Histone H3 lysine 36 methylation targets the Isw1b remodeling complex to chromatin

Abstract

Histone H3 lysine 36 methylation is a ubiquitous hallmark of productive transcription elongation. Despite the prevalence of this histone posttranslational modification, however, the downstream functions triggered by this mark are not well understood. In this study, we showed that H3K36 methylation promoted the chromatin interaction of the Isw1b chromatin-remodeling complex in Saccharomyces cerevisiae. Similar to H3K36 methylation, Isw1b was found at the mid- and 3' regions of transcribed genes genome wide, and its presence at active genes was dependent on H3K36 methylation and the PWWP domain of the Isw1b subunit, Ioc4. Moreover, purified Isw1b preferentially interacted with recombinant nucleosomes that were methylated at lysine 36, and this interaction also required the Ioc4 PWWP domain. While H3K36 methylation has been shown to regulate the binding of numerous factors, this is the first time that it has been shown to facilitate targeting of a chromatin-remodeling complex.

Fig 1

Isw1b was enriched at the midregions and 3′ ends of transcriptionally active genes. (A) ChIP-on-chip analysis was performed to analyze localization of Ioc4HA genome wide. All open reading frames, independent of length, were split into 40 bins (see Materials and Methods). The average Ioc4HA model-based analysis of tiling arrays (MAT) scores for each bin were calculated and plotted. The gray bar represents the coding region, and the dashed lines represent the translation start and end sites. The 5′ and 3′ intergenic regions (IGRs) represent 500 bp up- and downstream of the translation start and end sites, respectively. B) CHROMATRA-T plot for Ioc4 ChIP-on-chip. MAT scores were binned into segments of 150 bp, and the average enrichment value for each bin is color coded and plotted as a heat map. Genes were ordered by gene length and grouped into five classes according to their number of transcripts per hour (9). (C) Genes enriched for Ioc4 were more frequently transcribed than all other genes. The all-genes column shows all yeast genes binned based on transcriptional frequency (9), and the Ioc4-enriched column shows genes with at least 50% of all probes over the open reading frame having an enrichment score above a threshold of 1.5.

Fig 2

Isw1b preferentially interacted with H3K36-methylated chromatin. (A and B) Isw1b was purified from a whole-cell extract (WCE) of a strain expressing TAP-tagged Ioc4 (+), and the associated histones were subjected to immunoblot analysis. An untagged strain (−) was used as a negative control. Primary antibodies against histone H3 and H3K36me3 or H3K4me3 were used as indicated. Signals were generated using IRDye-labeled secondary antibodies and overlaid. (C) Immunoblots shown in A and B were repeated in four independent experiments and the results quantified. Error bars indicate means ± standard errors of the means (SEM). An unpaired t test comparing the relative enrichment of H3K4me3 and H3K36me3 in the Ioc4-associated histones revealed a P value of 0.0029. (D) Isw1b was purified from IOC2 and ioc2Δ strains expressing TAP-tagged Ioc4 (+), and the associated histones were subjected to immunoblot analysis for histone H3. An untagged strain (−) was used as a negative control.

Fig 3

SET2, which encodes the sole H3K36 methyltransferase, was required for Ioc4 association with chromatin. (A) The levels of Ioc4HA at the 5′ and 3′ ends of the active MET16 gene were measured relative to input by ChIP-qPCR. IgG represents negative-control ChIPs with rabbit IgG antibody in lieu of HA antibody. (B) Ioc4HA ChIP in the indicated strains at the 3′ end of an active MET16 gene. Results are shown relative to the wild type (WT), which was set to 1. (C) Quantitative immunoblots for levels of TAP-tagged Met16 or HA-tagged Ioc4 in the indicated strains, with the WT set to 1. For all experiments, error bars indicate the means ± SEM from three independent experiments.

Fig 4

Histone H3K36 was required for Ioc4 association with chromatin. (A) The levels of Ioc4HA at the 3′ end of the active MET16 gene were measured relative to input by ChIP-qPCR in the indicated strains. Results are shown relative to the WT, which was set to 1. (B) Quantitative immunoblots for TAP-tagged Met16 or HA-tagged Ioc4 in the indicated strains. (C) The levels of Sas3HA at the 5′ end of the COX10 gene were measured relative to input by ChIP-qPCR in the indicated strains. Results are shown relative to the WT. (D) Purified Isw1b was incubated with immobilized mononucleosomes generated from recombinant histones with the indicated methyl-lysine analogs at H3K36. Samples were immunoblotted for TAP-tagged Ioc4 or histone H3 as indicated. Input represents 10% of the Isw1b added to the binding assays. For panels A to C, error bars indicate the means ± SEM from three independent experiments.

Fig 5

The PWWP domain of Ioc4 was required for the interaction of Isw1b with chromatin. (A) ClustalW2 multiple-sequence alignments of the PWWP motif of Ioc4 with other known methyl-histone binding PWWP motifs from S. cerevisiae (S.c.), S. pombe (S.p.), and human (H.s.). Residues predicted to form the methyl-lysine binding pocket are highlighted in gray. Arrows indicate residues subjected to site-directed mutagenesis. Asterisks indicates positions that have a single, fully conserved residue, a colon indicates conservation between groups of strongly similar properties, and a period indicates conservation between groups of weakly similar properties. (B) Whole-cell extracts (WCE) and TAP-purified Isw1b from strains expressing Isw1TAP (+) and wild-type or mutant versions of HA-tagged Ioc4 were immunoblotted for levels of Isw1 and Ioc4. A untagged strain (−) was used as a negative control. (C) ChIP for Ioc4 at the 3′ end of the active MET16 gene in strains expressing wild-type or mutant versions of Ioc4HA was measured relative to input by qPCR. Results are shown relative to a WT strain, which was set to 1. Error bars indicate the means ± SEM from three independent experiments. (D) Isw1b was TAP purified from strains expressing Isw1TAP and either wild-type (WT) or mutant (Ioc4W21A) HA-tagged Ioc4. The purified complex was incubated with immobilized mononucleosomes generated from recombinant histones with a trimethyl-lysine analog at H3K36. Samples were immunoblotted for HA-tagged Ioc4 or histone H3 as indicated. Input represents 10% of the Isw1b added to the binding assay.