Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin

Abstract

In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC domain alone results in severe heterochromatin defects and depletion of siRNA, whereas overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, crossregulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We suggest that Lid2 enzymatic activity in euchromatin is regulated through a dynamic interplay with other histone-modification enzymes. Our findings provide mechanistic insight into the coordination of H3K4 and H3K9 methylation.

Figure 1. Dos1/Clr8 interacts with the JmjC-domain protein Lid2

A) TAP-tagged Clr8 retains wild-type function. The Tap-tag was constructed at the N-terminus of Clr8 and transformed into the clr8Δ mutant containing GFP-Swi6. GFP-Swi6 in the transformants appeared as 3–4 spots instead of diffuse in the nucleus indicating that TAP-tagged Clr8 rescued the mutant phenotype. B) Silver-stained gel showing the TAP-tag purification of Clr8 and a control purification from an untagged strain. Lid2 was identified in the extract by mass spectrometry. As predicted, Clr8 and Cul4 were also present in the complex. C) Schematic representation of domain organization of Lid2: it contains a JmjC domain, three PHD fingers and a JmjN domain. D) The interaction of Lid2 with Clr8 was confirmed by Co-IP. Extracts were prepared from the control strain which contains only Myc-Clr8 and the experimental strain which contains both Myc-Clr8 and Lid2-TAP. The extracts were subjected to IP with anti-Myc agarose beads. Input extracts and immunoprecipitates were analyzed by Western blotting using the antibody against S-Tag, which is part of the TAP-tag.

Figure 2. Lid2 is an essential chromatin-binding protein. Deletion of JmjC domain results in chromosome missegregation

A) in situ chromatin binding assay for GFP-Lid2. Mcm4-GFP, which associates with chromatin at anaphase/G1 phase, was used as a control. After washing with 1% Triton X-100 in a low-salt buffer, the Mcm4-GFP signal was observed only at anaphase/G1 phase (lower panel), but GFP-Lid2 was detected in the nucleus during all stages of the cell cycle (upper panel), indicating it is a chromatin-binding protein. B) Lid2 is associated with the centromere and mating-type region. DNA was isolated from whole cell crude extracts (WCE) of indicated strains or immunoprecipitated (ChIP) by IgG agarose, and was analyzed by multiplex PCR amplifying centromere (upper panel) or mating-type region (lower panel). An actin-like gene, alp5⁺, was used as an internal control (ctrl). WT, a control strain without carrying Lid2-TAP. The fold-enrichment of Lid2-TAP was normalized to alp5⁺. The value for WCE was set to 1.0. C) The lid2 mutant, lid2-j, in which the JmjC domain was completely removed, is isolated. This figure shows the bright-field microscopic images of WT and lid2-j; note that lid2-j displays defective cell shapes. D) The chromatin in lid2-j was disorganized as demonstrated by DAPI staining. E) lid2-j is hypersensitive to the microtubule-destabilizing drug, TBZ, suggesting it has a chromosome segregation defect. A serial dilution of indicated strains was plated on YES media containing 10 µg/ml TBZ. The left panel shows the growth of the strains on a control plate without TBZ.

Figure 3. Lid2 is required for heterochromatin silencing

A) Heterochromatin silencing is lost in lid2-j as shown by a silencing assay. lid2-j and clr8Δ containing the ura4⁺ marker at a otr (upper panel) or mating-type region (lower panel) were plated using a serial dilution on minimal medium without uracil or with the counter-selective FOA media. Ctrl, a control plate containing uracil. B) H3K9 methylation is lost at centromeres in lid2-j. A ChIP assay was carried out with the indicated strains using the antibody against H3K9me2. The centromere fragment from otr region and control (alp5⁺) were amplified from immunoprecipitated DNA by multiplex PCR. WCE, whole-cell extracts before immunoprecipitation; ChIP, immunoprecipitated DNA. The ratios of heterochromatin signals versus control signals between ChIP and WCE fractions were used to calculate the relative enrichment of precipitated DNA, shown below each lane. The value for WT was set to 1.0. C) H3K4 methylation is increased in heterochromatin in lid2-j. ChIP assay was performed as in B) using the antibody against H3K4me3. D) Swi6 binding is abolished at the centromere in lid2-j as shown by ChIP assay using anti-Swi6 antibody. E) Lid2 is required for recruitment of Clr4 to the centromere. A ChIP assay was carried out with the indicated strains carrying Flag-Clr4 using the antibody against Flag.

Figure 4. Lid2 is a trimethyl H3K4 histone demethylase

A) The JmjC domain of a histone demethylase contains two essential binding sites: α-ketoglutarate binding sites as indicated by blue; the conserved iron binding sites as indicated by red. B) Overexpression of WT Lid2 did not affect the H3K4me2 methylation but greatly reduced H3K4me3 methylation as shown by immunostaining with antibodies as indicated. However, overexpression of a point mutant of Lid2^H512A, in which the conserved iron-binding histine residue was mutagenized to alanine, did not cause a significant reduction of the H3K4me3 level. These results indicate that Lid2 is an H3K4me3 histone demethylase and needs the conserved iron binding sites for maximum enzymatic activity. C) Co-overexpression of Lid2 with Set1 or Lsd1 suppressed Lid2’s histone demethylase activity. Immunostaining was carried out as in B) using the antibody indicated. D) Western blot confirmed that overexpressing WT Lid2, but not the Lid2^H512A mutant, resulted in a significant decrease in H3K4me3 methylation. Total cell lysates were blotted using the indicated antibodies. tub, Tubulin used as a control. E) In vitro demethylation assay using TAP-tag purified Lid2. Purified S. pombe chromatin was used as a substrate. Reaction was analyzed by SDS-PAGE followed by Western blotting using antibodies indicated. Left: Analysis of TAP-tag purifed Lid2 by SYPRO Ruby staining.

Figure 5. Lid2 acts in the RNAi pathway and regulates Clr4 methylation in a dose-dependent manner by functioning cooperatively with Clr8

A) Centromere transcripts accumulated in lid2-j as demonstrated using strand-specific RT-PCR. alp5⁺ serves as a control (ctrl). For, forward centromere transcript; Rev, reverse centromere transcript; -RT, no reverse transcriptase. B) The genomic expression distribution at centromere II in lid2-j is shown using Genome Browser analysis. Genes (yellow and gray), dg repeats (green), dh repeats (red), imr repeats (purple), and tRNA genes (black) are indicated by different colors. Note that transcripts from heterochromatin region are significantly increased in lid2-j. C) A small RNA northern blot showed that centromeric siRNA was reduced in lid2-j. Small RNAs spanning 22 to 26 nucleotides corresponding to the centromeric dh repeat were readily detected in the WT. These small RNAs were lost in the lid2 and dcr1 mutants. D) The association of Ago1 with the centromere is reduced in lid2-j. A ChIP assay was conducted in WT and lid2-j using antibody against Ago1. The centromere fragment from otr region and control (alp5⁺) were amplified. E) Clr8 and Lid2 mutually affect each other’s recruitment to heterochromatin. ChIP was carried out for lid2-j and WT containing Myc-Clr8 using antibody against the Myc tag. ChIP also was performed for clr8Δ and WT containing Lid2-TAP using IgG sepharose. The centromere fragment and control (alp5⁺) were amplified. F) Expressing Lid2 in clr8Δ can not rescue the mutant’s silencing defect, and neither can expressing Clr8 in lid2-j; the silencing assays also show that the point mutant, lid2^H512A, can cause significant loss of silencing. The strains indicated containing the ade6⁺ marker in the otr region were plated by a serial dilution on a YES medium without supplementing adenine. Repression of ade6⁺ expression results in pink color. G) Overexpression of Lid2 significantly enhanced heterochromatic silencing. The strains containing ade6⁺ in the otr region were plated on a minimal medium lacking adenine. H) H3K9 methylation in the otr region was increased in the strain overexpressing Lid2. ChIP assay was performed as in Figure 3B.

Figure 6. Lid2 regulates euchromatin by nucleating H3K9 histone demethylase, Lsd1 and H3K4 histone methyltransferase, Set1

A) An unusual number of GFP-Swi6 spots was detected in lid2-j during mitotic interphase (left panel) and meiotic prophase (right panel). GFP-Swi6 is diffuse in clr8Δ but occurs as multiple spots in lid2-j, suggesting that ectopic heterochromatin is induced in lid2-j. B) Euchromatic region near mating-type regions became heterochromatic in lid2-j, same as in the lsd1Δ mutant (Lan et al., 2007); however, the region is not affected in lid2^H512A. The strains containing the ura4⁺ marker inserted in euchromatin as indicated were plated by a serial dilution on a minimal medium without uracil or on the FOA media. Ctrl, a control plate containing uracil. C) Genome-wide expression profile in lid2-j. Global expression profile of open reading frames between about 2.1 to 3 Mb from Chromosome I of the mutant is shown. The transcription profile of lsd1Δ (Lan et al., 2007) is presented for comparison. D) Lsd1 association with chromatin is abolished in lid2-j. A ChIP assay was performed with the indicated strains carrying Lsd1-Myc by using the antibody against Myc-tag. The promoter region of SPCC1620.06⁺ and control (alp5⁺) were amplified. E) Lid2 interacts with Lsd1 as shown by Co-IP. Extracts prepared from the control strain which contains only Lsd1-Myc and the experimental strain containing both Lsd1-Myc and Lid2-TAP were subjected to IP with anti-Myc agarose beads. Input extracts and immunoprecipitates were analyzed by Western blotting using the antibody against TAP-Tag. F) Set1 binding to chromatin is reduced in lid2-j. Cells expressing Set1-Myc were used for ChIP. IP and Multiplex PCR were carried out same as in E). G) Co-IP showed that Lid2 interacts with Set1 and the interaction was reduced if the JmjC domain is removed. Extracts were prepared from strains which carry either Set1-Myc and Lid2-TAP or Set1-Myc and the JmjC-domain deleted version of Lid2-TAP (Lid2 ^JmjCΔ-TAP). Immunoprecipitates were analyzed by the anti-Myc antibody. The strain which contains only Set1-Myc was used as a control.

Figure 7. PHD domains are important for Lid2’s function

A) PHD domains affect heterochromatin silencing as shown by a silencing assay. Left panel shows the schematic representation of Lid2 and its PHD domain deletion mutants. Strains containing the ura4⁺ marker at centromere imr region as indicated were plated by serial dilution on the FOA media. B) PHD2 and PHD3 domains affect GFP-Swi6 distribution in the nucleus. Aberrant GFP-Swi6 patterns were observed in lid2-phd2 and lid2-phd3, similar to lid2-j, suggesting that these PHD domains may also involve in euchromatin assembly. C) PHD2 domain preferentially binds to H3K9me3 methylated peptide. PHD domains tagged with TAP-tag were purified and incubated with biotinylated histone peptides methylated at different lysine residues, as indicated. Bound proteins are then eluted from the avidin beads, and resolved by SDS–PAGE followed by Western blotting using antibody against TAP-tag. D) Model for Lid2 function: Lid2 demethylates heterochromatic H3K4me3 and also mediates H3K9 methylation by Clr4 through interaction with Clr8-Rik1 complex (upper panel). This stabilizes the RITS association with chromatin which in turn recruits RDRC complex to synthesize the double strand RNA for dicer. At euchromatin (lower panel), Lid2 recruits Set1 and Lsd1 to maintain H3K4 methylation and at the same time removes H3K9 marks in actively transcribing genes. During the attenuation phase of transcription , Lid2 H3K4 demethylase activity could be triggered to reduce the H3K4 methylation level.