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Originally published In Press as doi:10.1074/jbc.M703034200 on May 24, 2007

J. Biol. Chem., Vol. 282, Issue 29, 20827-20835, July 20, 2007
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Two Saccharomyces cerevisiae JmjC Domain Proteins Demethylate Histone H3 Lys36 in Transcribed Regions to Promote Elongation*Formula {diamondsuit}

TaeSoo Kim and Stephen Buratowski1

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, April 10, 2007 , and in revised form, May 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Histone methylation is a reversible modification regulated by the antagonistic functions of residue-specific histone methyltransferases and demethylases. Although methylation of histone H3 at lysines 4 and 36 is linked to transcription, the roles of histone demethylases in transcription regulation are not understood. Here we show that overexpression of either Jhd1 or Rph1, two JmjC-domain proteins, bypasses the requirement for the positive elongation factor gene BUR1. Biochemical analysis and chromatin immunoprecipitation experiments indicate that Rph1 functions as a specific demethylase for H3 K36me3 and K36me2, directly regulating Lys36 methylation in transcribed regions. Both Jhd1 and Rph1 are required for normal levels of RNA polymerase II cross-linking to genes. Taken together, these findings indicate that a general function of histone demethylases for H3 Lys36 is to promote transcription elongation by antagonizing repressive Lys36 methylation by Set2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Posttranslational modifications of histones, including phosphorylation, acetylation, ubiquitylation, and methylation regulate gene expression by affecting chromatin architecture (1–5). Histone methylation has been implicated in diverse biological processes including X-chromosome inactivation, heterochromatin formation, and gene silencing (6–9). Methylations occur on both lysine (Lys) and arginine (Arg) residues. Five lysine residues (Lys4, Lys9, Lys27, Lys36, and Lys79) of histone H3 and Lys20 of histone H4 are methylated by specific histone methyltransferases (5, 8). These marks can be binding sites for effector proteins that possess domains that recognize and bind to methylated lysines, such as chromodomains, tudor domains, WD40-repeats, and PHD fingers (9–12). Furthermore, methylation states (mono-, di-, and tri-) within the same residue can produce different biological and transcriptional consequences.

Specific histone methylations have been correlated with either activation or repression of transcription (4, 6, 9). Histone methylations at Lys4, Lys36, and Lys79 are generally associated with active transcription. In contrast, transcriptionally inactive regions are methylated at H3 Lys9 and Lys27 as well as H4 Lys20. In yeast, H3 Lys4, Lys36, and Lys79 are methylated by Set1, Set2, and Dot1 methyltransferases, respectively (13–15). Methylations at Lys4 and Lys36 are closely linked to C-terminal domain (CTD) phosphorylation of RNA Pol II2 subunit Rpb1 (13, 16–18). Kin28, a catalytic subunit of TFIIH, phosphorylates CTD serine 5, and this modification recruits the Set1 COMPASS complex to 5' ends of genes (13). During elongation, Ctk1 phosphorylates CTD serine 2 (in addition to serine 5), which targets Set2 methyltransferase and H3 Lys36 methylation to the body of genes (16, 18–21). Generally, H3 Lys4 trimethylation is enriched at the 5' end of genes, while Lys36 trimethylation peaks at the 3' end of genes (18, 22).

Although histone methylation was originally thought to be a stable mark, recent studies show that methylation can be reversed by histone demethylases. Mammalian lysine-specific demethylase 1 (LSD1) specifically reverses mono- and dimethylation of H3 Lys4 and functions as a transcriptional repressor (23, 24). The chemical mechanism of Lsd1 precludes demethylation of trimethyl lysines. Recently, a family of histone demethylases characterized by the presence of a JmjC domain was identified. Unlike LSD1, JmjC demethylases are found from bacteria to humans and can theoretically reverse all three lysine methylation states by a Fe(II)- and {alpha}-ketoglutarate-dependent mechanism (25). Mammalian JHDM1 and JHDM2A have been shown to antagonize mono- and dimethylation of H3 Lys36 and H3 Lys9, respectively, and the JMJD2/JHDM3 family preferentially reverses di- and trimethylation of both H3 Lys36 and Lys9 (25–29). The function of JHDM1 in transcription has not been studied, but JHDM2A-dependent demethylation of H3 Lys9 positively affects transcription (26). In contrast, JMJM2A/JHDM3A, a trimethyl-specific demethylase for Lys9 and Lys36, negatively regulates ASCL2 transcription (28).

The downstream functions of H3 Lys36 methylation in yeast have been partially elucidated. This mark acts as a docking site for the chromodomain of Eaf3, a component of the Rpd3C(S) histone deacetylase complex (30–32). Histone deacetylation by Rpd3C(S) inhibits transcription initiation by RNA Pol II at cryptic start sites within open reading frames (30). The Set2/Rpd3C(S) pathway also inhibits elongation by RNA Pol II, and this inhibition is counteracted by the positive elongation factor Bur1 (31). Although BUR1 is a nearly essential gene (33–35), the severe growth defect of bur1{Delta} can be suppressed by deletions of either SET2 or genes encoding components of Rpd3C(S) (31, 36).

To further understand connections between H3 Lys36 methylation and transcription, we isolated high copy suppressors of bur1{Delta}. We reasoned that such suppressors may have a positive role in transcription and/or antagonize the Set2-Rpd3C(S) pathway. Two isolated suppressors were JHD1 and RPH1, both of which have a JmjC domain motif for histone demethylases. Jhd1 has previously been shown to specifically demethylate H3 Lys36 (25). Here we show that Rph1 can reverse both tri- and dimethylation at Lys36. We present evidence that the Jhd1 and Rph1 Lys36 demethylases promote transcription by RNA Pol II through repressive chromatin generated by Set2 and Rpd3C(S).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The following histone antibodies were used: anti-H3K36Me1 (Abcam 9048), anti-H3K36Me3 (Abcam 9050), anti-H3 (Abcam 1791), and anti-H3K36Me2 (Upstate%20Biotechnology">Upstate Biotechnology 07-369). Anti-Rpb3 was from Neoclone. Protein A- and protein G-Sepharose-4 Fastflow were from Amersham Biosciences, and IgG-agarose was from Sigma.

Yeast Strains and Plasmids—Yeast strains used in this study are listed in supplemental Table S1 and plasmids in supplemental Table S2. To generate pRS424 plasmids containing JHD1, RPH1, YJR119c/JHD2, GIS1, or ECM5, the entire ORF and 1kb of upstream region was amplified by PCR using oligonucleotide primers that create terminal NotI/SmaI (for RPH1, YJR119c/JHD2, and ECM5) or NotI/BamHI (for JHD1 and GIS1) sites. To construct pRS424-JHD1-3XHA, the JHD1 fragment digested with NotI and BamHI was cloned with a 500-bp BamHI/XhoI fragment containing 3x HA and SSN6 terminator from pBSSK(+)-3XHA/SSN6 terminator into the NotI/XhoI sites of pRS424. For cloning of RPH1, YJR119c/JHD2, ECM5 and GIS1, PCR fragments were gel-purified, digested with the appropriate enzymes for the new terminal sites, and cloned into the corresponding sites of pRS424-JHD1-3XHA. The downstream primers removed the stop codon and produced an in frame fusion to express triple-HA epitope tagged proteins. JHD1 and RPH1 point mutants were constructed by PCR using Pfu polymerase (Stratagene) and confirmed by sequencing. The sequences of oligonucleotides used in this study are listed in supplemental Table S3.

Phenotype Analyses—To isolate high copy suppressors of bur1{Delta}, the BUR1 shuffle strain YSB787 was transformed with pRS424 plasmids (2 micron, TRP1) containing different genes, and the resulting transformants were patched on synthetic complete media lacking uracil (as a positive growth control) or SC media containing 5-FOA (to select against the BUR1/URA3 plasmid). The plates were incubated for 2–6 days as indicated. Spotting analyses were performed as previously described (35).

Chromatin Pull-down Assay—Chromatin pull-down assays were carried out as described by Howe et al. (37). Whole cell extracts were made from wild type or set2{Delta} cells and nucleosomes were isolated via an Hhf2-TAP fusion protein by precipitation with IgG-agarose. A non-tagged strain served as a negative control. The bead-bound nucleosomes were incubated with whole cell extracts from cells containing either Jhd1-HA or Rph1-HA tagged proteins. After overnight incubation at 4 °C, the complexes were washed four times with lysis buffer (10 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A) and resolved by SDS-PAGE followed by immunoblotting analysis with the indicated antibodies.

Chromatin Immunoprecipitations—Chromatin immunoprecipitations were carried out as previously described with minor modifications (35, 38). For histone H3 and H3K36Me IPs, 1 µl of anti-H3 or 0.5 µl of anti-H3 K36Me3 were bound to protein A-agarose beads and used to precipitate chromatin. For K36Me3 antibody, binding was done overnight in FA lysis buffer (50 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 1% Triton X-100, 0.01% deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) containing 1 M NaCl. The precipitates were washed with the same buffer, once with FA lysis buffer containing 1.5 M NaCl, once with buffer containing 10 mM Tris-HCl (pH 8.0), 0.25 M LiCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and once with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Elution and decross-linking was performed as previously described (35, 38). The sequences of oligonucleotides for PCR amplification are in supplemental Table S3.

In Vitro Demethylase Assay—Recombinant Rph1 proteins were expressed as hexahistidine fusion proteins in Escherichia coli and purified using nickel-agarose. 4–6 µg of purified wild-type or mutated Rph1 proteins were incubated with 10 µgof calf thymus type II-A histones (Sigma) in reaction buffer (50 mM Tris-Cl (pH 7.5), 50 µM Fe(NH4)2(SO4)2, 1 mM {alpha}-ketoglutarate, and 2 mM ascorbate) for 2 h at 37 °C. The reaction was stopped by adding SDS-PAGE sample buffer and boiling. The reaction mixtures were subject to SDS-PAGE (15%) followed by Western blot analysis with the indicated antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of JmjC-domain Proteins Jhd1 or Rph1 Suppresses bur1{Delta}—Yeast cells grow poorly or not at all if they lack the Bur1/Bur2 kinase complex, but this requirement can be bypassed by mutation of H3 Lys36 to alanine, deletion of the SET2 methyltransferase gene, or by deletions of genes encoding components of the Rpd3C(S) HDAC complex (31, 36). These deletion mutants also exhibit increased resistance to 6-azauracil (6-AU) and mycophenolic acid (MPA), two chemicals that inhibit RNA Pol II transcription elongation by reducing nucleotide pools (16, 20, 31). These results indicate that Set2-dependent Lys36 methylation is a repressive mark that negatively regulates transcription. We therefore predicted that histone demethylases that can reverse Lys36 methylation should have a positive role in transcription. Furthermore, overexpression of histone demethylases for Lys36 might also suppress the growth defect of bur1{Delta}.

To explore this possibility, we tested whether overexpression of JmjC domain containing proteins could suppress the poor growth phenotype of a bur1{Delta} strain. Budding yeast has five JmjC proteins: Jhd1, Rph1, Gis1, Ecm5, and Yjr119c/Jhd2 (supplemental Fig. S1). Among these, two genes were considered likely candidates for bur1{Delta} suppressors. Jhd1 is most similar to mammalian JHDM1A, which antagonizes Lys36 mono- and dimethylation. Jhd1 can remove methyl groups from Set2 methylated H3 Lys36 in vitro, although no in vivo activity has been assigned (25). Recently, the PHD (plant homeodomain) finger of Ecm5 was reported to bind to trimethylated Lys36 in vitro (39). Since the Eaf3 subunit of Rpd3C(S) also binds methylated Lys36, Ecm5 overexpression might also suppress bur1{Delta} by competing with Eaf3 for binding to nucleosomes.


Figure 1
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  		FIGURE 1.
Overexpression of catalytically active Jhd1 or Rph1 suppresses bur1{Delta} lethality. A, high copy plasmids (based on pRS424) containing the indicated genes were transformed into a BUR1 plasmid shuffling strain (YSB787). The transformants were streaked onto synthetic complete (SC) media alone or containing 5-FOA to select against the BUR1/URA3 plasmid. SC-Ura plate is shown after 2 days; SC + 5-FOA after 5 days. B, strains were constructed as in A. Plasmids contained either wild-type JHD1 or RPH1 or the same genes mutated at a residue critical for demethylase activity in JmjC domain proteins. Cells were spotted in 3-fold dilutions onto an SC plate (shown after 2 days) or an SC + 5-FOA plate (shown after 6 days). A positive control for suppression (set2{Delta}) is shown for comparison. C, catalytically active and inactive demethylases are expressed at similar levels. Whole cell extracts prepared from strains containing the indicated pRS424 plasmids were analyzed by immunoblot analysis with anti-HA (the demethylases were epitope-tagged) and anti-Rpb3 antibodies (used as a loading control).

 
High copy plasmids expressing Jhd1, Rph1, Gis1, Ecm5, or Yjr119/Jhd2 were introduced into a BUR1-shuffling strain, and transformants were grown on synthetic complete (SC) media or media containing 5-FOA to test the ability to grow in the absence of BUR1. As predicted from its in vitro activity, Jhd1 overexpression allowed bur1{Delta} cells to grow (Fig. 1A), suggesting that Jhd1 acts as a H3 Lys36 demethylase in vivo. Surprisingly, high copy RPH1, but not GIS1, ECM5 or YJR119c/JHD2, also bypassed the requirement for BUR1 (Fig. 1A and data not shown). Rph1 was originally identified as a transcriptional repressor for the PHR1 gene (40). Interestingly, this protein has sequence similarity to mammalian JHDM3A/JMJD2A, a trimethyl-specific histone demethylase for H3 Lys9 and H3 Lys36 (27, 28, 41). These findings suggest that both Jhd1 and Rph1 may bypass the requirement of BUR1 by removing the repressive Lys36 methylation.

To test whether the histone demethylase activities of Jhd1 and Rph1 were required to bypass the requirement for BUR1, point mutants in a key catalytic histidine (41) were constructed. The Jhd1 H305A mutation abrogates demethylase activity in vitro (25), and this allele was unable to suppress bur1{Delta} (Fig. 1B). Similarly, overexpression of the H235A mutant Rph1 protein also failed to bypass the requirement for BUR1 (Fig. 1B). The mutant proteins were expressed at levels comparable with wild type (Fig. 1C), arguing that the loss of suppression is due to the loss of catalytic activity rather than defects in protein folding or stability. Neither protein suppressed as strongly as deletion of SET2 (Fig. 1B), suggesting that some Lys36 methylation persists even when the demethylases are overexpressed (see below).

Rph1 Reverses Trimethylation of H3 Lys36 in Vivo and in Vitro—To test whether overexpression of Jhd1 or Rph1 results in reduction of Lys36 methylation in vivo, immunoblot analysis with antibodies against different methylated forms of H3 Lys36 was carried out on chromatin fractions. Unfortunately, mono- and dimethylated Lys36 were not detectable under these conditions. However, trimethylation of H3 Lys36 was readily detected, and this signal was completely absent in set2{Delta} cells (Fig. 2A). Triple HA-tagged Jhd1 or Rph1 was overexpressed from the GAL10 promoter and equal amounts of chromatin fractions from the indicated strains were analyzed. Levels of Rpb3, histone H3, and dimethylated Lys4 were unaffected by overexpression of Jhd1 or Rph1. In contrast, Lys36 trimethylation levels were greatly reduced upon overexpression of Rph1 (Fig. 2B). Interestingly, overexpression of Jhd1 at best caused only a small decrease in Lys36 trimethylation.

To determine whether Rph1 directly acts as a Lys36 histone demethylase, recombinant Rph1 protein expressed in E. coli (shown in Fig. 2E) was tested for in vitro demethylase activity on purified bulk histones. Rph1 catalyzed demethylation of both the di- and trimethylated H3 Lys36, while mono-methylated Lys36 was unaffected (Fig. 2, C and D). This observation is consistent with previous reports that mammalian JHDM3A/JMJD2A is also capable of removing di- and trimethylation of H3 Lys36 in vitro (27, 28). An Rph1 protein mutated in a key residue for Fe(II) binding (H235A) was also tested for histone demethylase activity. This mutant protein had no effect on methylated Lys36 levels (Fig. 2, C and D).


Figure 2
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  		FIGURE 2.
Rph1 demethylates H3 Lys36 trimethylation in vivo and in vitro. A, specificity of anti-K36Me3 antibody for methylated H3 Lys36. Equal amounts of chromatin fractions prepared from wild type and set2{Delta} cells were analyzed by immunoblotting with anti-H3, anti-H3K36Me3, anti-Rpb3, and anti-H3-K4Me2 antibodies. B, overexpression of Rph1 reduces H3-K36Me3 in vivo. The indicated plasmids were transformed into wild-type strain and the transformants were cultured in medium containing galactose. Equal amounts of chromatin fractions from the indicated strains were analyzed by immunoblotting with the indicated antibodies. C, purified recombinant Rph1-HA and mutant Rph1-HA (H235A) proteins were incubated with purified bulk histones. The reaction mixtures were analyzed by immunoblot analysis with anti-H3, anti-H3-K36Me1, anti-H3-K36Me2, anti-H3-K36Me3, and anti-HA antibodies. D, the results from C were quantitated by densitometric scanning and methylation levels were plotted as a percentage of wild type. E, purified recombinant Rph1 and mutant Rph1(H235A) proteins were analyzed by gel electrophoresis and Coomassie staining. Size markers (M) are shown in the first lane of the gel; numbers are kilodaltons.

 


Figure 3
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  		FIGURE 3.
Association of Jhd1 and Rph1 with chromatin is independent of Set2-dependent methylation of histones. Nucleosomes were isolated via TAP-tagged Hhf2 (histone H4), while a negative control strain had no TAP tag. Beads carrying these nucleosomes were incubated with whole cell extracts containing either Jhd1-HA or Rph1-HA. The precipitated proteins were analyzed by immunoblot analysis with anti-PAP (to detect the TAP tagged histone) and anti-HA antibodies.

 
Jhd1 and Rph1 Associate with Nucleosomes Independently of H3 Lys36 Methylation—In most cases it is unclear how histone demethylases are recruited to their substrates. Some may be recruited to specific genes by interacting with sequence-specific DNA-binding proteins. Alternatively, they could be recruited directly or indirectly by other histone modifications such as acetylation, phosphorylation, ubiquitylation, or methylations at different residues. It was recently shown that JMJD2A/JHDM3A binds to methylated H3 Lys4 through its double Tudor domain (42). Jhd1 contains a PHD finger and Rph1 has two zinc fingers (supplemental Fig. S1). The Rph1 zinc domains have been implicated in sequence-specific binding (40).

To monitor the association of Jhd1 and Rph1 with histones, a chromatin binding assay was performed. Nucleosomes were isolated via a Hhf2-TAP protein from SET2 or set2{Delta} strains and incubated with whole cell extracts from cells expressing Jhd1-HA or Rph1-HA. Both Jhd1 and Rph1 co-precipitated with nucleosomes regardless of whether Lys36 methylation by Set2 was present (Fig. 3).

We performed chromatin immunoprecipitation to map the positions of Jhd1 and Rph1 along transcribed and transcriptionally inactive regions. However, no enrichment of Jhd1 or Rph1 was observed with either TAP-tagged or triple HA-tagged proteins (data not shown). Therefore, the association of these two JmjC proteins with histones might be equivalent throughout the genome or else too weak or transient to detect by ChIP.

Since these two demethylases might be working together, we tested whether Jhd1 and Rph1 are associated. However, precipitations of TAP-tagged Rph1 did not co-precipitate Jhd1. The converse experiment also gave negative results (data not shown).

Rph1 Regulates H3 Lys36 Methylation at Actively Transcribed Regions—Set2-dependent di- and trimethylation of H3 Lys36 generally peak near 3' ends of genes (18, 22). To test whether Rph1 affects H3 Lys36 methylation levels in transcribed regions of genes, we performed ChIPs with antibody against H3K36me3. The H3K36me3 signal was normalized for total H3. As seen in Fig. 4, A–C, trimethylation at the PMA1, ADH1, and YEF3 genes was completely abolished in a set2{Delta} strain. Overexpression of Rph1 partially decreased trimethylation within the body of genes but less so near promoters (Fig. 4, A–C). Interestingly, trimethylation levels were not affected by Jhd1 overexpression (data not shown). To confirm the effect of Rph1 on Lys36 methylation, we monitored trimethylation levels in deletion mutants for the Lys36 demethylase proteins. Wild type and jhd1{Delta} strains showed a normal pattern of Lys36 methylation, but increased levels of Lys36 trimethylation were seen in an rph1{Delta} mutant (Fig. 4, D–F).


Figure 4
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  		FIGURE 4.
Rph1 modulates H3-Lys36 trimethylation in transcribed regions. A, chromatin was prepared from wild-type strains containing pRS325-GALpro or pRS325-GALpro-RPH1-HA or from a set2{Delta} strain, all grown on galactose-containing media. DNA co-precipitated with anti-H3 or anti-H3-K36Me3 antibodies was analyzed by PCR using primers across the PMA1 gene (locations shown schematically at the top, PCR products in the middle). Signals from the gene-specific primers were normalized to those from a nontranscribed internal control (*) to generate a specific signal/background ratio. The signal for H3-K36Me3 was then further normalized to the total H3 signal and the ratios were graphed (bottom). Error bars show the standard deviation from three PCR reactions with two independent chromatin preparations. B, ChIP analysis for ADH1 gene was done as in part A. C, ChIP analysis for YEF3 gene was done as in A. D, chromatin from wild type, jhd1{Delta}, or rph1{Delta} strains was precipitated with anti-H3 or anti-H3-K36Me3 antibody. PCR analysis was performed as in A. E, ChIP analysis for ADH1 gene was done as in D. F, ChIP analysis for YEF3 gene was done as in D.

 
At least one function of H3 Lys36 methylation in transcribed regions is to recruit Rpd3C(S) to promote histone deacetylation. Therefore, cells lacking SET2 or components of Rpd3C(S) exhibit increased histone acetylation within the body of genes (30–32). Since our data indicate that Jhd1 and Rph1 antagonize this pathway, we tested for increased histone acetylation within transcribed regions in cells overexpressing Jhd1 or Rph1. As reported previously, acetylation of H3 and H4 at 3' end of genes was increased in set2{Delta}, but similar changes were not seen in Jhd1 or Rph1 overexpressing cells (data not shown). The changes in acetylation upon overexpression of the two JmjC proteins may be too subtle to detect by ChIP. Alternatively, the remaining Lys36 methylation in Jhd1 and Rph1 overexpressing cells may be sufficient for recruiting Rpd3C(S). In particular, monomethyl Lys36 may suffice because the Eaf3 chromodomain can bind to the monomethylated Lys36 in vitro (32).

Jhd1 and Rph1 Increase RNA Pol II Occupancy to Transcribed Regions—Given that the Set2/Rpd3C(S) pathway is repressive for transcription, the Lys36 demethylases should positively affect transcription. To test the effects of Jhd1 and Rph1 on transcribing RNA Pol II, cross-linking of polymerase subunit Rpb3 to actively transcribed regions was measured by ChIP. RNA Pol II binding to three actively transcribed genes, PMA1, ADH1, and YEF3, was significantly decreased in an rph1{Delta} strain and slightly reduced in a {Delta} jhd1 strain (Fig. 5, A–C). This effect is not due to differences in cell growth since the deletion mutants grow at the same rate as wild-type at different temperatures as well as on plates containing different carbon sources (Fig. 5D).

To further confirm the positive effect of Jhd1 and Rph1 on RNA Pol II transcription, a similar Rpb3 ChIP was carried out in a bur2{Delta} strain, which exhibits significantly reduced cross-linking of RNA Pol II. RNA Pol II occupancy in cells lacking Bur2 can be restored by deletions of genes for SET2 or components of Rpd3C(S) (31). As shown in Fig. 6, A and B, Rpb3 cross-linking to the PMA1 and ADH1 genes was increased in the bur2{Delta} background when Jhd1 or Rph1 is overexpressed. In contrast, TBP binding to promoters was unaffected.


Figure 5
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  		FIGURE 5.
jhd1{Delta} and rph1{Delta} reduce RNA Pol II cross-linking to transcribed regions. Cross-linked chromatin fractions from wild type, jhd1{Delta}, and rph1{Delta} strains were precipitated with anti-Rpb3 antibody. PCR analysis of the precipitated DNA was performed on the PMA1 (A), ADH1 (B), and YEF3 (C) genes. Signals from specific primer products were normalized to those from input, and the quantitated results are graphed at the bottom. D, the indicated strains were spotted onto YPD (1% yeast extract, 2% peptone, 2% dextrose) or YP (1% yeast extract, 2% peptone)-galactose plates at the indicated temperatures. YPD plates were photographed after 2 days; YP-galactose after 3 days.

 
The jhd1{Delta} and rph1{Delta} mutants were not sensitive to 6-AU or MPA (data not shown). However, overexpression of Rph1, but not Jhd1, conferred some resistance to 6-AU or MPA, as is also seen upon deletion of SET2 (Fig. 6C). This phenotype is consistent with the Rph1 demethylase playing a positive role in transcription elongation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It was recently discovered that at least some JmjC proteins can act as histone lysine demethylases, and the challenge is now to assign biological targets and functions to these enzymes. Here we show that the Saccharomyces cerevisiae Rph1 protein acts on H3 Lys36 tri- and dimethylated species both in vitro and in vivo. Similar results have been recently reported by two other groups (43, 44). Rph1 is the second yeast Lys36 demethylase described, since the Jhd1 protein has also been shown to act upon dimethylated Lys36 (25). Our results indicate that both of these proteins can antagonize Set2-dependent methylation, which is targeted to regions transcribed by RNA Pol II (16, 18, 19, 21). Methylation at Lys36 in transcribed regions leads to deacetylation and a more repressive chromatin structure that suppresses cryptic transcription initiation (30, 32).

In addition to repressing cryptic initiation, several lines of evidence indicate that methylation of Lys36 and subsequent deacetylation inhibit elongation by RNA polymerase II. Yeast strains lacking Set2 or the Rpd3(C)S complex grow better than wild-type cells on media containing 6-AU or MPA (16, 20, 31), two drugs that slow elongation by limiting nucleotide triphosphate pools. We show here that overexpression of Rph1 also confers this phenotype. Furthermore, deletion of the genes for the Set2/Rpd3(C)S pathway bypass the requirement for the positive elongation factor Bur1 (31, 36).

Here we show that overexpression of either Rph1 or Jhd1 also allows cells to grow in the absence of Bur1 and that this phenotype is dependent upon demethylase activity (Fig. 1). The relevant substrate for Bur1 kinase activity is still unknown, but candidates include the RNA pol II CTD, Rad6, or an unidentified elongation factor (35, 45, 46). It is unknown whether Bur1 might have a direct role in H3 Lys36 methylation, but we suspect that Bur1 function is necessary to allow RNA Pol II to transit through the repressive chromatin marked by Set2 methylation.

This suppression of bur1{Delta} lethality by demethylase overexpression is due to antagonism of the Set2/Rpd3(C)S pathway, because combining demethylase overexpression with deletion of Set2 or Rpd3(C)S provides no additional growth benefit (data not shown). In contrast, although deletion of CHD1 weakly suppresses a BUR1 deletion (31), there is further improvement in growth when Rph1 or Jhd1 is overexpressed (data not shown). ChIP experiments show that cells lacking Bur2 (the cyclin partner to the Bur1 kinase) have drastically reduced levels of RNA Pol II crosslinking, but these can be partially restored by deletion of SET2 (31) or overexpression of Rph1 or Jhd1 (Fig. 6). In wild-type cells, deletion of RPH1 or JHD1 leads to a reduction in levels of RNA Pol II crosslinking (Fig. 5). For all phenotypes tested, RPH1 seems to be stronger than JHD1, suggesting it may play the more significant in vivo role for H3 Lys36 demethylation in transcribed regions. However, it appears that both histone demethylases for H3 Lys36 can promote RNA Pol II transcription by removing the repressive Lys36 methylation.


Figure 6
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  		FIGURE 6.
Jhd1 or Rph1 overexpression restores RNA Pol II cross-linking in a bur2{Delta} strain. A, chromatin fractions from bur2{Delta} strains containing pRS325-GALpro, pRS325-GALpro-JHD1-HA, or pRS325-GALpro-RPH1-HA were incubated with anti-Rpb3 antibody and precipitated. PCR analysis of the precipitated DNA was performed on the PMA1 gene. The quantitated results are graphed at the right. B, chromatin immunoprecipitation was carried out as in part A except on the ADH1 gene. Chromatin was precipitated with anti-Rpb3 or anti-TBP antibody. C, cells overexpressing Rph1 or Jhd1 were spotted in 3-fold dilutions on SC plates (2 days) or SC plates containing 6-AU or MPA (3 days). For comparison, strains containing vector alone (pRS424) or a set2{Delta} are shown as negative and positive controls, respectively.

 
Interestingly, Rph1 was originally isolated as a transcriptional repressor for PHR1, the gene that encodes the DNA repair enzyme photolyase (40). This repression appears to be mediated by sequence specific binding of the Rph1 zinc fingers to a repressor element within the PHR1 promoter. Also, Rph1 is phosphorylated by Rad53 upon DNA damage (47). It is not yet known whether Rph1 phosphorylation by Rad53 affects Rph1 demethylation activity or whether demethylase activity is necessary for repression of PHR1. Nonetheless, these observations suggest the possibility that histone methylation/demethylation may be somehow linked to DNA repair.

In addition to Rph1, the JmjC protein Gis1 was also isolated as a transcriptional repressor of the PHR1 gene (40). The two proteins have significant sequence identity, but Gis1 lacks a key histidine that makes up part of the Fe(II) binding site and is therefore unlikely to have demethylase activity (41). In fission yeast, the Epe1 protein also lacks a key residue in the Fe(II) binding site and, unlike its mammalian homolog JHDM1, fails to catalyzes demethylation of Lys36 or Lys9 methylation in vitro (25). Nonetheless, the JmjC domain of Epe1 is involved in regulating H3 Lys9 methylation levels (48), suggesting that JmjC domains can have additional functions independent of histone demethylation.

Budding yeast has five JmjC-domain containing proteins (supplemental Fig. S1) and their functions are becoming clearer. Both Jhd1 and Rph1 are H3 Lys36 demethylases. As discussed above, Gis1 is very similar to Rph1, but its target or even whether it has demethylase activity is unclear. The two remaining JmjC proteins are Yjr119c/Jhd2 and Ecm5. Recently, it has been shown that Yjr119c/Jhd2 demethylates trimethylated Lys4 (49–51). Ecm5 lacks some conserved amino acids necessary for binding the cofactors Fe(II) and {alpha}-ketoglutarate, so its enzymatic function remains to be determined.

Is there a correspondence between the yeast and mammalian demethylases? Jhd1 is most similar to the mammalian JHDM1 family, while Rph1 and Gis1 are closest to JHDM3A/JMJD2A (41). JHDM1A predominantly catalyzes demethylation of mono- and dimethylated Lys36 (25). JHDM3A/JMJD2A is able to reverse all three methylation states of H3 Lys9 and H3 Lys36 in vitro and siRNA knockdown suggests this protein acts as a transcriptional repressor for ASCL2 (28). Yeast Yjr119c/Jhd2 most resembles the JARID subfamily, of which several members have recently been shown to demethylate H3 Lys4 (52–55). Based on the similarities in protein sequences and target residues, it seems likely that at least some biological functions of these enzymes are also conserved over eukaryotic evolution.

One important question that remains to be answered is how the H3 Lys36 demethylases are targeted. The demethylases could randomly target histones, functioning to generally promote turnover of methylation marks. Alternatively, JmjC proteins could be associated with elongating RNA Pol II, but so far there are no reports of association between polymerase and either Jhd1 or Rph1. We were unable to detect either demethylase by ChIP across several genes (data not shown). Finally, there could be targeting to specific genes, or locations within genes, through the additional domains typically found in JmjC proteins. These include Tudor, PHD finger, ARID, and JmjN domains. Rph1 contains a JmjN domain and two zinc finger motifs. The zinc fingers mediate sequence-specific interactions with DNA (40), while the function of the JmjN domain is not understood. Interestingly, the JmjN domain is very similar to the dimerization domain of the pyrimidine nucleotide biosynthetic regulator PyrR (56).

We showed that Jhd1 and Rph1 can associate with nucleosomes in vitro independently of Lys36 methylation by Set2 (Fig. 3). JHDM3A/JMJD2A binds to methylated Lys4 of H3 via its double Tudor domain (42). A recent study showed that the PHD finger of Jhd1 can bind to trimethylated Lys4 in vitro (39), although the biological meaning of this interaction is unclear. One interesting possibility is that H3 Lys36 demethylases are targeted to 5' regions of genes via binding to H3 Lys4 methylated histones. This might function to cleanly separate the activating Lys4 methylation from repressive Lys36 methylation and thereby promote transcription.


   FOOTNOTES
 
* This work was supported by Grant GM46498 (to S. B.) from the National Institutes of Health. This work was also supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-214-C00065). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1, Tables S1–S3, and additional references. Back

{diamondsuit} This article was selected as a Paper of the Week. Back

1 To whom correspondence should be addressed. Tel.: 617-432-0696; Fax: 617-738-0516; E-mail: SteveB{at}hms.harvard.edu.

2 The abbreviations used are: Pol II, polymerase II; CTD, C-terminal domain; HA, hemagglutinin; 5-FOA, 5-fluoroorotic acid; 6-AU, 6-azauracil; MPA, mycophenolic acid; PHD, plant homeodomain; ChIP, chromatin immunoprecipitation. Back


   ACKNOWLEDGMENTS
 
We thank Fred Winston, Gerry Fink, and LeAnn Howe for strains and plasmids and Yang Shi and Johnathan R. Whetstine for helpful discussions.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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