Phosphorylation of an HP1-like protein is a prerequisitefor heterochromatin body formation in TetrahymenaDNA eliminationKensuke Kataokaa,1, Tomoko Notoa, and Kazufumi Mochizukia,1

aInstitute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), A-1030 Vienna, Austria

Edited by Jasper Rine, University of California, Berkeley, CA, and approved June 27, 2016 (received for review April 14, 2016)

Multiple heterochromatic loci are often clustered into a higherorder nuclear architecture called a heterochromatin body in diverseeukaryotes. Although phosphorylation of Heterochromatin Protein 1(HP1) family proteins regulates heterochromatin dynamics, its role inheterochromatin bodies remains unknown. We previously reportedthat dephosphorylation of the HP1-like protein Pdd1p is required forthe formation of heterochromatin bodies during the process ofprogrammed DNA elimination in the ciliated protozoan Tetrahymena.Here, we show that the heterochromatin body component Jub4p isrequired for Pdd1p phosphorylation, heterochromatin body forma-tion, and DNA elimination. Moreover, our analyses of unphosphory-latable Pdd1p mutants demonstrate that Pdd1p phosphorylation isrequired for heterochromatin body formation and DNA elimination,whereas it is dispensable for local heterochromatin assembly. There-fore, both phosphorylation and the following dephosphorylation ofPdd1p are necessary to facilitate the formation of heterochromatinbodies. We suggest that Jub4p-mediated phosphorylation of Pdd1pcreates a chromatin environment that is a prerequisite for subsequentheterochromatin body assembly and DNA elimination.

heterochromatin body | HP1 phosphorylation | Tetrahymena |DNA elimination

Heterochromatin is a compacted state of chromatin that playsan important role in gene silencing. In some cell types, lo-

cally compacted heterochromatic loci are further assembled intohigher ordered nuclear compartments called heterochromatinbodies, such as the chromocenter, which is mainly composed ofcentromeric regions and repetitive sequences in mice, flies, andplants (1–3); the Barr body, which is composed of an entire inactiveX chromosome in mammalian females (4); and the senescence-associated heterochromatin foci, which are formed by the assemblyof preexisting heterochromatic loci in mammalian cells (5). Het-erochromatin body is formed during cell differentiation (6) and isoften dissociated in cancer cells (7). These examples imply thatheterochromatin bodies may have important functions for regulat-ing the underlying sequence beyond the local heterochromatin.Heterochromatin Protein 1 (HP1) is one of the most exten-

sively characterized components of heterochromatin (8). HP1family proteins contain two conserved domains: a chromodo-main (CD), which recognizes methylated histone H3 at lysine9 (H3K9me), and a chromoshadow domain (CSD), which formsa homotypic dimer; in addition, an unconserved hinge region islocated between the CD and CSD that interacts with nucleicacids. HP1 family proteins in a wide variety of eukaryotes aremultiply phosphorylated, and many of the identified phosphor-ylation sites are located outside of the CD and CSD (9–11). Inyeast, phosphorylation of the HP1 homolog Swi6 is required fortranscriptional gene silencing by recruiting a histone deacetylasecomplex (12). In mammals, phosphorylation of serine (Ser)clusters at the N-terminal region of HP1α increases its affinity toH3K9me, presumably by inhibiting the DNA binding propertyof this protein (13, 14). Consistently, in flies, substitutions ofphosphorylated Ser residues of HP1a with alanine compromiseheterochromatic gene silencing (15, 16). These lines of evidence

suggest that phosphorylation of HP1 family proteins regulatesheterochromatin dynamics. However, the involvement of HP1phosphorylation in the process of heterochromatin body for-mation has not been reported.During the process of programmed DNA elimination in the

ciliated protozoan Tetrahymena, heterochromatin and hetero-chromatin bodies are formed de novo (17), and the phosphory-lation status of the HP1-like protein Pdd1p changes dynamically(18). Tetrahymena maintains two distinct nuclei in a single cell:the diploid micronucleus (MIC), which has a 150-Mb genome,and the polyploid macronucleus (MAC), which has a 100-Mbgenome. In the sexual reproduction process called conjugation(Fig. S1), the MIC in each mating cell undergoes meiosis, andthe mating pair reciprocally exchanges one of the haploid nuclei.The exchanged and the remaining nuclei fuse to form a diploidzygotic nucleus. The zygotic nucleus divides twice and differen-tiates into both the new MICs and the new MACs, whereas theparental MAC is degraded. When the new MAC differentiates,∼10,000 internal eliminated sequences (IESs), consisting of one-third (∼50 Mb) of the MIC genome and many relating totransposons, are removed by DNA elimination (19). At the earlystage of conjugation, ∼29-nt small RNAs, called Early-scnRNAs,are produced from IESs in the MIC by an RNAi-related mech-anism. Subsequently, these Early-scnRNAs travel to the newMAC and recruit the histone methyltransferase Ezl1p to IESs. Ezl1pleads to accumulation of heterochromatin-associated H3K9me and

Significance

In various eukaryotes, heterochromatin is cytologically visiblebecause multiple heterochromatic loci are assembled into higherorder structures called heterochromatin bodies. Although het-erochromatin bodies are dynamically assembled and disassembledduring development, aging, and carcinogenesis, their role and themechanism of formation remain to be clarified. In this report, byinvestigating the role of the heterochromatin body componentJub4p in the de novo heterochromatin body formation process inthe ciliated protozoan Tetrahymena, we reveal that the phos-phorylation and subsequent dephosphorylation of the Hetero-chromatin Protein 1-like protein Pdd1p are prerequisites forheterochromatin body formation and the following programmedDNA elimination. This study demonstrates the biochemical andbiological importance of a temporally ordered phosphorylation–dephosphorylation cycle of a heterochromatin component inheterochromatin body formation.

Author contributions: K.K. and K.M. designed research; K.K., T.N., and K.M. performedresearch; K.K. and K.M. analyzed data; and K.K. and K.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE83252).1To whom correspondence may be addressed. Email: Kazufumi.Mochizuki@imba.oeaw.ac.at or kensuke.kataoka@imba.oeaw.ac.at.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606012113/-/DCSupplemental.

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methylation at histone H3 lysine 27 (H3K27me), and the Pdd1pthat binds to these methylated histones (20, 21) nucleates het-erochromatin. Then, another class of small RNAs, which are also∼29 nt in length and are called Late-scnRNAs, are producedfrom IESs in the new MAC in an Ezl1p- and Pdd1p-dependentmanner, and they reinforce robust heterochromatin formation onIESs (22). The heterochromatinized IESs are eventually excised bythe domesticated transposase Tpb2p (23, 24).Heterochromatinized IES loci are assembled into hetero-

chromatin bodies before or during DNA elimination. Previousstudies showed that Pdd1p is phosphorylated at the time of denovo heterochromatin formation and is then dephosphorylatedwhen heterochromatin bodies are formed (18, 25). We alsoshowed that the unconserved regions of Pdd1p are multiplyphosphorylated upon heterochromatin establishment on IESs,and subsequent dephosphorylation of Pdd1p is required for theassembly of heterochromatin bodies (25). Because phosphomi-metic mutations of Pdd1p inhibit its interaction with RNAin vitro, we have proposed that the dephosphorylation of Pdd1ppromotes heterochromatin body formation by reducing the netnegative charge of Pdd1p and thus facilitating its interaction withRNA. However, it is unclear how Pdd1p is initially phosphory-lated and whether the phosphorylation of Pdd1p plays a role inheterochromatin dynamics. Here, we show functional analyses ofthe novel heterochromatin body component Jub4p and demon-strate that the phosphorylation of Pdd1p is a prerequisite forheterochromatin body assembly.

ResultsJub4p Is Required for Heterochromatin Body Formation. Jub4p is oneof the eight heterochromatin body components that we identi-fied previously (25). The heterochromatin body localization ofJub4p was confirmed by expressing hemagglutinin epitope-taggedJub4p (HA-Jub4p) from the conjugation-specific PDD1 promoter(Fig. 1A, Top). HA-Jub4p was detected exclusively in the newMAC at 8 hours post-mixing (hpm) (Fig. 1A) and localized tonuclear foci, which also contained the known heterochromatinbody component Pdd1p in the new MAC at 14 hpm (Fig. 1A).To analyze the function of Jub4p, we generated JUB4 knock-

out (KO) strains, in which the entire protein-coding sequences ofJUB4 in both MIC and MAC were replaced by a drug-resistantmarker cassette (Fig. S2A, Top). PCR amplification of the locus,followed by a restriction enzyme digestion, confirmed the com-plete replacement of the wild-type (WT) allele with the KOconstruct in the JUB4 KO strains (Fig. S2A, Bottom). Northernblot analysis further demonstrated these strains lost JUB4 ex-pression (Fig. S2B).We first compared developmental profiles of WT and JUB4

KO cells (Fig. S2C). JUB4 KO cells mated almost normally, buttheir progress into the late stage (i.e., new MAC differentiationstage; see also Fig. S1) was delayed, and a small fraction of pairsseparated prematurely before the new MAC development (Fig.S2C, between 8 and 10 hpm). The significance of this prematureseparation is not clear. Nonetheless, because most of the matedcells formed new MACs and MICs, at the cytological level, nucleardifferentiation appeared to occur normally in the absence of Jub4p.We then tested whether Jub4p is required for heterochromatin

body formation. We categorized the formation and turnover ofheterochromatin bodies in exconjugants (progeny postpairing)into three stages based on the localization of Pdd1p (Fig. 1B,Top): In stage 1, Pdd1p is localized throughout the new MAC; instage 2, Pdd1p is localized in discrete foci (heterochromatinbodies); and in stage 3, Pdd1p disappears completely. In WT cells,more than two-thirds of the exconjugants were in stage 2 at 12hpm, and upon DNA elimination, which occurs at ∼14 hpm inWTcells, most of the exconjugants reached stage 3 by 18 hpm (Fig. 1B,WT). By contrast, all exconjugants from the JUB4 KO cellsremained in stage 1 even at 24 hpm (Fig. 1B, ΔJUB4), indicatingthat Jub4p is essential for heterochromatin body formation.Because the heterochromatin body formation is suggested to

be required for DNA elimination and eventually for the formation

of viable progeny (25–27), we next tested whether Jub4p is re-quired for these processes. DNA elimination was analyzed byDNA-FISH using probes complementary to the two moderatelyrepeated IESs, Tlr1 and REP2 (28, 29). In the exconjugants fromWT cells at 36 hpm, IESs complementary to these probes weredetected only in the new MICs but not in the new MACs (Fig. 1Cand Fig. S2D, WT, na). In contrast, the IESs were detected in thenew MACs in all exconjugants from JUB4 KO cells (Fig. 1C and

Fig. 1. Jub4p is required for heterochromatin body formation. (A, Top) TheWT JUB4 and HA-JUB4 loci. (A, Bottom) The cells expressing HA-Jub4p at 8and 14 hpm were stained with anti-HA (green), anti-Pdd1p (red), and anti-bodies and DAPI (blue). Arrowheads indicate the MIC (i), new MAC (na), andparental MAC (pa). (Scale bar,10 μm.) (B) Three stages of heterochromatinbody formation (stage 1, preheterochromatin body; stage 2, heterochro-matin body; stage 3, postheterochromatin body) according to Pdd1p local-ization (green). DNA was stained with DAPI (magenta). (Scale bar, 2 μm.)Exconjugants (n = 200) at indicated time points from WT and JUB4 KO(ΔJUB4) cells were analyzed, and the averaged fractions from two in-dependent experiments are shown. (C) WT and JUB4 KO cells at 36 hpmwere hybridized with probes complementary to Tlr1-IES (green). DNA wasstained with DAPI (magenta). Arrowheads indicate the MIC (i) and new MAC(na). (Scale bar, 5 μm.) The average percentages of exconjugants withoutTlr1-IES signal in the new MAC from three independent crosses are given.

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Fig. S2D, ΔJUB4, na), indicating that Jub4p is required for DNAelimination. In addition to the DNA elimination defect, we no-ticed that the amount of DNA in the new MACs was lower,probably caused by a block of DNA endoreplication, and theelimination of one of the two newMICs did not occur in JUB4KOcells (Fig. 1C and Fig. S2D). These defects are most probably aconsequence of the block of DNA elimination because variousDNA elimination defective mutants show similar phenotypes (30–32). Consistent with the previous observations that DNA elimi-nation is required for viable progeny production (26, 27, 31), noneof the pairs from JUB4 KO strains produced viable progeny,whereas ∼80% of the pairs isolated from WT cells produced vi-able progeny (Fig. S2E). Altogether, we conclude that Jub4p isrequired for heterochromatin body formation, DNA elimination,and progeny viability.

Jub4p Is Dispensable for the Establishment of Heterochromatin onIESs. Heterochromatin body formation relies on the establish-ment of heterochromatin on individual IES loci before theiraggregation. Therefore, heterochromatin on IESs was analyzedin JUB4 KO cells. Heterochromatin establishment includes theaccumulation of trimethylated histone H3 at lysine 9 (H3K9me3)and lysine 27 (H3K27me3), which provide binding sites for Pdd1p(20, 21). We analyzed accumulation of H3K9me3 and H3K27me3by immunofluorescent staining using an anti-H3K9me3 and ananti-H3K27me3 antibody, respectively. Similar to WT cells, bothH3K9me3 (Fig. 2A) and H3K27me3 (Fig. S3A) were detected inthe new MACs upon their formation at 8 hpm in JUB4 KO cells.However, as we observed for Pdd1p (Fig. 1B), this H3K9/K27me3-containing heterochromatin did not form heterochromatin

bodies in JUB4 KO cells (Fig. 2A and Fig. S3A, 14 hpm). Theseresults indicate that, in the absence of Jub4p, heterochromatinis established, but the loci are not assembled into heterochro-matin bodies.We next examined the localization of heterochromatin by

chromatin immunoprecipitation followed by DNA sequencing(ChIP-seq) using the new MACs that were purified by fluores-cent activated cell sorting (FACS) from cells at 12 hpm, whenmost of the IESs are heterochromatinized in WT cells but mostlyremain in the chromosomes (25, 33). In the new MACs from WTcells, Pdd1p as well as H3K9me3 and H3K27me3 were specifi-cally accumulated on most of the IESs (Fig. 2B and Fig. S3B,Left; IESs are marked with red) as well as on a modeled IES inwhich all predicted 1–5 kb IES loci (5,606 loci total) werecompiled (Fig. 2B and Fig. S3B, Right). Similarly, in the newMACs from JUB4 KO cells, ChIP-seq analysis using the anti-Pdd1p antibody showed that Pdd1p was specifically accumulatedon the IESs (Fig. 2B, Bottom). Altogether, we conclude thatheterochromatin is formed properly on IESs in the absence ofJub4p and thus Jub4p acts downstream of heterochromatin es-tablishment for heterochromatin body formation.

Jub4p Facilitates Phosphorylation of Pdd1p. We and others pre-viously identified at least 33 phosphorylated serine (Ser)/threonine(Thr) residues in Pdd1p from WT cells before heterochromatinbody formation (25, 34), and a mutagenesis study revealed that thesubsequent dephosphorylation of Pdd1p is required for the het-erochromatin body formation and DNA elimination (25). There-fore, we analyzed the phosphorylation–dephosphorylation cycle ofPdd1p in JUB4 KO cells. At 8 hpm in WT cells, several Pdd1pspecies were detected by Western blot analysis using an anti-Pdd1p antibody (Fig. 3, WT); the fastest migrating species cor-responds to the unphosphorylated Pdd1p, whereas the otherslower migrating species are phosphorylated Pdd1p (25). Thephosphorylated Pdd1p was then gradually dephosphorylated at10–14 hpm, when the heterochromatin body is formed, and Pdd1pmostly disappeared by 20 hpm, when DNA elimination is com-pleted. In contrast, in JUB4 KO cells, the slower migrating Pdd1pwas not detected at 8 hpm, and only a slower migrating Pdd1p,likely corresponding to lowly phosphorylated Pdd1p, was accu-mulated at the later stages (Fig. 3, ΔJUB4). Pdd1p did not dis-appear even at 20 hpm in JUB4 KO cells, which probably reflectsthe inhibition of DNA elimination in the absence of Jub4p (Fig.1C and Fig. S2D).The loss of Pdd1p phosphorylation in JUB4 KO cells was

confirmed using an antibody against phosphorylated Ser317 ofPdd1p (p-S317), which recognizes the slowest migrating (hyper-phosphorylated) species of Pdd1p in WT cells (Fig. S4). In WTcells, p-S317 appeared at 8 hpm and disappeared at later stages,whereas we did not detect any p-Ser317 in JUB4 KO cells (Fig. 3,phos-Pdd1p). Altogether, we conclude that Jub4p is required forthe accumulation of phosphorylated Pdd1p. Because Jub4p has no

Fig. 2. Jub4p is dispensable for local heterochromatin establishment.(A) WT and JUB4 KO (ΔJUB4) cells at 8 and 14 hpm were stained with anti-H3K9me3 (green) and anti-Pdd1p (red) antibodies. DNA was stained withDAPI (blue). Arrowheads indicate the MIC (i), new MAC (na), and parentalMAC (pa). (Scale bar, 10 μm.) (B) ChIP-seq with an anti-Pdd1p antibody wasperformed using the new MACs from WT and JUB4 KO cells at 12 hpm.Reads were mapped to a 100-kb representative MIC locus (Left; LMR locus)or to a modeled IES locus (Right), which consisted of all predicted 1- to 5-kbIESs (red) and their flanking regions (blue). Fold enrichment relative to inputis shown.

Fig. 3. Pdd1p phosphorylation is inhibited in JUB4 KO cells. Proteins fromWT and JUB4 KO (ΔJUB4) cells in exponentially growing (E) and conjugation(0; 4–20 hpm) were analyzed by Western blot with anti-Pdd1p and anti-phosphorylated Pdd1p antibodies. Phosphorylated (phos) and unphos-phorylated (unphos) Pdd1p are indicated. Twi1p and α-tubulin were ana-lyzed as mating and loading controls, respectively.

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identifiable kinase-related domains, Jub4p may facilitate Pdd1pphosphorylation by recruiting some kinase(s) or protect phosphor-ylated Pdd1p from some phosphatase(s). Alternatively, Jub4pmay regulate some other activity that indirectly promotes Pdd1pphosphorylation.

Phosphorylation of Pdd1p Is a Prerequisite for Heterochromatin BodyFormation. The loss of Jub4p disturbs both Pdd1p phosphoryla-tion (Fig. 3) and heterochromatin body formation (Fig. 1B),suggesting that the phosphorylation of Pdd1p may be requiredfor heterochromatin body formation. However, this result mayalso suggest that Jub4p is required for these two events in-dependently. Therefore, we analyzed the importance of Pdd1pphosphorylation in heterochromatin body formation by directlymutating the phosphorylated residues of Pdd1p.We generated a series of constructs to express WT Pdd1p

(WT) or Pdd1p carrying unphosphorylatable mutations, in which10, 22, or 26 of the identified phosphorylated Ser/Thr residues inthe N terminus and hinge regions of Pdd1p (25, 34) weresubstituted with alanines (MUT10, MUT22, and MUT26, re-spectively; Fig. 4A). Then, the PDD1 KO locus of PDD1 KO cellswas replaced by these constructs to express the WT and mutantPdd1p from the PDD1 promoter (Fig. S5A). As we previouslyreported (25), the expression of WT in PDD1 KO cells fullyrestored heterochromatin body formation, based on the locali-zation of Pdd1p and H3K9me3 at 14 hpm (Fig. 4B, Left, +WT).Cells expressing MUT10 and MUT22 formed heterochromatinbodies containing both Pdd1p and H3K9me3 at 14 hpm, whereasMUT26 did not restore the formation of heterochromatin bodies(Fig. 4B, Left). The size of heterochromatin bodies in the cellsexpressing MUT22 were smaller than those in cells expressingWT, and the number of cells showing heterochromatin bodiesdecreased as the number of mutations in the expressed Pdd1p in-creased. Consistently, although MUT10 fully restored DNA elimi-nation of Tlr1 IESs, MUT22 did so only in a limited fraction of cellsand MUT26 did not support DNA elimination (Fig. 4C). Fur-thermore, the number of produced viable progeny decreased as thenumber of mutations in the expressed Pdd1p increased (Fig. S5B).We believe that the inabilities of the MUT26 protein to supportheterochromatin body formation, DNA elimination, and progenyproduction are not due to the loss of its structural integrity because(i) Western blot analysis showed that MUT26 andWT accumulatedsimilarly (Fig. S5C); (ii) ChIP-seq analysis showed that MUT26 andWT were similarly accumulated on the IESs (Fig. S5D); (iii) pull-down assays showed that WT and MUT26 bound similarly to thepeptides corresponding to H3K9me3 and H3K27me3 (Fig. S5E);(iv) WT and MUT26 showed similar affinity to RNA in vitro (Fig.S5F), which we previously suggested to be essential for hetero-chromatin body formation (25); and (v) Pdd1p-dependent pro-duction of Late-scnRNA (22) occurred normally in the cellsexpressing MUT26 (see Phosphorylation of Pdd1p Is Dispensablefor Late-scnRNA Production). Altogether, we conclude that, toform functional heterochromatin bodies for DNA elimination,certain numbers of Ser/Thr residues, or at least a few particularresidues, need to be accumulatively phosphorylated.

Phosphorylation of Pdd1p Is Dispensable for Late-scnRNA Production.We previously reported that Pdd1p is required for the pro-duction of ∼26–32-nt small RNAs, called Late-scnRNAs, andthat Late-scnRNAs are important for DNA elimination of asubset of IESs (22). Because the accumulation of both Late-scnRNAs and phosphorylated Pdd1p start at the onset of new MACdevelopment (∼8 hpm), the involvement of Pdd1p phosphorylation

Fig. 4. Unphosphorylatable mutations of Pdd1p inhibit heterochromatinbody formation. (A) The identified phosphorylated Ser (S)/Thr (T) residues ofPdd1p are shown as open circles (Top). The N-terminal region (NT) and hingeregions (HNG1 and HNG2) are indicated. The WT, PDD1 KO (ΔPDD1) andrescued loci, and the proteins expressed from the indicated constructs areshown (Bottom). Green circles indicate the introduced unphosphorylatablemutations. (B, Left) Exconjugants at 14 hpm were stained with anti-Pdd1p (red)and anti-H3K9me3 (green) antibodies and DAPI (blue). Arrowheads indicate new

MACs (na). (Scale bar, 10 μm.) (Right) The appearance of heterochromatinbodies was analyzed as in Fig. 1B. (C) Exconjugants at 36 hpm from indicatedstrains were hybridized with a Tlr1-IES probe (green). DNA was stained withDAPI (magenta). Arrowheads indicate newMACs (na). (Scale bar, 10 μm.) Theaverage percentages of exconjugants without Tlr1-IES signals in the newMAC from more than two independent mating pairs are given.

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in Late-scnRNA production was tested. Small RNAs were extractedfrom cells expressing the unphosphorylatable Pdd1p mutant MUT26at the late stage of conjugation (10.5 hpm) and were analyzed byhigh-throughput sequencing (Fig. 5). The sequenced reads weremapped to representative 300-kb MIC loci containing two types ofIESs: type A IESs, which produce both Early- and Late-scnRNAs,and type B IESs, which produce only Late-scnRNAs (22) (Fig. 5A).We also analyzed the occurrence of scnRNA production from typeA and type B IESs in the whole genome (Fig. 5B). As reportedpreviously (22), in the absence of Pdd1p, the accumulation of smallRNAs complementary to type B IESs (Late-scnRNAs) was severelyreduced (Fig. 5 A and B, 10.5 hpm, ΔPDD1). In both PDD1 KOcells rescued with WT and MUT26, the accumulation of Late-scnRNAs was restored similarly (Fig. 5 A and B, 10.5 hpm, +WTand +MUT26). These results suggest that Pdd1p phosphorylation isdispensable for Late-scnRNA production, and thus the failure ofrestoring heterochromatin body formation by MUT26 (Fig. 4B) isnot due to the loss of Late-scnRNA production. Similarly, we foundthat, in the cells expressing the phosphomimetic Pdd1p mutantMIM22 in which 22 of the phosphorylated Ser/Thr residues weresubstituted with glutamate residues (25), Late-scnRNAs also accu-mulated normally (Fig. 5 A and B, 10.5 hpm, +MIM22). Therefore,

we conclude that the phosphorylation–dephosphorylation cycle ofPdd1p is not required for Late-scnRNA production.

Phosphorylation of Pdd1p May Prevent the Degradation of Pdd1p.WT protein became undetectable from the new MACs of mostexconjugants by 18 hpm (Fig. 4B, Right, +WT, stage 3), whenDNA elimination is mostly completed in WT cells (25, 33).Therefore, the degradation of WT Pdd1p coincides with DNAelimination as previously noticed (35). However, although only4.5% and 0% of exconjugants completed DNA elimination incells expressing MUT22 and MUT26, respectively (Fig. 4C),these proteins disappeared from the new MACs by 18 hpm (Fig.4B, Right, stage 3). These results suggest that DNA eliminationand Pdd1p degradation can occur independently in some con-ditions and raise the possibility that the phosphorylation ofPdd1p prevents the degradation of Pdd1p. In WT cells, DNAelimination and degradation of Pdd1p may be coupled by somemechanism that maintains Pdd1p phosphorylation until cells areready for DNA elimination. In JUB4 KO cells, both DNAelimination and the disappearance of Pdd1p were inhibited (Fig.1 B and C and Fig. S2D), which could be because Pdd1p waslowly but constitutively phosphorylated in these cells (Fig. 3).Therefore, some phosphorylations at particular residues ofPdd1p, which is phosphorylated in JUB4 KO cells but is mutatedin MUT22 and MUT26, might play a role in preventing thedegradation of Pdd1p. This idea is consistent with our previousobservation that the degradation of Pdd1p was inhibited byphosphomimetic mutations (25). Phosphorylation of Pdd1p atsome residue may directly prevent Pdd1p from being recognizedby protein degradation machinery. Alternatively, it may tightenthe Pdd1p–chromatin interaction, and dephosphorylation ofPdd1p may promote dissociation of Pdd1p from chromatin fordegradation. In any case, the results above indicate that thedephosphorylation of Pdd1p may trigger not only the hetero-chromatin body formation but also degradation of Pdd1p.

DiscussionFrom our previous observations that the phosphorylation ofPdd1p prevents the interaction between Pdd1p and RNA in vitroby increasing the net negative charge of Pdd1p and that thedephosphorylation of Pdd1p triggers the formation of hetero-chromatin bodies in vivo, we hypothesized that the Pdd1p–RNAinteraction acts as a molecular glue for heterochromatin bodyassembly, and the phosphorylation of Pdd1p merely regulates thetiming of the formation of heterochromatin bodies. Therefore,we expected that the loss of phosphorylation of Pdd1p causespremature formation of heterochromatin bodies in new MACs.However, we found that both the loss of Jub4p, which preventsphosphorylation of Pdd1p, and unphosphorylatable mutations ofPdd1p caused a complete loss of, but not premature formationof, heterochromatin body. Therefore, the phosphorylation ofPdd1p by itself has an important function in heterochromatinbody formation. Because Pdd1p phosphorylation precedes het-erochromatin body formation and is not involved in local het-erochromatin establishment, the results shown in this study indicatethat the phosphorylation of Pdd1p provides a prerequisite envi-ronment for subsequent heterochromatin body formation. How-ever, the molecular nature of such a prerequisite environmentremains unclear. Below we discuss possible biochemical roles ofPdd1p phosphorylation.One of the simplest scenarios for the role of Pdd1p phos-

phorylation might be that it promotes the accumulation of longnoncoding RNAs from heterochromatic IESs in the new MACthat we believe later interacts with dephosphorylated Pdd1p toinduce heterochromatin body formation. Because Late-scnRNAs,which are processed from the IES transcripts (22), accumulatednormally in the cells expressing the unphosphorylatable Pdd1pmutant (Fig. 5), Pdd1p phosphorylation is unlikely to be involvedin the transcription of long noncoding RNAs from IESs. However,Pdd1p phosphorylation may still be required for stable accumulationand/or proper localization of the long IES transcripts. Future studies

Fig. 5. The phosphor-regulation of Pdd1p is dispensable for Late-scnRNAproduction. (A) Normalized numbers (reads per kilobase per million reads,RPKM) of sequenced 26- to 32-nt small RNAs from the indicated strains thatmap uniquely to 300-kb windows of the MIC genome are shown as histo-grams with 100-nt bins. The magenta and blue bars indicate the positions oftype A and type B IESs, respectively. (B) All possible 25 mers extracted fromall predicted IESs (genome) or the small RNA reads from the indicated cells at3 or 10.5 hpm were classified as sequences complementary to only type AIESs (magenta), only type B (blue), or both (purple).

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should identify Pdd1p-associated RNAs in vivo and analyze theirbehaviors in the presence and absence of Pdd1p phosphorylation.Another possible role of Pdd1p phosphorylation might be the

recruitment of other heterochromatin-associated proteins. HP1in other eukaryotes can recruit various effector proteins to het-erochromatin (36), and phosphorylated Swi6, the HP1 homologof fission yeast, selectively recruits the histone deacetylasecomplex SHREC (12). Phosphorylation of Pdd1p in Tetrahymenamight also regulate its interaction with other proteins thatare required for subsequent heterochromatin body formation.Alternatively, because phosphorylation and/or RNA bindingof HP1 modulate its affinity to chromatin in some eukaryotes(13–15, 37), Pdd1p phosphorylation may also modulate thephysical dynamics of Pdd1p, which may be required for otherheterochromatin proteins to be properly arranged on chroma-tin for heterochromatin body formation. Several proteins areknown to be required for the formation of heterochroma-tin bodies in Tetrahymena (26, 27, 32), and the phosphorylationof Pdd1p may be essential for the chromatin localization ofsuch proteins.Because Pdd1p is highly phosphorylated, the phosphorylation of

Pdd1p could change the dynamics of not only individual hetero-chromatin proteins but also chromatin within the new MAC. Suchan alteration might result in localizing heterochromatinized IESsto the perinuclear region, where a heterochromatin body is laterformed, or might promote a specific chromatin conformation,such as chromatin looping, which may be required for subsequent

heterochromatin body assembly. It will be important to clarify theinvolvement of Pdd1p phosphorylation in 3D localizations of het-erochromatinized IESs in the nucleus with high spatiotemporalresolution and their conformations by chromatin conformationcapture (3C)-based techniques.

Materials and MethodsStrains and Culture Conditions. The production of HA-JUB4, JUB4 KO, andPDD1 mutant strains are described in SI Materials and Methods. Cultureconditions and progeny viability test were described previously (25).

ChIP-Seq and Small RNA-Seq. ChIP-seq and small RNA-seq were performed asdescribed (22, 25). The data were deposited at the National Center forBiotechnology Information (NCBI) Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession no. GSE83252.

Cytology and Biochemistry. Detailed procedures for immunostainings, DNAFISH, production of recombinant proteins, EMSA, and histone peptide pull-down assay are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank the Next Generation Sequencing unit of theVienna BioCenter Core Facilities for sequencing. This work was supported by aNaito Memorial Grant from the Naito Foundation, a European Research CouncilStarting Grant (204986) under the European Community’s 7th Framework Pro-gram, a Stand-Alone Grant (P26032-B22) and a Special Research Program(F4307-B09) from the Austrian Science Fund, a Grant for Basic Science ResearchProjects from the Sumitomo Foundation (150112), and core funding from theAustrian Academy of Sciences.

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