Histone H3 lysine 36 dimethylation (H3K36me2) is sufficient to recruit the Rpd3s histone deacetylase complex and to repress spurious transcription - PubMed (original) (raw)
Histone H3 lysine 36 dimethylation (H3K36me2) is sufficient to recruit the Rpd3s histone deacetylase complex and to repress spurious transcription
Bing Li et al. J Biol Chem. 2009.
Abstract
Histone methylation is associated with both transcription activation and repression. However, the functions of different states of methylation remain largely elusive. Here, using methyl-lysine analog technology, we demonstrate that the histone deacetylase complex, Rpd3S, can distinguish the nucleosomes methylated to different extents and that K36me2 is sufficient to target Rpd3S in vitro. Through a genome-wide survey, we identified a few mutants in which the level of K36me3 is significantly reduced, whereas the level of K36me2 is sustained. Transcription analysis and genome-wide histone modification studies on these mutants suggested that K36me2 is sufficient to target Rpd3S in vivo, thereby maintaining a functional Set2-Rpd3S pathway.
Figures
FIGURE 1.
Rpd3S preferentially binds to H3K36 di- and trimethylated nucleosomes. A, mass spectra (ESI-oaTOF) analysis demonstrates that the majority of end products are correct forms of Lys-36 methyl-lysine mimic histones. The y axis denotes relative intensity. The* denotes a mass spectrometry artifact peak at +41 that does not appear on other spectrometers and is present in both the starting material and the methylated histone products. B, Rpd3S preferentially binds to the di- and trimethylated nucleosomes. Recombinant MLA nucleosomes are reconstituted through the serial dilution method followed by gel purification.SON, short oligonucleosomes, represents the nucleosomes made through the octamer transfer method using extracted native HeLa nucleosomes (Nuc). C, Chd1-TAP served as a negative control that cannot distinguish nucleosomes methylated at Lys-36 or that are unmethylated. The* marks represent the partially dissociated nucleosome population.
FIGURE 2.
Identification of Paf1 gene product to be required for histone H3 lysine 36 trimethylation by GPS. Yeast whole cell extracts were made from each of the non-essential gene deletion mutants of the Saccharomyces cerevisiae genome and were resolved on SDS-PAGE. The histone H3K36me3 antibody was used to carry out the GPS screen. Blue arrows indicate empty wells as place markers. The red arrow indicates the position of the PAF1 deletion mutant at plate location D7.
FIGURE 3.
Multiple genes affect the states of methylation at lysine 36 of histone H3. A, roles of the Rad6-Bre1 and Paf1 complexes (A) in H3 Lys-36 trimethylation. Cell extracts were obtained from several components of each complex and subjected to SDS-PAGE followed by Western blotting using antibodies specifically against different methylation states of histone H3. Anti-acetyl H3 was also applied as a load control. WT, wild type.B, Lge1 is required for proper H3 Lys-36 trimethylation. Lys-36 trimethylation was restored after transforming of a plasmid carrying_LGE1_, which contains a FLAG tag at their C terminus and is under the control of the GAL1 promoter, to strains that are lacking Lge1. Cell extracts were subjected to SDS-PAGE and Western blotting using the indicated antibodies. Anti-acetyl H3 was used as a loading control, and anti-FLAG was used to detect expression of targeted proteins.
FIGURE 4.
Dimethylation of H3K36 in various mutants is sufficient for normal suppression of cryptic promoters at the STE11 gene. Yeast strains grown exponentially were subjected to Northern blot analysis using probes against STE11 (5′ ORF or 3′ ORF). FL denotes full-length transcript; short transcripts refer to transcripts initiated from cryptic promoters. WT, wild type.
FIGURE 5.
Deletion of PAF1 does not lead to genome-wide increase of histone H4 acetylation level at the 3′ end of coding region, a typical phenotype seen in the Set2-Rpd3S defective mutants. ChIP-chip was performed using Agilent high resolution tiling arrays. The log2 ratio of acetylation of H4 (AcH4) in mutants over AcH4 in wild type was subjected to a modified average gene analysis (17). All genes were divided into multiple subclasses based on either their ORF length or their transcription frequency in complete mediums. The averages of each subclass were plotted, with the number of genes in each class being indicated within the parentheses in the white box.
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