chapter 4 · deletions of not4 and bur2 display similar ... but not with not3, caf40 and caf130...

Chapter 4: The Ccr4-Not complex regulates histone methylation

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Chapter 4The Ccr4-Not and Bur1/2

complexes cooperate to regulatehistone H3K4 tri-methylation

Submitted for publication to Molecular and Cellular Biology


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The Ccr4-Not and Bur1/2 complexes cooperate toregulate histone H3K4 tri-methylationKlaas W. Mulder1, Arjan B. Brenkman2, Akiko Inagaki1, Niels J.F. van den Broek2

and H. Th. Marc Timmers11Department of Physiological Chemistry, 2Department of Physiological Chemistry Mass Spectrometry Unit,University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

ABSTRACTEfficient transcription is directly linked to modification of chromatin. For instance, tri-methylation of lysine 4 on histone H3 (H3K4) strongly correlates with transcriptionalactivity and is regulated by the Bur1/2 kinase complex. Here, we find that theevolutionary conserved Ccr4-Not complex is critical for tri-methylation of H3K4 inSaccharomyces cerevisiae. We observe synthetic lethal interactions of Ccr4-Notcomponents with BUR1 and BUR2 . Deletions of NOT4 and BUR2 display similarphenotypes and are defective in transcriptional induction of RNR3 following HUtreatment. Further analysis indicates that the genes encoding the Not-proteins areessential for efficient regulation of H3K4me3, but not H3K4me1/2, H3K36me2 orH3K79me2/3 levels. Moreover, Not4p localization coincides with occurrence of theH3K4me3 mark at the 5’-region of the PYK1 gene. Finally, we find NOT4 to be importantfor ubiquitylation of histone H2B via recruitment of the PAF complex, but not forrecruitment of the Bur1/2 complex. These results suggest a mechanism in which theCcr4-Not complex functions downstream of the Bur1/2 kinase to facilitate H3K4me3 viaPAF complex recruitment.

INTRODUCTIONEfficient transcription in eukaryotesrequires interplay between the RNApolymerase II (pol II) transcriptionmachinery and factors regulatingmodification of the chromatin (reviewed in:(Berger, 2002). Tri-methylation of lysine 4on histone H3 (H3K4) occurs co-transcriptionally (Ng et al., 2003). Inaddition, it is strongly correlated withtranscriptional activity (Santos-Rosa et al.,2002) and is mediated by the Set1phistone methyltransferase (HMT) complexin yeast (Krogan et al., 2002; Roguev etal., 2001). In recent years, several factorsrequired for this modification have beenidentified. It is now apparent thatubiquitylation of histone H2B is necessaryfor methylation of H3K4 by Set1p (Sunand Allis, 2002) and of H3K79 by Dot1p(Ng et al., 2002). H2B ubiquitylation ismediated by the Bre1p-Rad6p E3-E2 pair(Hwang et al., 2003; Wood et al., 2003a).The Bur1/2 kinase complex is involved intranscription elongation. This CDK/cyclin

pair can phosphorylate the C-terminaldomain (CTD) of the largest subunit of polII in vitro (Murray et al., 2001), but it doesnot seem to contribute to CTDphosphorylation in vivo (Keogh et al.,2003). Interestingly, this kinase complex isrequired for H2B ubiquitylation and H3K4methylation (Laribee et al., 2005), whichmay involve direct phosphorylation ofRad6p (Wood et al., 2005). Like theBur1/2 complex, the PAF complex isimplicated in transcription elongation andis essential for efficient H2B ubiquitylationand H3K4 methylation, but not forrecruitment of Rad6p (Krogan et al.,2003a; Wood et al., 2003b). The PAFcomplex consists of five subunits (Paf1p,Rtf1p, Ctr9p, Leo1p and Cdc73p) andinteracts with pol II (Mueller and Jaehning,2002; Squazzo et al., 2002). BUR2 isrequired for efficient PAF complexrecruitment to chromatin, but themechanism by which this occurs remainsunclear (Laribee et al., 2005; Wood et al.,2005).


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The evolutionary conserved Ccr4-Notcomplex is composed of nine coresubunits and has been implicated invarious steps of mRNA production andprocessing (reviewed in (Collart andTimmers, 2004; Denis and Chen, 2003).The genes encoding the Not-proteins(N O T 1 - 5 ) were initially identified asnegative regulators of transcriptioninitiation. Support for such a role wasprovided by the observation that severalmutations in N O T genes suppress atemperature sensitive allele of SRB4 (Leeet al., 1998), which encodes an essentialsubunit of the Mediator co-activatorcomplex (Holstege et al., 1998). However,various reports also indicate a positive rolefor the Ccr4-Not complex (Denis et al.,2001; Liu et al., 1998; Mulder et al., 2005).For example, this complex is required fortranscription of RNR genes following DNAdamage or replication stress (Mulder et al.,2005). Genetic and physical interactionsbetween Ccr4-Not complex componentsand transcription initiation and elongationfactors have been described (reviewed in(Collart and Timmers, 2004; Denis andChen, 2003)). Besides this, Ccr4p andCaf1p represent the major mRNAdeadenylases in yeast (Tucker et al.,2001).To further investigate the role of the Ccr4-Not complex, we performed a genome-wide screen to find non-essential genedeletion mutants that display syntheticgenetic interactions with a deletion ofN O T 4 . Here, we describe geneticinteractions of components of the Ccr4-Not complex with BUR2 and BUR1. Wefound that the NOT genes are required tospecifically facilitate tri-methylation, but notdi-methylation, of H3K4. Deletion of NOT4reduced both histone H2B ubiquitylationand PAF complex recruitment, but did notaffect Bur1/2 recruitment.. Together, ourresults show a novel role for the Ccr4-Notcomplex in chromatin modification andsuggest a mechanism by which itcontributes to positive regulation oftranscription.

MATERIALS AND METHODSYeast genetics, media and plasmidsThe yeast strains used in this study andtheir relevant genotypes are indicated inTable 1. Knock-out, TAP- and mycAVI-tagged strains were constructed byhomologous recombination of a PCRproduct and verified by PCR, phenotypicand/or Western blot analysis. Cells wereroutinely cultured in YPD or SC mediumlacking the appropriate amino acids. TheGAL1-LacZ fusion and pGR422 plasmidshave previously been described (Gardneret al., 2005; Rondon et al., 2003). ThepRS306 based NOT4 integrative vectorwas previously published (Mulder et al.,2005).

Phenotypic assaysTen or 5-fold serial dilutions of theindicated strains were spotted on SC-U -/+6-azauracil (100 µg/ml), YPD -/+ 100 mMHU or SC -/+ 5-FOA (0.1%). For the 6-AUsensitivity assay, cells were transformedwith pRS316. Growth at 30°C wasassessed after 3-4 days.

In vivo elongation assayCells transformed with the GAL1-LacZplasmid were grown in SC-U mediumcontaining 2% raffinose overnight,collected and subsequently shifted tomedium containing 2% galactose.Samples were taken at the indicated time-points. RNA extraction and Northern blotanalysis were performed as describedpreviously (Mulder et al., 2005). PCRproduct probes spanning the ORFs wereradio labeled using the Redi-prime II kit(InVitrogen).

Western blotting and antibodiesSamples were taken from cells grown inYPD and extracts were prepared asdescribed previously (Kushnirov, 2000).Proteins were separated by SDS-PAGEand analyzed by Western blot. Antibodiesagainst H3K4me1 (Ab8895), H3K4me2(Ab7766) , H3K4me3 (Ab8580) ,H3K79me3 (Ab2621) and the C-terminusof H3 (Ab1791) were obtained from


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Abcam. H3K36me2 (#07-369) andH3K79me2 (#07-366) antibodies werefrom Upstate Biotechnology. TBPantiserum was a kind gift from P.A. Weil.Antibody 8WG16 was used to detect theCTD of RNA pol II. TAP-tagged proteinswere detected using IgG-peroxidaseconjugates. Antibodies against the HA-tag(12CA5 and 3F10) or the FLAG-tag (M2,sigma) were used to detect Ctr9-HA, Bur1-HA, Bur2-HA and FLAG-H2B,respectively.

Chromatin Immunoprecipitation (ChIP)Cell cultures and extract preparation wasessentially performed as describedpreviously with minor modifications(Mulder et al., 2005). Briefly, proteinA-coupled agarose beads were incubatedwith the appropriate antibody for 30 min at4°C, washed and subsequently incubatedwith extracts of cross-linked cells for 2-3hours at 4°C. Beads were washed, DNAeluted, and cross-links reversed at 65°Covernight. DNA was isolated using a PCRpurification kit (Qiagen) and analyzed bySYBR-green based quantitative PCRanalysis. Not4-mycAVI was purified usingstreptavidin coated Dyna-beads (Dynal)and subjected to stringent washingconditions (3% SDS in TE) before reversecross-linking. Further analysis wasperformed as above. Data is representedas percentage of input with a region fromthe HMR locus as a control.

Northern blot analysis and reversetranscriptase qPCRRNA extraction and Northern blots wereperformed as described previously (Mulderet al., 2005). Equal amounts of total RNAwere used to prepare cDNA using randomhexamers and the SuperScript II kit( InV i t rogen) accord ing to themanufacturers protocol. A dilution series ofgenomic DNA was used to determine thecDNA levels by SYBR-green basedquantitative PCR analysis.

RESULTSThe Ccr4-Not complex is involved intranscription elongationTo obtain more insight into the function ofthe Ccr4-Not complex, we performed agenome-wide survey to find syntheticgenetic interactions with a null allele ofNOT4. We identified several interactionswith genes implicated in transcriptionelongation by RNA polymerase II (K.W.M.,A.I. and H.T.M.T., manuscript inpreparation). Most striking was theinteraction between NOT4 and BUR2 .Subsequent tetrad analysis of doubledeletions of B U R 2 and Ccr4-Notcomponents showed additional syntheticlethal interactions with NOT2 and CCR4,but not with NOT3, CAF40 and CAF130(Figure 1A). Notably, deletion of the geneencoding Bur1p, the cyclin dependentkinase partner of Bur2p, results in a stronggrowth defect and this strain was notrepresented in the library used for thegenetic screen. To test a geneticinteraction between NOT4 and BUR1, weintroduced a plasmid-based WT ortemperature sensitive mutant of BUR1(bur1-23) into either NOT4 or not4Δ strainsin which the sole copy of B U R 1 wasexpressed from a URA3 plasmid(pRS316). Plasmid shuffle analysisdemonstrated synthetic lethality between adeletion of NOT4 and bur1-23, already atthe permissive temperature (Figure 1B).Considering the fact that the Bur1/2complex was found to play a role intranscription elongation (Keogh et al.,2003; Murray et al., 2001), not4Δ andbur2Δ strains were tested for sensitivity to6-azauracil (6AU). In agreement withobservations linking the Ccr4-Not complexto transcription elongation (Denis et al.,2001), cells lacking NOT4 or BUR2 were6AU-sensitive (Figure 1C). Strikingly, astrict correlation was observed betweenthe 6AU-sensitivity of Ccr4-Not genedeletions and synthetic lethality with BUR2(Denis et al., 2001). This suggests that thissynthetic lethality is due to defects intranscription elongation. To extend theseobservations, we investigated requirement


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Figure 1. Ccr4-Not complex components geneticallyinteract with BUR1 and BUR2 and play a role intranscription elongation. (A) Tetrad analysis ofbur2Δ (KMY201) and BY4741 Ccr4-Not deletion ornot4Δ (KMY40) BY4741bur2Δ diploids. Circlesindicate double knock-out strains. Relevantgenotypes of the obtained haploid strains areindicated (B) Plasmid-shuffle analysis of bur1-23and not4Δ double mutant strains containingpRS316-BUR1 . Ten-fold serial dilutions of theindicated strains were spotted on SC and SCcontaining 0.1% 5-FOA. (C) 6-azauracil sensitivityassay. Ten-fold serial dilutions of the indicatedstrains were spotted on SC-U and SC-U containing6-AU (100 µg/ml). (D) In vivo transcriptionelongation analysis of a long GC-rich reporter.BY4741 and not4Δ (KMY58) strains transformedwith a GAL1pr-LacZ plasmid (pGR422) were grownin medium containing raffinose and shifted tomedium containing galactose for 0, 45 and 90minutes. Northern blot analysis on LacZ andendogenous GAL1 mRNA was performed using 18SrRNA as a control.

of NOT4 for transcription of a long andGC-rich reporter gene. This experimentalset-up has previously been used to studyelongation defects (Krogan et al., 2003b;Rondon et al., 2003). Figure 1D showsthat in cells lacking NOT4 , the G A L 1

promoter driven LacZ reporter gene wasinefficiently transcribed, whereas theendogenous GAL1 gene was expressed toWT levels. Together, these experimentsshow that Ccr4-Not complex componentsgenetically interact with both BUR2 andBUR1 and confirm a role for this complexin transcription elongation (Denis et al.,2001).

Efficient H3K4 tri-methylation isdependent on the Ccr4-Not complexThe Bur1/2 complex has been implicatedin regulation of tri-methylation of H3K4(Laribee et al., 2005; Wood et al., 2005).Based on the genetic interactions betweenNOT4, BUR2 and BUR1, involvement ofthe Ccr4-Not complex in this process wasinvestigated by analyzing global levels ofH3K4me1, H3K4me2 and H3K4me3 usingspecific antibodies. Strains deleted forNOT4 or NOT5 displayed a loss of H3K4tri-methylation, but not of mono- or di-methylation (Figure 2A). However, deletionof NOT3 , CCR4 or any of the Ccr4-associated factors (CAFs) did not give riseto this effect (Figure 2A). It is worth notingthat several CCR4-NOT gene deletionphenotypes are not evident in not3Δ cells(Liu et al., 1998; Mulder et al., 2005),Figure 1A and data not shown).To extend these observations, two mutantalleles of the essential NOT1 gene anddeletions of the other NOT genes wereanalyzed in a different geneticbackground. Under permissive conditions,both no t1 alleles displayed a cleardecrease in overall H3K4me3 levels (50-70% as shown by quantification of theresults). Moreover, deletion of NOT2 ,NOT4 or NOT5 reduced H3K4me3 levels(86-94%; Figure 2B). In contrast,H3K4me2 levels were not affected to thesame extent (Figure 2B). Set1p complexeslacking Spp1p are unable to tri-methylateH3K4 (Morillon et al., 2005; Schneider etal., 2005). Deletion of NOT4 or NOT5resulted in H3K4me3 levels comparable todeletion of SPP1 (Figure 2C). In addition,H3K79me2 and H3K79me3 levels inextracts of CCR4-NOT deletion strains


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Figure 2. Specific requirement for the NOT genesfor global H3K4 tri-methylation. (A) Histone H3K4mono-, di- and tri-methylation levels weredetermined in lysates from logarithmically growingBY4741-based CCR4-NOT gene deletion strains.Protein extracts of the indicted strains wereseparated by SDS-PAGE (15%) and subjected toWestern blotting using H3K4me1, H3K4me2 andH3K4me3 specific antibodies. TBP levels weredetermined as a loading control. (B) MY1-basedN O T mutant strains were used to determineH3K4me2, H3K4me3 and total H3 levels as in (A).Quantified H3K4me3 and H3K4me2 levels areshown as relative to WT after normalization to totalH3 levels. (C) Direct comparison of H3K4me2 andH3K4me3 levels in BY4741, not4Δ , not5Δ andspp1Δ strains. Analysis was performed as in (A).(D) H3K79me2 and H3K79me3 levels in theindicated BY4741 deletion strains. Analysis wasperformed as in (A) (E ) Silencing assay usingstrains, containing URA3 in the telomeric region ofthe left arm of chromosome 7, lacking NOT4 orDOT1. Cells were spotted in 5-fold serial dilutionson SC plates or on SC plates containing 5FOA.

were not affected. As expected deletion ofR A D 6 led to a severe decrease inH3K79me2 and H3K79me3 levels. Theseresults suggest a specific requirement forthe NOT genes for H3K4 tri-methylation

(Figure 2D). Dot1p, the histonemethyltransferase specific for methylationof H3K79 plays an important role insilencing of genes near telomeres (vanLeeuwen et al., 2002). Deletion of SET1also results in a defect in this process(Krogan et al., 2002). Notably, integrity oftelomeric silencing was not affected bydeletion of N O T 4 (Figure 2E). Thisobservation is consistent with the notionthat H3K4me3 is not required for telomericsilencing (Schneider et al., 2005).Together, these results show that the NOTgene encoded module of the Ccr4-Notcomplex is essential for H3K4 tri-methylation but not for H3K79 methylationor telomeric silencing.

NOT4 and BUR2 are critical for efficienttranscription and H3K4 methylation ofthe RNR3 geneThe Not-module of the Ccr4-Not complexis essential for tolerance to hydroxyurea(HU) by facilitating transcription of RNRgenes (Mulder et al., 2005). In agreementwith previous observations (Wood et al.,2005), deletion of BUR2 also resulted insensitivity to HU (Figure 3A). In addition,we found that BUR2 was required fortranscription of R N R 3 following HUtreatment, as previously found for NOT4(Figure 3B).The H3K4 methylation status of the RNR3locus was analyzed by chromatin-immunoprecipitation (ChIP) using extractsfrom WT, not4Δ and bur2Δ cells, beforeand after induction with HU (Figure 3C).As expected, H3K4me2 and H3K4me3levels were elevated and pol II wasrecruited following HU treatment in WTcells (Figure 3D). Interestingly, noinduction of H3K4 di- or tri-methylation orrecruitment of pol II was observed in cellslacking NOT4, BUR2 (Figure 3E and F) orR A D 6 (data not shown). Theseexperiments suggest that NOT4 andB U R 2 collaborate to regulate H3K4methylation and induction of RNR3transcription following HU treatment.


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Figure 3. Transcription and H3K4 methylation of theRNR3 gene are dependent on both NOT4 andBUR2. (A) HU sensitivity of cells lacking NOT4 orBUR2. BY4741 not4Δ and bur2Δ strains werespotted in 10-fold serial dilutions on YPD or YPDcontaining 100 mM hydroxyurea (HU). (B) HUinduced RNR3 transcription in cells lacking NOT4 orBUR2. Exponentially growing BY4741, not4Δ andbur2Δ strains were (mock-) treated with 200 mM HUfor 2 hours in YPD. RNA was extracted andsubjected to quantitative reverse-transcriptase PCR.(C) Schematic representation of the RNR3 locusand the amplicons used in panels (D-F). ( D )Chromatin immunoprecipitation (ChIP) analysis ofthe RNR3 locus. Exponentially growing BY4741,not4Δ and bur2Δ strains were (mock-) treated with200 mM HU for 2 hours in YPD. Chromatinimmunoprecipitation (ChIP) was performed usinghistone H3K4me3 specific antibodies. (E) ChIPanalysis as in (B) using histone H3K4me2 specificantibodies. (F) ChIP analysis as in (B) using CTDantibodies.

H3K4 tri-methylation on the PYK1 locusrequires NOT4To investigate a role for N O T 4 inregulation of histone H3 methylation of theconstitutively active and highly expressedPYK1 gene, ChIP experiments usingextracts of WT and no t4Δ cells wereperformed. H3K4me3, H3K4me2,H3K36me2 and H3K79me2 levels at the 5’and the middle region of the PYK1 ORFwere examined (Figure 4A-E). Not4Δ cellsdisplayed a clear decrease of the level ofH3K4me3 at the 5’ end of the PYK1 ORFcompared to WT (Figure 4B), whereasH3K4me2 levels were not significantlyaffected by deletion of NOT4 (Figure 4C).ChIP analysis of an spp1Δ strain showed areduction of the H3K4me3 levels at thePYK1 locus similar to deletion of NOT4(Supplemental Figure 1A and B). Furtheranalysis showed that H3K36me2 andH3K79me2 levels were not decreased innot4Δ cells (Figure 4D and 4E). Inaddition, pol II occupancy was determinedusing antibodies against the CTD ofRpb1p as a measure for transcriptionalactivity. In parallel, P Y K 1 and TUB1mRNA levels were measured byquantitative RT-PCR in WT and not4Δcells. Both pol II association to the PYK1ORF and PYK1 transcript levels werediminished in the absence of NOT4(Figure 4F and G). In agreement with this,we found that deletion of SPP1 affectedPYK1 transcript levels to the same extentas deletion of NOT4 (Supplemental Figure1C). Extending on these results, wedetermined the H3K4 methylation status ofcells deleted for N O T 2 . Indeed,examination of not2Δ cells confirmed arequirement for NOT2 in regulation ofH3K4me3 specifically (Figure 4H and I).Notably, essentially identical results wereobtained when analyzing the PGK1 locus(Supplemental Figure 2A-H), suggesting abroad role for N O T 4 in regulatingH3K4me3 levels.To assess direct involvement of Not4p inregulation of PYK1 expression, ChIPanalysis using a chromosomally


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expressed tagged form of NOT4 wasperformed. The encoded protein (Not4p-mycAVI) was biotinylated in vivo by co-expression of the E. coli derived BirAbiotin ligase (van Werven and Timmers,2006). Streptavidin coupled beads wereused to capture the biotinylated Not4proteins from chromatin extracts of cellscontaining either a plasmid expressingBirA or an empty vector. Interestingly,Not4p was detected on the 5’ end of thePYK1 ORF and the region spanning theTATA-box (Figure 4J), coinciding with theregion targeted for tri-methylation by theSet1p complex (Figure 4C). Takentogether, these ChIP experiments confirmthe specificity of the H3K4me3 defect andindicate that Not4p is physically present onthe 5’ region of the PYK1 ORF. In addition,deletion of NOT4 resulted in decreasedpol II occupancy and transcript levels,suggesting direct regulation of PYK1expression by Not4p through tri-methylation of H3K4.

Figure 4. NOT4 is required forH3K4 tri-methylation but not di-methylation of the PYK1 ORF. (A)Schematic representation of theP Y K 1 locus and the ampliconsused in (B-H). (B) ChIP analysis ofthe PYK1 ORF in BY4741 andno t4Δ strains. Exponentiallygrowing cells were subjected toChIP analysis using H3K4me3antibodies. (C) As in (A), usingH3K4me2 antibodies. (D) As in(A), using H3K36me2 antibodies.(E) As in (A), using H3K79me2antibodies. (F) As in (A), usingCTD antibodies. (G) Quantitativereverse- t ranscr ip tase PCRanalysis of PYK1 and T U B 1mRNA levels in BY4741 WT andnot4Δ strains. (H) ChIP analysis ofH3K4me3 levels in MY1 WT andnot2Δ cells. (I) ChIP analysis ofH3K4me2 levels in MY1 WT andnot2Δ cells. (J) Not4p is recruitedto the 5’ region of the PYK1 ORF.Strains expressing mycAVI-taggedNot4p were transformed with aBirA expression plasmid andsubjected to ChIP analysis.Signals were normalized to anempty plasmid control.

NOT4 is critical for ubiquitylation ofhistone H2B and recruitment of the PAFcomplexH2B ubiquitylation is required for efficientH3K4 methylation (Wood et al., 2005). Toinvestigate a role for Not4p inubiquitylation of H2B, plasmid-basedFLAG-tagged H2B was expressed innot4Δ cells. Ubiquitylated H2B could bedetected in logarithmically growing WTcells, but was decreased in cells lackingNOT4 and absent from cells expressingFLAG-tagged H2B-K123R (Figure 5A).H2B ubiquitylation is dependent onrecruitment of the PAF complex tochromatin (Wood et al., 2003b). To assessa role for Not4p in recruitment of the PAFcomplex, strains expressing HA-taggedCtr9p and containing or lacking NOT4were constructed. Ctr9-HA was expressedto equal levels in the two strains, whereasH3K4me3 levels were reduced in thenot4Δ cells as expected (Figure 5B).Extracts were prepared from exponentially


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growing cells and subjected to ChIPanalysis. Whereas Ctr9-HA was readilydetected in the ORFs of PYK1 and PGK1in WT cells, binding was severely reducedin cells lacking NOT4 (Figure 5C and D).Together, these results show arequirement for N O T 4 in H2Bubiquitylation and a role in regulation ofPAF complex recruitment to the PYK1 andPGK1 genes to facilitate tri-methylation ofH3K4.

The Ccr4-Not complex functionsdownstream of the Bur1/2 kinaseChromatin association of the PAF complexdepends on the Bur1/2 complex (Laribeeet al., 2005; Wood et al., 2005). Therequirement for NOT4 for efficient PAFcomplex recruitment places the Ccr4-Notcomplex in the pathway containing theBur1/2 kinase. To investigate this further,strains expressing HA-tagged Bur1p orBur2p, containing or lacking NOT4, wereconstructed to determine occupancy ofBur1p and Bur2p. Expression of the HA-tagged versions of Bur1p and Bur2p was

Figure 5. Deletion of NOT4diminishes H2B ubiquitylation andPAF complex recruitment. (A) NOT4is required for efficient H2Bubiquitylation. Extracts of theindicated strains carrying a FLAG-HTB1 plasmid were separated on a12.5% SDS-PAGE gel andsubjected to Western blot analysisusing FLAG antibodies. The lowerpanel shows a shorter exposure ofthe same blot, indicating equalloading. (B) Ctr9-HA is equallyexpressed in NOT4 and not4Δ cells.Strains expressing HA-tagged Ctr9p,containing or lacking NOT4, wereused to determine Ctr9-HA levels byWestern blotting. H3K4me3 andTBP levels were used as controls.(C, D) NOT4 is required for PAFcomplex recruitment to the PYK1and PGK1 ORFs. Strains from (B)were subjected to ChIP analysis ofthe PYK1 locus (amplicons areschematically depicted in upperpanel). Exponentially growing cellswere cross-linked and ChIPs wereperformed using anti-HA (12CA5)

antibodies.

equal between WT and not4Δ cells (Figure6A). ChIP analysis with these strainsshowed that both Bur1p and Bur2p werefound to associate with the PYK1 andPGK1 loci with similar efficiencies (Figure6B and C). These results place the Ccr4-Not complex downstream of the Bur1/2kinase and upstream of the PAF complexin the pathway leading to H3K4 tri-methylation.

DISCUSSIONThe Ccr4-Not complex is involved inmRNA biogenesis at different levels(reviewed in: (Collart and Timmers, 2004;Denis and Chen, 2003)). Here, we showthat the NOT genes, in contrast to CCR4and the CAF genes, are required for globaland gene specific tri-methylation of H3K4(Figure 2, 3 and 4). In addition, we foundthat the Ccr4-Not complex is functionallylinked to the Bur1/2 kinase complex(Figure 1 and 3), which is also implicatedin H3K4 tri-methylation. The mechanismby which the Ccr4-Not complex is involvedin this histone modification is independent


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Figure 6. The Ccr4-Not complex functionsdownstream of the Bur1/2 kinase. (A) Bur1-HA andBur2-HA are equally expressed in WT and not4Δcells. Extracts of cells expressing HA-tagged Bur1por Bur2p containing or lacking NOT4 , or a non-tagged control, were subjected to Western blotanalysis using antibodies against the HA-tag,H3K4me3 and TBP. (B) Recruitment of the Bur1/2complex to the PYK1 locus does not depend onNOT4. WT and not4Δ cells expressing HA-taggedBur1p or Bur2p, and a no-tag control, weresubjected to ChIP analysis using anti-HA (12CA5)antibodies (amplicons are schematically depicted inthe upper panel). (C) As in (B), except that theanalysis was performed for the PGK1 locus.

of direct regulation of the Set1p histonemethyltranferase complex (discussedbelow, Supplemental Figure 3). Instead,the Ccr4-Not complex functionsdownstream of Bur1/2p at the level of PAF

complex recruitment, resulting indecreased ubiquitylation of H2B, in cellslacking NOT4 (Figure 5 and 6). Together,our results provide a detailed study of therole of the Ccr4-Not complex in regulationof global H3K4 tri-methylation. Further,they confirm a functional distinctionbetween the cytoplasmic deadenylaseactivity of the Ccr4p-Caf1p module and thenuclear function of the Not-proteins of theCcr4-Not complex (Bai et al., 1999).

Regulation of H3K4 tri-methylation bythe Ccr4-Not complex and involvementof the PAF complexOur data show that the Ccr4-Not complexfacilitates tri-methylation of H3K4 on aglobal scale by regulation of H2Bubiquitylation through PAF complexrecruitment. Interestingly, Not4p containsa RING-finger motif and displaysubiquitylation activity in vitro (K.W.M., A.I.and H.T.M.T., manuscript in preparation).Therefore, the H3K4me3 status wasdetermined in cells expressing an inactive(L35A) mutant of Not4p. However, theH3K4me3 defect of not4Δ cells wascomplemented by the L35A mutant ofNot4p, suggesting that its RING-finger isnot required for ubiquitylation of histoneH2B (data not shown). Anotherexplanation for the observed effect onH3K4me3 levels would be that NOT4 isrequired for either transcription of genesencoding or incorporation of subunits ofthe Set1p complex. For instance, deletionof SPP1 results in formation of a Set1pcomplex that is unable to tri-methylatelysine 4 on histone H3 (Wood et al., 2005).However, neither transcript levels of itssubunits nor composition of the Set1pcomplex were significantly affected bydeletion of NOT4 (Supplemental Figure 3Aand B). In addition, using soluble histonesor (pre-methylated) synthetic peptides assubstrates, no intrinsic defect in themethylation potential of Set1p complexespurified from not4Δ cells could be detected(Supplemental Figure 3C and D). Themechanism underlying the H3K4me3


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Figure 7. The Ccr4-Not complex functionsdownstream of the Bur1/2 kinase in the pathwayleading to tri-methylation of H3K4. In short, theCcr4-Not complex functions downstream of theBur1/2 complex and facilitates recruitment (or stableassociation) of the PAF complex to chromatin. Thisis required for subsequent efficient H2Bubiquitylation by Bre1p and Rad6p leading to tri-methylation of H3K4. Since mutants of the Ccr4-Notcomplex do not affect H3K79 methylation, thissuggest bifurcation of these pathways upstream ofthe Ccr4-Not complex, but upstream of Bur1/2. Inaddition, the spatial restriction of Ccr4-Not complexrecruitment might aid localization of the H3K4me3mark.

defect in not4Δ cells is therefore distinctfrom deregulation of the Set1p complex atthe level of transcription, composition orintrinsic activity and specificity. Inagreement with this conclusion is theobservation that Set1p is not recruited tothe RNR3 locus upon stimulation with HU(Mulder et al., 2005). In addition, thisobservation can account for the observedeffect on H3K4me2 levels at the RNR3locus in cells lacking NOT4 (Figure 3).Moreover, the observation that Not4p isphysically present at the 5’ region of thePYK1 ORF, coinciding with the H3K4methylation mark and the PAF complex,

suggests a direct role for the Ccr4-Notcomplex in regulation of this modification.Possibly, recruitment of the Ccr4-Notcomplex to this region aids localization ofthe H3K4me3 mark. Another importantinsight into the mechanism by which theCcr4-Not complex controls H3K4me3levels comes from the observation thatPAF complex recruitment is severelydecreased in the absence of N O T 4 ,whereas Bur1p and Bur2p recruitment isnot affected (Figure 5 and 6). Given thatthe Bur1/2 kinase is required forassociation of the PAF complex tochromatin, this result places the Ccr4-Notcomplex downstream or Bur1/2, butupstream of the PAF complex in thepathway leading to tri-methylation of H3K4(Figure 7). Possibly, regulation of theCcr4-Not complex by the Bur1/2 kinase isinvolved in recruitment and/or retention ofthe PAF complex to transcribed genes. Inline with such a proposal, and thesuggestion that the Not-module and Ccr4pplay distinct roles in transcriptionelongation (see above), is the observationthat CCR4, but none of the NOT genes, issynthetic lethal with CDC73 and PAF1(Maillet et al., 2000). In addition, it isinteresting to note that previousexperiments have physically linked thePAF complex and Ccr4p (Chang et al.,1999). However, we, and others havefailed to detect PAF complex members orBur1p/2p in Ccr4-Not purifications (Gavinet al., 2002) and data not shown). Inaddition, deletion of PAF1 gives rise to anHU sensitivity phenotype and deregulationof RNR gene transcription (Betz et al.,2002). Intriguingly, this was also observedfor deletions of CCR4-NOT genes (Mulderet al., 2005) and deletion of BUR2 (Figure3), suggesting a functional interplaybetween these complexes. Alternatively,alteration of other components of thehistone code might also contribute to theregulation of H3K4me3 levels by the Ccr4-Not complex. Irrespective of this, it isclear that further experiments are requiredto elucidate the connections between theCcr4-Not and PAF complexes.


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Positive regulation of transcription bythe Ccr4-Not complexThe Bur1/2 complex was shown toregulate H3K4 methylation (Laribee et al.,2005) and th is may invo lvephosphorylation of Rad6p (Wood et al.,2005). The genetic interactions betweengenes encoding Ccr4-Not complexsubunits (NOT2, NOT4 and CCR4) andBUR1 and BUR2, suggest a role for thiscomplex in transcription elongation (Figure1A). Interestingly, deletions of severalCcr4-Not components were shown toexhibit 6-AU sensitivity, suggesting such arole (Denis et al., 2001). Notably, a strictcorrelation between the published 6-AUsensitivity for Ccr4-Not components andsynthetic lethality with B U R 2 wasobserved. Since the Bur1/2 complex wasshown to be involved in regulation ofH3K4me3 levels (Laribee et al., 2005), wedetermined the levels of various histoneH3 lysine methylation marks in cellslacking genes encoding Ccr4-Notcomponents. We found a striking reductionin global H3K4 tri-methylation levels inNOT gene deletion strains (Figure 2, 3 and4). In contrast, decreased H3K4me3 levelswere not observed in ccr4Δ cells,suggesting that Ccr4p and the Not-modulecontribute to transcription elongation bydistinct mechanisms. An alternativeexplanation would be that deletion of Ccr4-Not components and BUR2 results inpartial loss of subsequent activities withinthe same pathway, ultimately resulting inthe observed synthetic lethality.Insight into the mechanism by which theNot-proteins function in the pathwayleading to H3K4 tri-methylation comesfrom the observations that PAF complexrecruitment and subsequent efficientubiquitylation of H2B, but not Bur1/2complex recruitment, is dependent on thepresence of NOT4 (summarized in Figure7). However, other modifications such asH3K4 mono and di-methylation or H3K79di- and tri-methylation were not affected bydeletion of NOT4. These results suggest amodel in which the pathways leading toH3K4 and H3K79 methylation bifurcate

upstream of the Ccr4-Not complex (Figure7) and are in agreement with a recentreports showing that BUR2 is required forH3K4 methylation (Laribee et al., 2005;Wood et al., 2005). Alternatively, theresidual degree of ubiquitylated H2B thatis observed in cells lacking NOT4 may besufficient to sustain H3K79me2/3 andH3K4me2 but not H3K4me3 levels. Thissupport for this hypothesis comes from theobservation that cells deleted for PAF1 orC D C 7 3 show some residual H3K4methylation ((Wood et al., 2003b) and datanot shown).Our results support a global role for theCcr4-Not complex in positive regulation oftranscription. In addition, mutation ofcomponents of this complex has beenshown to result in derepression of avariety of genes. These seeminglycontradicting observations can bereconciled by the observation thatrecruitment of ING2 to tri-methylatedH3K4me3, via its PHD domain, is involvedin gene repression in human cells (Shi etal., 2006). Furthermore, the PHD domainof a homologous protein from yeast,Pho23p, binds H3K4 methylated peptidesin vitro (Shi et al., 2006; Wysocka et al.,2006). Notably, Pho23p was found to beassociated with the Rpd3p containinghistone deacetylase complex (Loewith etal., 2001) and to be required for repressionof the PHO5 gene (Lau et al., 1998).These observations suggest that Ccr4-Notcomplex mediated repression could befacilitated by its role in H3K4 methylation.In summary, we have identified a role forthe Ccr4-Not complex in regulation H3K4tr i -methylat ion levels by act ingdownstream of the Bur1/2 kinase tofacilitate PAF complex recruitment andsubsequent H2B ubiquitylation. Theseresults provide a connection between therole of Ccr4-Not complex in positiveregulation of transcription and activatinghistone methylation marks.

ACKNOWLEDGEMENTSWe thank Drs M. Collart, F. van Leeuwen,J. Jaehning, W.W. Pijnappel, S.


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Buratowski, P.A. Weil, G.P. Prelich, A.Aguilera and B. Strahl for generouslysharing reagents, Drs G.S. Winkler andF.C. Holstege for critical reading of themanuscript and members of the Timmerslaboratory for helpful discussions,technical advice and assistance. Drs N.J.Krogan and B. Strahl are gratefullyacknowledged for sharing results prior topublication. This work was supported bygrants from The Netherlands Organizationfor Scientific Research (NWO-MW Pionier#900-98-142 and NWO-CW #700-50-034),European Union (Improving HumanPotential RTN2-2001-00026 and STREPLSHG-CT-2004-502950) and EMBO(ASTF 184.00-02).

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SUPPLEMENTAL INFORMATIONMATERIALS AND METHODSTandem affinity purificationTAP-tag mediated protein purificationswere performed essentially as described(Logie and Peterson, 1999). Briefly, 20liters of YPD culture was grown to OD600~2-3, washed and lysed in E-buffer (20mM HEPES-KOH pH 8, 350 mM NaCl,10% glycerol, 0.1% Tween-20). Lysateswere cleared by centrifugation in aBeckman 50.2Ti rotor (45,000 rpm, 45min, 4°C). An aliquot of lysate was usedfor purification over a 200 µ l IgGsepharose column (IgG-sepharose fastflow, Pharmacia). Proteins were bound byrotating at 4°C for 2 hours andsubsequently washed with 35 ml E-bufferand 10 ml TEV protease cleavage-buffer(10 mM Tris-HCl pH 8, 150 mM NaCl,0.1% Tween-20, 0.5 mM EDTA and 1 mMDTT). TEV protease (100 Units) cleavagewas performed in 1 ml at 18°C for 2 hours.The TEV eluate was bound to 100 µ lcalmodulin affinity resin (Stratagene) inbinding buffer (10 mM Tris pH 8, 150 mMNaCl, 1 mM MgAc, 1 mM imidazole, 2 mMCaCl2, 0.1% Tween-20, 10% glycerol and10 mM β-mercaptoethanol) rotating at 4°Cfor 1 hour. The column was washed with25 ml binding buffer and bound proteinswere recovered in elution buffer (10 mMTris-HCl pH 8, 150 mM NaCl, 1 mM MgAc,1 mM imidazole, 2 mM EGTA, 0.1%Tween-20, 10% glycerol and 10 mM β-mercaptoethanol). A fraction of the purifiedproteins was precipitated as described(Wessel and Flugge, 1984), separated ona 4-12% SDS-PAGE gradient gel(NuPage, Invitrogen), stained with Biosafe(BioRad) and processed for massspectrometric analysis.

Tandem Mass SpectrometryIn-gel proteolytic digestion of coomassie-stained bands was performed essentiallyas described (Kinter and Sherman, 2000),using trypsin (Roche). Samples weresubjected to nanoflow liquid (LC)

chromatography (Agilent 1100 series) andconcentrated on a C18 precolumn (100µm ID, 2 cm). Peptides were separated onan analytical column (75 µM ID, 20 cm) ata flow rate of 200 nl/min with a 60 minlinear acetonitrile gradient from 0 to 80%.The LC system was directly coupled to aQTOF Micro tandem mass spectrometer(Micromass Waters, UK). A survey scanwas performed from 400-1200 amu/s andprecursor ions were sequenced in MS/MSmode at a threshold of 150 counts. Datawere processed and subjected todatabase searches using ProteinlynxGlobal Server version 2.1 (Micromass,UK) or MASCOT software (Matrixscience)against SWISSPROT and the NCBInonredundant database, with a 0.25 Damass tolerance for both precursor ion andfragment ion. The identified peptides wereconfirmed by manual interpretation of thespectra.

In vitro methylation assaySet1p complexes (10-20 µl TEV eluate)from WT or not4Δ cells were incubatedwith 10 µg purified histones (Sigma) or 2.5µ g H3 K4 (methylated) tail peptides(Abcam, Ab7228, Ab1340, Ab7768 andAb1342, respectively), 1.5 µCi 3H-S-adenosyl methionine in buffer (50 mMTris-HCl pH 8, 10 mM MgCl2 and 10 mMβ-mercaptoethanol) at 30°C for 30 minAfter separation by SDS-PAGE, the gelswere dried and exposed to X-ray films.

REFERENCESLogie, C., and Peterson, C. L. (1999).Purification and biochemical properties ofyeast SWI/SNF complex. MethodsEnzymol 304, 726-741.Kinter, M., and Sherman, N. E. (2000).Protein Sequencing Identification UsingTandem Mass Spectrometry (New York,Wiley and Sons).Roguev, A., Schaft, D., Shevchenko, A.,Pijnappel, W. W., Wilm, M., Aasland, R.,and Stewart, A. F. (2001). TheSaccharomyces cerevisiae Set1 complex


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includes an Ash2 homologue andmethylates histone 3 lysine 4. Embo J 20,7137-7148.

Supplemental Figure 1. H3K4 tri-methylation andPYK1 transcription is affected by deletion of NOT4or SPP1. (A) Schematic representation of the PYK1gene and the amplicons. (B) Deletion of NOT4 orSPP1 leads to a similar decrease in H3K4me3levels on the PYK1 gene. Chromatin extracts ofBY4741, not4Δ and spp1Δ strains were subjected toChIP analysis using H3K4me3 specific antibodies.(C) PYK1 mRNA transcript levels are affected bydeletion of NOT4 or SPP1. PYK1 mRNA in totalRNA of BY4147, not4Δ and spp1Δ strains wasanalyzed by quantitative reverse-transcriptase PCR.

Supplemental Figure 2. NOT4 is required for H3K4tri-methylation but not di-methylation of the PGK1ORF. (A) Schematic representation of the PGK1locus and the amplicons used in (B-H). (B) ChIPanalysis of the PYK1 ORF in BY4741 and not4Δstrains. Exponentially growing cells were subjectedto ChIP analysis using H3K4me3 antibodies. (C) Asin (A), using H3K4me2 antibodies. (D) As in (A),using H3K79me2 antibodies. (E) As in (A), usingCTD antibodies. (F) Quantitative reverse-transcriptase PCR analysis of PGK1 and TUB1mRNA levels in BY4147 WT and not4Δ strains. (G)ChIP analysis of H3K4me3 levels in MY1 WT andnot2Δ cells. (H) ChIP analysis of H3K4me2 levels inMY1 WT and not2Δ cells.


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Supplemental Figure 3. The H3K4me3 defect innot4Δ cells is independent of direct regulation of theSet1p complex. (A) mRNA levels of Set1p complexcomponents. RNA was extracted from exponentiallygrowing BY4741 and not4Δ and subjected toNorthern blot analysis using the indicated probes.(B ) Subunit composition of purified Set1pcomplexes. Strains containing or lacking NOT4 andexpressing a TAP-tagged Bre2p were used to purifySet1p complexes (upper panel). Equal expressionof Bre2-TAP proteins was checked by Western blotanalysis (lower panel). The indicated proteins wereidentified using LC-MS/MS. Several additionalproteins were present in the not4Δ purification whencompared to the WT. Mass spectrometry showedthat these bands contained non-related lysosomalproteins. We consider this an artifact of thepurification, likely caused by differences inconcentration of the lysates of the WT and not4Δcells. (C) Histone methyl transferase assay usingpurified Set1p complexes. Increasing amounts ofSet1p complex or a mock-purification control wereuse to methylate soluble histones (from calfthymus) in vitro. Samples were separated on a 15%SDS-PAA gel, stained with coomassie, dried andexposed to an X-ray film. (D) In vitro methylation ofsynthetic pre-methylated histone H3-tail peptides.Pre-methylated peptides were used as substratesfor in vitro methylation using Set1p complexespurified from WT or not4Δ cells. Samples wereseparated on a 20% SDS-PAA gel, dried andexposed to an X-ray film. Signals were quantifiedusing ImageQuant software and represented asarbitrary units after background correction.


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chapter 4 · deletions of not4 and bur2 display similar ... but not with not3, caf40 and caf130...

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