Codependency of H2B Monoubiquitination and Nucleosome Reassembly on Chd1
Total Page：16
File Type：pdf, Size：1020Kb
Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION H3K79 trimethylation by Set1/COMPASS and Dot1, a pro- Codependency of H2B cess termed histone cross-talk (Dover et al. 2002; Sun and monoubiquitination and Allis 2002; Hwang et al. 2003; Wood et al. 2003a; Schneider et al. 2004; Smith and Shilatifard 2010). Other factors were nucleosome reassembly identified by GPS to be required for proper H3K4 and/or H3K79 methylation, including the Paf1 complex consist- on Chd1 ing of Paf1, Rtf1, Cdc73, and Ctr9 (Krogan et al. 2003; Jung-Shin Lee,1 Alexander S. Garrett,1 Wood et al. 2003b); the CTD kinases Bur1/Bur2 and Ctk1/ Ctk2/Ctk3 (Wood et al. 2005, 2007); the cell cycle regulator Kuangyu Yen,2 Yoh-Hei Takahashi,1 Deqing Hu,1 3 1 SBF (Schulze et al. 2009); and the N-terminal acetyl- Jessica Jackson, Christopher Seidel, transferase Ard1 (Takahashi et al. 2011). B. Franklin Pugh,2 and Ali Shilatifard1,4 Collectively, these studies using antibodies recognizing histone H3K4 and/or H3K79 methylation showed numer- 1Stowers Institute for Medical Research, Kansas City, Missouri 2 ous connections between the implementation of histone 64110, USA; Center for Eukaryotic Gene Regulation, H2B monoubiquitination and H3K4 methylation. In this Department of Biochemistry and Molecular Biology, The study, we used a recently generated antibody recognizing Pennsylvania State University, University Park, Pennsylvania 3 yeast H2B monoubiquitination and identified the chro- 16802, USA; Department of Biochemistry, St. Louis University matin remodeler Chd1 as a novel factor required for the School of Medicine, St. Louis, Missouri 63104, USA maintenance of high levels of histone H2B monoubiqui- tination. Importantly, the status of H3K4 and H3K79 Monoubiquitination of histone H2B on Lys 123 (H2BK123ub) methylation was unaffected in the chd1D background, is a transient histone modification considered to be essen- explaining why Chd1 was not identified in the earlier tial for establishing H3K4 and H3K79 trimethylation by GPS screens using H3K4- and H3K79-specific anti- Set1/COMPASS and Dot1, respectively. Here, we identified bodies. Despite the earlier studies suggesting integra- Chd1 as a factor that is required for the maintenance of tion of H2B monoubiquitination and H3 methylation high levels of H2B monoubiquitination, but not for H3K4 machineries, this study demonstrates that only low and H3K79 trimethylation. Loss of Chd1 results in a sub- levels of monoubiquitination are required for proper stantial loss of H2BK123ub levels with little to no effect on levels of H3K4 and H3K79 methylation. Also, recent the genome-wide pattern of H3K4 and H3K79 trimethyla- studies suggested other possible roles associated with histone H2B monoubiquitination, distinct from histone tion. Our data show that nucleosomal occupancy is reduced cross-talk (Pavri et al. 2006; Tanny et al. 2007; Fleming in gene bodies in both chd1D and, as has been shown, et al. 2008; Batta et al. 2011; Schulze et al. 2011), K123A mutant backgrounds. Wealsodemonstratedthat including effects on nucleosome positioning (Batta Chd1’s function in maintaining H2BK123ub levels is con- et al. 2011). We therefore used genome-wide expression served from yeast to humans. Our study provides evidence and nucleosome occupancy analysis in a chd1D strain that only small levels of H2BK123ub are necessary for full and a strain defective for H2B monoubiquitination and levels of H3K4 and H3K79 trimethylation in vivo and propose an evolutionarily conserved function for Chd1 points to a possible role for Chd1 in positively regulating in positively regulating gene expression through pro- gene expression through promoting nucleosome reas- moting nucleosome reassembly coupled with H2B sembly coupled with H2B monoubiquitination. monoubiquitination. Received January 9, 2012; revised version accepted March Results and Discussion 26, 2012. Loss of H2B monoubiquitination with normal levels of H3K4 and H3K79 methylation As fundamental components of chromatin, the histone Prior GPS screens using antibodies specific to either proteins are subjected to a variety of post-translational H3K4 or H3K79 methylation identified a requirement modifications that function in diverse processes (Bhaumik for numerous factors connected to H2B monoubiquitina- et al. 2007; Smith and Shilatifard 2010). In order to un- tion in proper H3 methylation (Smith and Shilatifard derstand the machinery that implements these modifica- 2010). Here, we instead performed a GPS-style screen tions and the cellular contexts in which they function, we with antibodies specific to histone H2B monoubiquitina- developed a biochemical screen called global proteomic tion and identified Chd1 as a novel factor required for analysis in Saccharomyces cerevisiae (GPS) (Schneider maintaining proper levels of histone H2B monoubiquiti- et al. 2004). This screen and other genetic screens identi- nation (Fig. 1A, lane 12). Although H2B monoubiquitina- fied numerous factors required for the proper implemen- tion levels are significantly reduced, H3K4 and H3K79 tation of different H3K4 and H3K79 methylation states. methylation levels were unchanged (Fig. 1B), indicating Notably, these include the histone H2B monoubiquitinase why Chd1 was not previously identified in screens probing complex Rad6/Bre1’s requirement for histone H3K4 and the methylation status of these residues. In the absence of Bre1, the E3 ligase required for H2B monoubiquitination by Rad6 (Wood et al. 2003a), we cannot detect any histone H2B [Keywords: chromatin; transcription; ubiquitination; histone] monoubiquitination or any histone H3K4 and/or H3K79 4Corresponding author. E-mail [email protected] trimethylation (Fig. 1B, lanes 7–10), validating the specific- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.186841.112. ity of our antibodies. 914 GENES & DEVELOPMENT 26:914–919 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Chd1 and H2B monoubiquitination immunoprecipitation (ChIP) coupled with deep sequenc- ing (ChIP-seq) (Fig. 2A-C). Our data show that the global patterns of both H3K4 and H3K79 trimethylation were not significantly altered, although histone H2B mono- ubiquitination levels were decreased in these cells. An overview of the ChIP-seq signal on the entire ;230-kb chromosome I (Fig. 2A) indicates that the patterns of enrichment for the given histone marks are not grossly altered in level or intensity upon CHD1 deletion. In order to more quantitatively determine the extent of genome- wide changes in these marks, we computed an occupancy enrichment metric as the ratio of the sum of normalized read levels of each histone mark in the region of 6500 base pairs (bp) surrounding each gene start site divided by the normalized read sum of the associated whole-cell ex- tract control sample (input) in the same region. We then compared the occupancy enrichment ratios between the wild-type and chd1D strains for both marks (Fig. 2B,C) and found that the Pearson correlation coefficients for H3K4 Figure 1. Chd1 is required for the maintenance of high levels of and H3K79 trimethylation were 0.928 and 0.787, respec- H2B monoubiquitination but not H3K4 or H3K79 trimethylation. Cell extracts from the nonessential yeast gene deletion mutants tively. When we assessed only the normalized read sums were subjected to SDS-PAGE and analyzed by Western blotting surrounding gene start sites (without dividing by the using H2B monoubiquitination-specific antibodies. Lane H of plate whole-cell extract sample), both marks had a correlation #148 of the collection is shown in A. The red arrow indicates the coefficient >0.95 between the wild-type and chd1D strains. position in the plate containing the chd1D strain. The green arrow points to the empty well in the collection, used as the plate marker. (B) Cell extracts from wild-type (WT) and chd1D and bre1D strains Chd1, histone H2B monoubiquitination, nucleosomal were prepared and used in a titration analysis. Each extract was occupancy, and gene expression analyzed by its application to SDS-PAGE and tested for the presence of modified H3K4 and H3K79 mono-, di-, and trimethylation To determine the effect of the loss of Chd1 on overall gene (H3K4me1–3 and H3K79me1–3) and H2B monoubiquitination expression in yeast cells, we performed RNA sequencing (ubH2B) or total H4 using the appropriate antibodies. (C) A direct (RNA-seq) analyses in wild-type and chd1D strains (Fig. role for Chd1 in the regulation of histone H2B monoubiquitination 2D–G). We first checked the expression of the CHD1 was demonstrated by complementation of the chd1D strains with a vector carrying the CHD1 coding sequence under the GAL1 locus in the chd1D strain and confirmed that the deletion promoter. In the presence of galactose, Chd1 is expressed (shown resulted in complete loss of CHD1 expression (Fig. 2D). in lanes 7,8), as evident by Western signal with anti-Flag, and yeast Using an adjusted P-value threshold of 0.05, we found cells are restored for monoubiquitinated H2B. (D) CHD1 was also that 31 genes were down-regulated and 118 genes were deleted in a parental background that contains a sole copy of Flag- up-regulated after CHD1 deletion (Fig. 2E). A visual tagged histone H2B (to detect both wild-type and monoubiquitinated example of loss of expression for some highly expressed H2B). Extracts prepared from wild-type and chd1D strains were analyzed by SDS-PAGE and Western with the indicated antibodies. genes in the wild-type strain—namely, HOM3 (Fig. 2F) and ARN2—are shown (Fig. 2G). Our RNA-seq studies and a recent microarray study on the gene expression We confirmed a specific role for Chd1 in regulating regulatory and nucleosome organization function of Chd1 histone H2B monoubiquitination levels by complement- (Gkikopoulos et al. 2011) demonstrate that the expression ing the chd1D strain with a vector carrying FlagTCHD1 of very few genes are altered in the absence of Chd1. under the GAL1 promoter (Fig.