ORF. (B) Pol II occupancy at the GAL1 promoter, 5′ and 3′ ORF. Pol II occupancy was normalized to wild-type, and histone occupancy was normalized to the ORF-free region, INT-V. The results represent the means from three to four independent experiments, with bars depicting SEM. Asterisks indicate a statistically significant difference between the double and single mutants as determined by Student's t test (p < 0.05). Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 H2B Ubiquitylation and Spt16 Functionally Interact to Repress Internal Transcription Initiation (A) Total RNA was extracted from wild-type, htb-K123R, or hht-K36A cells grown at 30° in YPD. RT-qPCR reactions were performed with primers corresponding to the 3′ and 5′ coding regions of the YLR454w, FLO8, and VPS72 genes, and the ratio of 3′/5′ ORF products was calculated. (B) Pol II occupancy at 2 kb intervals across the GAL-YLR454w gene after 2 hr of galactose induction at 30°. (C) Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown in YPD for 1 hr at 37°. RT-qPCR reactions were performed as described in (A). The data represent the means from three to six independent experiments after normalization to wild-type, with bars depicting SEM. Black asterisks highlight a significant difference between the single mutants and wild-type (∗p < 0.05; ∗∗ p < 0.005), and the gray asterisk indicates a significant difference between spt16 htb-K123R and spt16 (p < 0.05) as determined by Student's t test. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 H2B Ubiquitylation and Spt16 Interact to Regulate Chromatin Structure Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown in YPD for 1 hr at 37°. Nuclei were incubated with MNase over time, and the extracted DNA was analyzed by sequential Southern blot analysis using labeled probes corresponding to the Yα region of the silent HMLα locus and the transcriptionally active FLO8 and STE11 genes. The MNase digestion pattern of total genomic DNA was visualized by ethidium bromide staining. Exposure times for the HMLα blot were adjusted to normalize the overall band intensities between wild-type and htb-K123R, and spt16 and spt16 htb-K123R samples. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 H2B Ubiquitylation and Spt16 Functionally Interact during Histone Reassembly at GAL1 Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown at 30° in YP + 2% raffinose for 2 hr, shifted to YP + 2% galactose for 2 hr, followed by an additional 1 hr at 37°. Glucose was added to 2%, and H2B or H3 occupancy was measured at the GAL1 5′ and 3′ ORF. Occupancies were normalized to the levels present in galactose-induced cells (set as 1.0 for each strain). The results represent the average of three to four independent experiments, with bars depicting SEM. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 H2B Ubiquitylation and Spt16 Regulate the Kinetic Properties of Elongating Pol II (A) Pol II occupancy was measured at the GAL1 5′ ORF in the samples described in Figure 5, and normalized to the level present in galactose-induced cells (set as 1.0 for each strain). The results represent the average of three to four independent experiments, with bars depicting SEM. Asterisks indicate a statistically significant difference between wild-type and spt16 K123R double mutant (p < 0.05) as determined by Student's t test. (B) Data from Figure 2B were used to determine the ratio of Pol II occupancy at the GAL1 3′ ORF relative to the 5′ ORF. The results were further normalized to Pol II occupancy in the wild-type strain. The asterisk represents a statistically significant difference between spt16 htb-K123R and spt16 (p < 0.05). There was no significant difference between wild-type and spt16 htb-K123R. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Regulatory Interplay between H2BK123ub1 and Spt16 (A) Wild-type, htb-K123R, spt16, and spt16 htb-K123R cells were grown in YPD at 30° or shifted to 37° for 1 hr. Whole-cell lysates were separated on 15% acrylamide gels, and blots were probed with the indicated antibodies. Representative results from three to four independent experiments are shown. (B) Wild-type, htb-K123R, and spt16 cells were grown at 30° in YP + 2% raffinose for 1.5 hr and transferred to 37° for 30 min; galactose was then added to 2%, and cells were kept at 37°. H2BK123ub1 levels were measured by sequential ChIP. The data represent the average of two independent experiments. (C) Spt16 ChIP was performed on the samples described in (B). The data represent the means from two to three independent experiments, with bars depicting SEM. Molecular Cell 2008 31, 57-66DOI: (10.1016/j.molcel.2008.04.025) Copyright © 2008 Elsevier Inc. Terms and Conditions