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RESEARCH ARTICLE SUMMARY ◥ lease of the TetR-Clr4 initiator (TetR-Clr4-I)from DNA.
RESULTS: Cells containing the reporter gene in combination with the expressionof TetR-Clr4-I formed pink colonies on low- Epigenetic inheritance uncoupled adenine medium lacking tetracycline, in-dicating ade6+ silencing. The establishment from sequence-specific recruitment of heterochromatin resulted in high levelsof H3K9 methylation (H3K9me), which was subsequently lost upon Kaushik Ragunathan, Gloria Jih, Danesh Moazed* tetracycline-induced re-lease of TetR-Clr4-I within Read the full article INTRODUCTION: Changes in histone post- RATIONALE: The fission yeast Schizosaccharo- at http://dx.doi.
10 cell divisions, result- translational modifications are associated myces pombe contains chromosomal domains ing in the appearance of with epigenetic states that define distinct pat- that share many features with heterochroma- white colonies. Whereas terns of gene expression. Whereas sequence- tin in multicellular eukaryotes, such as meth- perturbations to path- specific DNA binding proteins play essential ylation of histone H3 lysine 9 (H3K9), catalysis ways that altered the rate of histone exchange roles in establishing an epigenetic state, their by the human Suv39h homolog Clr4, asso- or eliminating competition from endoge- contributions to maintenance remain unclear.
ciation with HP1 proteins (Swi6 and Chp2), nous heterochromatic loci had subtle effects Previous attempts to separate the inheritance and histone hypoacetylation. We developed on epigenetic inheritance of ade6+ silenc- of epigenetic states from sequence-specific an inducible system for heterochromatin es- ing, deletion of the putative JmjC domain– establishment suggest that specific DNA se- tablishment in S. pombe by fusion of the Clr4 containing demethylase Epe1 resulted in quences and DNA binding proteins are con- methyltransferase catalytic domain to the bac- cells that retained ade6+ silencing for >50 tinuously required for epigenetic inheritance.
terial tetracycline repressor (TetR) protein. To generations after tetracycline-induced re- Moreover, in addition to DNA binding pro- generate a reporter locus, we introduced 10 lease of TetR-Clr4-I or deletion of the TetR teins, the establishment and maintenance of tetracycline operators upstream of the normal- module. Furthermore, the chromodomain of epigenetic states involves self-reinforcing in- ly expressed ade6+ gene (10XtetO-ade6+). The Clr4, which is involved in recognition of the teractions between histone modifications and silencing of ade6+ results in the formation H3K9me mark, was indispensable for main- RNA interference (RNAi) or DNA methylation.
of red or pink colonies upon growth on me- tenance, suggesting that a direct "read-write" Therefore, whether histone-based mechanisms dium with limiting adenine concentrations.
mechanism mediated by Clr4 propagates can transmit epigenetic memory indepen- This system allowed us to determine whether histone modifications and allows histones dently of specific DNA sequences remains heterochromatin, once established, could be to act as carriers of epigenetic information.
maintained after tetracycline-mediated re- This mechanism allows epigenetic statesto be inherited during mitosis and meiosisand is also critical for maintaining low levels of H3K9me at native pericentromeric repeats.
CONCLUSION: Our findings indicate that even in the absence of any coupling to otherpositive-feedback loops, or in the absence of sequence-dependent initiation signals,H3K9me defines a silent state that can be epigenetically inherited. Maintenance ofthe OFF state is determined by the balance between the rate of H3K9me by the Clr4reader-writer module and the loss rate due to clr4+, epe1+ demethylation by an Epe1-dependent mech-anism, transcription-coupled nucleosome ex- clr4+, epe1∆ change, and dilution of histones during DNAreplication. The regulation of histone de- red colony = silent, white colony = expressed methylation activity may play a broad role indetermining the reversibility of epigenetic states. RELATED ITEMS IN SCIENCE P. N. C. B. Audergon et al., Restrictedepigenetic inheritance of H3K9 methylation.
Science 348, 132135 (2015).
H3K9me defines a silent state that can be epigenetically inherited. A direct read-write Department of Cell Biology, Howard Hughes Medical Institute,Harvard Medical School, 240 Longwood Avenue, Boston, MA mechanism involving the Clr4 H3K9 methyltransferase propagates histone modifications and allows histones to act as carriers of epigenetic information in the absence of any input from the DNA *Corresponding author: sequence, DNA methylation, or RNAi. Epe1, a putative demethylase, and other transcription- Cite this article as K. Ragunathan et al., Science 348, 1258699(2015). DOI: 10.1126/science.1258699 associated histone turnover pathways modulate the rate of decay of the epigenetic state.
3 APRIL 2015 • VOL 348 ISSUE 6230 SCIENCE RESEARCH ARTICLE that domains of H3K9me can be inherited for >50generations in the absence of sequence-specificrecruitment and define central roles for the pu-tative demethylase, Epe1, in the erasure of H3K9me and the chromodomain of the Clr4 methyltrans-ferase in its maintenance.
Epigenetic inheritance uncoupled Inducible establishmentof heterochromatin from sequence-specific recruitment Silent chromatin domains can be established byectopic recruitment of histone-modifying enzymesto chromatin via fusion with heterologous DNA Kaushik Ragunathan, Gloria Jih, Danesh Moazed* binding proteins (, ). To create an induciblesystem for heterochromatin formation, we fused Changes in histone posttranslational modifications are associated with epigenetic states a Clr4 protein lacking its N-terminal chromodo- that define distinct patterns of gene expression. It remains unclear whether epigenetic main (required for binding to methylated histone information can be transmitted through histone modifications independently of specific H3K9) while retaining its enzymatic methyltrans- DNA sequence, DNA methylation, or RNA interference. Here we show that, in the fission ferase activity to the bacterial TetR protein (de- yeast Schizosaccharomyces pombe, ectopically induced domains of histone H3 lysine signated "TetR-Clr4-I" for TetR-Clr4 initiator) 9 methylation (H3K9me), a conserved marker of heterochromatin, are inherited through (Fig. 1A). The TetR DNA binding domain facil- several mitotic and meiotic cell divisions after removal of the sequence-specific initiator.
itates protein targeting to a locus that harbors The putative JmjC domain H3K9 demethylase, Epe1, and the chromodomain of the H3K9 its cognate DNA binding sequence, and this re- methyltransferase, Clr4/Suv39h, play opposing roles in maintaining silent H3K9me cruitment activity is abrogated by the addition domains. These results demonstrate how a direct "read-write" mechanism involving of tetracycline (TetRoff system) We gener- Clr4 propagates histone modifications and allows histones to act as carriers of ated cells in which TetR-Clr4-I replaced the wild- type (WT) Clr4 (TetR-clr4-I) or in which WT Clr4 was intact and TetR-Clr4-I was inserted at an- n individual cell can give rise to progeny response element, which acts analogously to other locus (TetR-clr4-I, clr4+). Comparisons be- with distinct patterns of gene expression the yeast silencer, is continuously required for tween strains with or without clr4+ allowed us and phenotypes without change in its DNA maintenance of domains of histone H3 lysine 27 to evaluate the contribution of the Clr4 chromo- sequence. In eukaryotic cells, a major mech- (H3K27) methylation and reporter gene silencing domain to establishment and/or maintenance.
anism that gives rise to such phenotypic , ). The question therefore remains as to To generate a reporter locus, we replaced the eu- or epigenetic states involves changes in histone whether histones can act as carriers of epigenetic chromatic ura4+ locus with an ade6+ gene con- posttranslational modifications and chromatin information in the absence of any input from the taining 10 tetracycline operators immediately structure , ). The basic unit of chromatin is the underlying DNA sequence.
upstream of the promoter (10XtetO-ade6+) (Fig.
nucleosome, which is composed of 147 base pairs The fission yeast Schizosaccharomyces pombe 1A). ade6+ provides a convenient visual reporter, (bp) of DNA wrapped twice around an octamer contains extensive domains of heterochromatin as its silencing results in formation of red or pink composed of histones H2A, H2B, H3, and H4 ( at its pericentromeric DNA repeats, subtelomeric colonies upon growth on medium with limiting The highly conserved basic N termini and, to a regions, and the silent mating type loci (These adenine concentrations ( lesser extent, the globular domains of histones domains share many features of heterochromatin As shown in Fig. 1B, cells containing the contain a variety of posttranslational modifica- in multicellular eukaryotes, such as H3K9 meth- 10XtetO-ade6+ reporter in combination with the tions that affect nucleosome stability or provide ylation (H3K9me), which is catalyzed by the hu- expression of the TetR-Clr4-I fusion protein, but binding sites for effectors that activate or repress man Suv39h homolog Clr4; association with HP1 not those containing the fusion protein alone or proteins (Swi6 and Chp2); and histone hypoacet- the reporter alone, formed pink colonies on low- It has been established for nearly four decades ylation. Furthermore, S. pombe heterochromatin adenine medium lacking tetracycline (–tet). Con- that parental histones are retained and randomly displays epigenetic inheritance properties in which sistent with previous observations (silencing distributed to newly synthesized daughter DNA cells containing a reporter gene inserted within did not require clr4+—suggesting that the chromo- strands during DNA replication (It was heterochromatin display variegating reporter gene domain of Clr4 was not required for de novo therefore logical to propose that combinations expression The ON and OFF states of such heterochromatin establishment (Fig. 1B)—but of histone modifications, sometimes referred to reporter genes can be stably transmitted in cis depended on HP1 proteins (Swi6 and Chp2) and as a "histone code," are responsible for epigenetic through both mitotic and meiotic cell divisions histone deacetylases (Clr3 and Sir2), which act memory of gene expression patterns , How- (However, because these observations of downstream of H3K9me (fig. S1A). Colonies grown ever, previous attempts to separate sequence- epigenetic inheritance were made at native se- in the absence of tetracycline were then plated specific establishment from maintenance have quences, contributions arising from sequence- on medium containing tetracycline (+tet) to de- failed to provide unambiguous support for a pure- specific elements that stabilize heterochromatin termine whether the silent state could be main- ly histone-based inheritance mechanism in sys- could not be ruled out To determine whether tained upon release of TetR-Clr4-I from DNA.
tems that display stable epigenetic expression heterochromatin maintenance can be separated Tetracycline-dependent release of TetR-Clr4-I from states , ). Most notably, silencers (i.e., DNA from the sequences that initiate its establishment, tetO sites resulted in the loss of silencing, as in- sequences that mediate the establishment of we developed a system for inducible heterochro- dicated by the formation of white colonies (Fig.
hypoacetylated domains of silent chromatin in matin establishment in S. pombe by fusion of 1B, +tet). Chromatin immunoprecipitation (ChIP) the budding yeast Saccharomyces cerevisiae) are the Clr4 methyltransferase catalytic domain to experiments verified that tetracycline addition continuously required for maintenance of the si- the bacterial tetracycline repressor (TetR) pro- resulted in release of TetR-Clr4-I from the 10XtetO lent state ). Similarly, the Drosophila Polycomb tein. This allowed us to establish an extended sites, as the TetR ChIP signal in the presence of heterochromatic domain and study its initiator- tetracycline was near background levels sim- Department of Cell Biology, Howard Hughes Medical independent maintenance either by tetracycline- ilar to that observed for cells lacking TetR-Clr4-I Institute, Harvard Medical School, 240 Longwood Avenue, mediated release of TetR-Clr4 from DNA or after (Fig. 1C). Furthermore, ChIP combined with high- Boston, MA 02115, USA.
*Corresponding author: deletion of the TetR module. Our results indicate throughput sequencing (ChIP-seq) and ChIP SCIENCE 3 APRIL 2015 • VOL 348 ISSUE 6230

RESEARCH RESEARCH ARTICLE quantitative polymerase chain reaction (qPCR) but not the Dicer ribonuclease (Dcr1) or the resulted in the formation of only white colonies experiments showed that a 40- to 50-kb domain Argonaute protein (Ago1) (Fig. 3, B and C). Thus, on +tet medium (Fig. 3E). Therefore, rather than of H3K9 di- and trimethylation (H3K9me2 and maintenance requires the machinery that acts differences in clr4+ dosage, the appearance of red -me3) encompassing the 10XtetO-ade6+ region downstream of H3K9me but occurs indepen- or sectored colonies depends on the presence of was completely lost 24 hours ( 10 cell divisions) dently of an RNA interference (RNAi)–based Clr4 with an intact chromodomain.
after the addition of tetracycline to the growth mechanism. Consistent with the idea that Epe1 In an attempt to identify other features of medium (Fig. 1D and fig. S1, B to D). We obtained acts as a demethylase, the replacement of epe1+ chromatin that could be important for mainte- similar results with cells that carried a WT copy with alleles containing active-site mutations (epe1- nance, we deleted two genes that are associated of clr4+ in addition to TetR-clr4-I (Fig. 1D and K314A and epe1-H297A) , ) displayed a main- with transcription: mst2+, which encodes a his- fig. S1D, lower two rows). These results demonstrate tenance phenotype similar to its deletion (epe1D) tone acetyltransferase (), and set1+, which en- that a domain of H3K9me and the associated (Fig. 3D). Furthermore, consistent with a require- codes a histone H3K4 methyltransferase (In silent state are reversed within 10 cell divisions ment for the chromodomain of clr4+ in mainte- both cases, we observed establishment that was after release of the sequence-specific initiator nance (Fig. 2A), the replacement of clr4+ with a stronger than that of the wild type (–tet), as well clr4-ID allele (which lacks the chromodomain) as weak maintenance (+tet) effects (fig. S4, A and Inheritance uncoupled fromsequence-specific initiation A minimal mechanistic requirement for inheri-tance of a domain containing H3K9me marks involves the recognition of preexisting H3K9 methylated histones coupled to the modifica-tion of newly deposited histones. The loss of a 45-kb domain of H3K9 dimethylation after release of TetR-Clr4-I (Fig. 1D) may either be dueto nucleosome exchange processes associated FLAG ChIP (24 hours)
with transcription and chromatin remodeling or erasure of the methyl mark by a demethylase.
Alternatively, the rapid decay of methylation inthis domain may be facilitated by competition between the ectopic locus and native heterochro- ade6 TetR-clr4-I, clr4+
matic domains for a limiting pool of proteins that play critical roles in maintenance.
To test these different scenarios, we constructed TetR-clr4-I, 10XtetO-ade6+ cells (with or without clr4+) that carried deletions for genes involvedin various chromatin-maintenance pathways ). The deletion of epe1+, which encodes a domain size 45kb putative histone H3K9 demethylase (), didnot affect the establishment of silencing, as in- dicated by the appearance of red colonies on–tet medium for both TetR-clr4-I, epe1D cells and TetR-clr4-I, clr4+, epe1D cells (Fig. 2A, left side).
In contrast to epe1+ cells, epe1D cells formed red,white, and sectored colonies in the presence of TetR-clr4-I + tet
tetracycline, indicating that the silent state was maintained after release of the initiator and thatthis phenotype was observed only in the case TetR-clr4-I, clr4+ + tet
of cells that also contained a WT copy of clr4+(Fig. 2A, right side; compare last two rows withfirst two rows). ChIP experiments verified that tetracycline addition promoted the release of Fig. 1. Ectopic heterochromatin is lost after sequence-specific establishment upon tetracycline TetR-Clr4-I from tetO sites (Fig. 2B). Consistent addition. (A) Diagram of experimental scheme for TetR-Clr4-I (TetR-Clr4-I)mediated H3K9me at the with the colony color–silencing assays, ChIP-seq ura4D::10XtetO-ade6+ locus. Tetracycline (tet) promotes the release of TetR-Clr4-I from tetO sites so that and ChIP-qPCR experiments also indicated that initiator-independent maintenance could be tested. (B) Test of ade6+ silencing on low-adenine medium the large domain of H3K9 di- and trimethyla- in the absence (-tet) and presence (+tet) of tetracycline. Silencing of ade6+ results in formation of red colonies.
tion surrounding the 10XtetO-ade6+ locus was lost Centromeric ade6+ (otr1R::ade6) served as a positive control; the target locus alone (ura4D::10XtetO- in TetR-clr4-I, epe1D cells but was maintained ade6+) and ura4D::ade6+ cells served as negative controls. (C) ChIP-qPCR experiments assess the in TetR-clr4-I, clr4+, epe1D cells (Fig. 2C and fig.
association of FLAG-tagged TetR-Clr4-I with the 10XtetO-ade6+ locus in the presence and absence of S2, A to C) 24 hours after the addition of tetra- tetracycline. The dash on the x axis indicates background ChIP signal from cells that did not express TetR- cycline. Within this domain, the expression of Clr4-I. In the presence of tetracycline, TetR-Clr4-I occupancy is close to background levels. Error bars several other transcription units was silenced indicate SD. (D) ChIP-seq experiments show that TetR-Clr4-I induces a de novo H3K9me2 domain that on –tet medium, and this silencing was main- surrounds the tetO-ade6+ locus (highlighted in red) for 20 kb on either side, in cells with or without clr4+, tained for several hours after tetracycline addi- which is lost 24 hours after tetracycline addition. H3K9me2 ChIP-qPCR data and H3K9me3 for tion (fig. S3).
samples in (D) are presented in fig. S1. Chromosome 3 (site of insertion of 10XtetO-ade6+) and chro- Maintenance of the silent state in epe1D cells mosome 1 [centromere 1 left (cen1L)] coordinates are shown above the tracks, and read numbers (per required the HP1 proteins (Swi6 and Chp2) and million) are indicated on the right. H3K9me2 at DNA repeats of cen1L serves as an internal control for the Clr3 and Sir2 histone deacetylases (Fig. 3A) the ChIP-seq data.
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RESEARCH RESEARCH ARTICLE D). Although there were no obvious effects on not correlate with the strength of the initial si- for a subset of these time points are presented colony color after 3 days of growth on tetracycline- lenced state (establishment), indicating that per- in Fig. 4, B to E, and the results for all time points containing medium, we observed clear persist- turbations to each pathway results in the release are plotted in Fig. 4F. In general, upon tetra- ence of H3K9me coupled with the release of or recruitment of subsets of proteins that make dif- cycline addition, the pattern of silencing of the TerR-Clr4-I in mst2D cells 24 hours after the ad- ferent contributions to the establishment and/or 10XtetO-ura4-GFP reporter in either epe1+ or epe1D dition of tetracycline (fig. S4, B and C). We then maintenance of heterochromatin.
over 2 days was consistent with the 10XtetO- tested whether destabilizing endogenous het- Because the different deletions essentially al- ade6+ silencing results (Fig. 4, B and C). With the erochromatic regions could release factors that tered the rate of decay of ectopic H3K9me, we higher time resolution and sensitivity of detect- affect maintenance by deleting poz1+, a DNA bind- hypothesized that epigenetic maintenance at the ing phenotypic expression states, we found that ing protein required for heterochromatin forma- ectopic locus might also exist in WT cells over the OFF state persists in 10% of the TetR-clr4-I, tion at telomeres or dcr1+, which is required the time scales of a few cell divisions. To observe clr4+, epe1+ cells for up to 40 hours ( 20 cell divi- for RNAi-dependent heterochromatin formation the decay of silent chromatin with higher time sions) after transfer to tetracycline-containing at centromeres ((fig. S5). In these mutants, resolution, we generated cells in which a green medium (Fig. 4, D and F), whereas cells contain- effects on colony color were absent (dcr1D, fig.
fluorescent protein (GFP) reporter was silenced ing TetR-clr4-I, clr4+, epe1D displayed the most S5A) or subtle (poz1D, fig. S5D), but we clearly by TetR-Clr4-I. We modified the endogenous ura4+ stable maintenance patterns with 60% of the cells observed persistence of H3K9me 24 hours after gene to encode a Ura4-GFP fusion protein. In ad- maintaining the OFF state, even after 100 hours release of TetR-Clr4-I in both deletion backgrounds dition, we inserted the same 10XtetO sites used of growth ( 50 cell divisions) in tetracycline- (fig. S5, B, C, E, and F).
in the earlier experiments immediately upstream containing medium (Fig. 4E). These observa- These results indicate that a domain of H3K9 of a 114-bp fragment of the ura4+ promoter (Fig.
tions indicate that epigenetic maintenance was methylation and its associated silent state can 4A). As determined by fluorescence-activated cell not unique to epe1D cells and that its detection be maintained through mitotic cell divisions in sorting (FACS) analysis, 10XtetO-ura4-GFP was was normally masked by the rapid erasure of the absence of sequence-specific initiation. The silenced in a TetR-Clr4-I–dependent manner in H3K9me marks by Epe1. Although the decay decay rate of this epigenetic state is primarily in- the presence or absence of clr4+. To assess decay rate for cells containing TetR-clr4-I, epe1+ was fluenced by the erasure of the methyl mark by rates, we transferred cells that were grown in rapid (<6 hours), the deletion of Epe1, even in the putative demethylase Epe1 and, to a lesser ex- medium lacking tetracycline (GFP OFF cells) to this background, resulted in a slower decay rate tent, by pathways that promote transcription or medium containing tetracycline and harvested (Fig. 4E). We observed the GFP OFF state in TetR- heterochromatin assembly at endogenous loci.
samples at 0, 3, 6, 9, 12, 15, 23, 26, 32, 36, 39, 50, clr4-I, epe1D cells lacking clr4+, 6 hours after trans- We note that the efficiency of maintenance does 60, 77, and 100 hours for FACS analysis. FACS data fer to tetracycline medium with a complete shiftto the ON state occurring only at 23 hours aftertransfer (Fig. 4C). In these cells, upon elimina- FLAG ChIP (24 hours) tion of Epe1, the decay rate is probably defined primarily by the dilution of modified histones, which may maintain epigenetic states for a fewgenerations in the absence of Clr4-mediated It may be argued that maintenance of H3K9me and the silent state in the initiator-based experi-ments might arise from low-affinity binding of epe1 TetR-clr4-I, clr4+
TetR-Clr4-I to DNA in the presence of tetracy- cline (We sought to unequivocally rule out any role for sequence-dependent initiation in the inheritance we observed by using homologousrecombination to replace TetR-clr4-I with clr4- ID, which harbors a deletion of the TetR DNA domain size 45kb binding domain (Fig. 5A). After transformationto replace TetR-clr4-I with clr4-ID and plating on selective low-adenine medium (Fig. 5A), we ob- tained red, sectored, and white colonies, whichwere tested and confirmed for the replacement event by allele-specific PCR (fig. S6). The isola- TetR-clr4-I + tet
tion of red clr4-ID, epe1D cells confirmed that the silent state could be maintained in the complete absence of sequence-dependent Clr4 recruitment TetR-clr4-I,clr4+ + tet
to DNA. Furthermore, the plating of red clr4-ID,epe1D cells on low-adenine medium produced red,sectored, and white colonies (Fig. 5B), whereas plating of white clr4-I D, epe1D isolates produced only white colonies (Fig. 5C). In the absence of Fig. 2. Deletion of epe1+ allows maintenance of heterochromatin after release of TetR-Clr4-I.
the initiator and no other means of reestablish- (A) Color-silencing assays showing that in TetR-clr4-I, clr4+, epe1D cells, silencing is maintained on ment, the loss of the silent state was an irreversible +tet medium. (B) ChIP experiments showing that TetR-Clr4-I is released from tetO sites in +tet medium.
event (Fig. 5C). Consistent with the deletion of Error bars indicate SD. (C) ChIP-seq experiments showing that in epe1D cells, H3K9me2 is maintained the TetR domain, ChIP experiments showed a 24 hours after tetracycline addition in a clr4+-dependent manner. H3K9me2 ChIP-qPCR and H3K9me3 complete loss of the TetR occupancy signal at ChIP-seq data for samples shown here are presented in fig. S2. Chromosome 3 (site of insertion of 10XtetO- the 10XtetO-ade6+ locus (Fig. 5D). On the other ade6+) and chromosome 1 (cen1L) coordinates are shown above the tracks, and read numbers (per hand, we detected high levels of H3K9 dimethyl- million) are indicated on the right. H3K9me2 at DNA repeats of cen1L serves as an internal control for ation at the 10XtetO-ade6+ locus in red but not the ChIP-seq data.
white isolates (Fig. 5E). We conclude that, once SCIENCE 3 APRIL 2015 • VOL 348 ISSUE 6230

RESEARCH RESEARCH ARTICLE assembled, silent chromatin and histone H3K9me hypothesis, the introduction of an additional ment pathways, is primarily responsible for at the 10XtetO-ade6+ locus can be maintained in copy of WT clr4+, but not the clr4-W31G mutant, RNAi-independent silencing in these deletion the complete absence of the sequence-specific into ago1D cells boosted the residual H3K9me recruitment. To determine whether the initiator- levels approximately threefold (Fig. 5F). Togeth- independent silent state could also be inherited er, these results support a direct "read-write" through meiosis, we crossed red haploid cells of mechanism in which Clr4 binds to preexisting Our findings on mitotic and meiotic inheritance opposite mating type, which lacked the TetR DNA H3K9-methylated nucleosomes and catalyzes the of H3K9me and silent chromatin in the absence binding domain (clr4-ID, clr4+, epe1D), to obtain methylation of H3K9 on newly deposited nucleo- of sequence-specific recruitment—and without diploid cells (Fig. 6A). These diploid cells were somes to maintain heterochromatin independent- requirement for a small RNA (sRNA) positive- then sporulated, and after tetrad dissection, the ly of the initial signals that induced methylation.
feedback loop associated with RNAi, or other resulting haploid progeny were plated on low- It is noteworthy that, in RNAi mutant cells, dele- known modification systems such as DNA CpG adenine medium. As shown in Fig. 6B, the result- tions of epe1+, mst2+, and poz1+ boost H3K9me methylation—strongly suggest that histones modi- ing haploid cells formed mostly red or sectored levels and restore silencing to varying degrees fied by H3K9me can act as carriers of epigenetic colonies, indicating that the silent state was also at the pericentromeric repeats Fur- information. This conclusion is supported by (i) inherited through meiotic cell divisions.
thermore, consistent with its role in H3K9me our demonstration that deletion of the fission inheritance, the chromodomain of Clr4 is also yeast putative histone H3K9 demethylase, epe1+, Inheritance of H3K9me at required for the RNAi-independent spreading stabilizes the epigenetic OFF state and allows native heterochromatin of H3K9me at the mating type locus ). Our its transmission through >50 cell divisions and We next determined the extent to which epige- results suggest that the epigenetic maintenance (ii) the requirement for the Clr4 methyltrans- netic maintenance mechanisms akin to what is of H3K9me, rather than alternative establish- ferase chromodomain, a domain that recognizes observed for the ectopic locus might also operateat native S. pombe pericentromeric repeats. Al- Fig. 3. Requirements though RNAi is required for silencing of reporter for maintenance of the genes that are inserted within pericentromeric repeat regions, deletion of RNAi components does silent state. (A) not entirely eliminate H3K9me and silencing Establishment and (Fig. 7A). Residual H3K9me in RNAi de- maintenance require letions might arise from either epigenetic mainte- HP1 proteins (Swi6 nance mechanisms or weak RNAi-independent and Chp2) and histone establishment signals within centromeres deacetylases (Clr3 and To test for the presence of RNAi-independent sig- Sir2). (B) Maintenance nals that may operate at pericentromeric repeats, of ectopic silencing in we determined whether H3K9me could be es- epe1D cells does not tablished de novo at the repeats in cells with de- require Dicer (Dcr1), as letions of RNAi factors dcr1+ or ago1+. To perform indicated by the growth this experiment, we reintroduced clr4+ into clr4D, D cells on clr4D ago1D, or clr4D dcr1D cells (Fig. 7B) and used ChIP-seq and ChIP-qPCR to quantify H3K9 di- methylation levels. As shown in Fig. 7C, the re- ectopic silencing in introduction of clr4+ into clr4D cells fully restored ∆ dcr1 TetR-clr4-I, clr4+
epe1D cells does not H3K9 dimethylation at the pericentromeric dg require Argonaute and dh repeats of chromosome 1. In contrast, clr4+ (Ago1), as indicated by reintroduction into clr4D ago1D or clr4D dcr1D the growth of red ago1D double-mutant cells failed to promote any H3K9 cells on +tet medium.
dimethylation (Fig. 7, C and D). These results (D) Either deleting epe1 show that RNAi is the primary mechanism for (epe1D) or mutations sequence-specific establishment of pericentro- in its active site (epe1- meric H3K9me domains and suggest that the K314A or epe1-H297A) H3K9me observed at these repeats after deletion allow maintenance of the of RNAi components results from epigenetic off state after release of the TetR-Clr4-I initiator.
Our ectopic heterochromatin experiments es- (E) Replacement of tablished a role for the chromodomain of Clr4 clr4+ with clr4DCD, in epigenetic inheritance of H3K9me (Figs. 2 to encoding Clr4 lacking 5). Consistent with the hypothesis that RNAi- the chromodomain, independent H3K9me at pericentromeric re- abolishes initiator- peats is maintained by epigenetic mechanisms, residual H3K9me at the centromeric dg repeats was abolished in ago1D clr4-W31G double-mutantcells (Fig. 7E). Clr4-W31G contains a mutation inthe chromodomain that attenuates binding to H3K9me . The complete loss of H3K9me inago1D clr4-W31G double mutant cells therefore TetR-clr4-I, clr4+, epe1
suggests that the residual H3K9me marks are maintained by a mechanism that involves directchromodomain-dependent recruitment of Clr4 to preexisting marks. In further support of this 3 APRIL 2015 • VOL 348 ISSUE 6230 SCIENCE RESEARCH RESEARCH ARTICLE di- and trimethylated H3K9, suggesting a direct sequences or modulation of H3K9 demethyl- raising the possibility that a posttranslational read-write mechanism for this mode of epige- ase activity in mammalian cells. Also, in fission modification or cofactor may be required for netic inheritance.
yeast, a previous study reported that ectopic reconstitution of Epe1 demethylase activity.
Several recent studies have described the trans- heterochromatin-dependent silencing of an ade6+ Regulation of histone demethylation activity may generational inheritance of environmentally in- allele induced by a centromeric DNA fragment play a broad role in determining the reversibil- duced changes in gene expression from parent to is maintained in an RNAi-dependent manner ity of epigenetic states. Although it is unknown offspring ). The mechanism of this transgene- after excision of the centromeric DNA fragment whether Epe1 activity or levels regulate epige- rational inheritance has not been fully defined (suggesting that a sRNA amplification loop netic transitions in S. pombe, regulation of his- but appears to occur via both sRNA-dependent may somehow be established at the ade6+ locus, tone demethylase activity has been implicated in and -independent pathways. In Caenorhabditis which helps to maintain the silent state. How- control of developmental transitions in multicel- elegans, plants, fission yeast, and possibly other ever, these findings contradict other studies, which lular eukaryotes. In mouse embryonic stem cells, systems, the transmission of histone modifica- have demonstrated that silencing of ura4+ alleles the pluripotency transcription factor Oct4 acti- tion patterns is often coupled to sRNA generation by the generation of sRNA from a hairpin could vates the expression of Jumonji domain Jmjd1a and/or CpG DNA methylation The latter not be maintained in the absence of the inducing and Jmjd2c H3K9 demethylases, and this activa- pathways can form positive-feedback loops that hairpin Our findings indicate that even in tion appears to be important for stem cell self- help maintain histone modification patterns the absence of coupling to other positive-feedback renewal An attractive possibility is that as This coupling of positive-feedback loops would loops, or in the absence of sequence-dependent epigenetic states become established during tran- increase the rate of reestablishment of silent do- initiation signals, H3K9me defines a silent state sition from pluripotency to the differentiated state, mains and thus counteract the erasure activity that can be epigenetically inherited (fig. S7). Main- reduction in the expression of H3K9 demethyl- of enzymes such as Epe1 or mechanisms that in- tenance of the OFF state is probably determined by ases helps to stabilize the differentiated state crease the rate of histone turnover.
the balance between the rate of H3K9me by the In another example, down-regulation of the amine A previous study used a small-molecule dimer- Clr4 reader-writer module and the loss rate due to oxidase family histone demethylase LSD1 during ization strategy to show that ectopically induced demethylation by an Epe1-dependent mechanism, activation of individual olfactory receptor (OR) domains of H3K9me at the Oct4 locus in murine transcription-coupled nucleosome exchange, and genes in the mammalian nose has been suggested fibroblasts can be maintained after the removal of dilution of histones during DNA replication.
to create an epigenetic "trap" that prevents the the small-molecule inducer by a mechanism that Although demethylase activity for the S. pombe activation of additional OR genes (). More gen- is reinforced by CpG DNA methylation . How- Epe1 protein has not yet been demonstrated erally, H3K9 demethylases may act as surveillance ever, unlike the experiments presented here, the in vitro, key residues required for demethylase enzymes that prevent the formation of spurious use of the Oct4 locus, which is normally packaged activity in other Jumonji domain proteins are H3K9 methylated domains, which may lead to into heterochromatin in fibroblasts , precludes conserved in Epe1 and required for its in vivo epigenetic mutations and gene inactivation.
any conclusions about sequence-independent in- effects on silencing (, and its effect on heritance, as contributions from locus-specific se- initiator-independent inheritance of heterochro- Materials and methods quence elements that normally silence Oct4 in matin (this study, Fig. 4C). In addition, a recent differentiated cells cannot be ruled out. It there- study showed that the activity of human PHF2, Plasmids containing 10XtetO binding sites up- fore remains to be determined whether H3K9me another member of the Jumonji domain protein stream of ade6+ and ura4-GFP reporter genes can be inherited independently of specific DNA family, is regulated by phosphorylation (), were constructed by first synthesizing a plasmid 100 hours
TetR-clr4-I, clr4+, epe1+ TetR-clr4-I, clr4+, epe1∆ Fig. 4. Kinetics of decay of the silent state after release of TetR-Clr4-I using a GFP reporter gene reveals epigenetic maintenance in epe1+ cells.
(A) Schematic diagram of the 10XtetO-ura4-GFP locus. (B to E) FACS analysis of GFP expressionin the indicated strains at 0, 6, 23, and 100 hours after tetracycline addition shows the time evolu- tion of the distribution of GFP-OFF cells. a.u., ar- bitrary units. (F) Data for time points between 0 and 100 hours after addition of tetracycline were plotted to display the fraction of GFP-OFF Fluorescence (a.u.) cells as a function of time. Dose-response curvefitting was used as a guide.
SCIENCE 3 APRIL 2015 • VOL 348 ISSUE 6230 RESEARCH RESEARCH ARTICLE containing 10 tetO sites flanked by 200-bphomology sequences to facilitate reporter in-sertion at the ura4 locus. The reporter genes werecloned downstream of the tetO binding sites using PacI and AscI restriction sites that were incorporated during the initial synthesis of the deletion of TetR DNA plasmid. The ade6+ reporter construct consists of the full-length WT ade6+ gene with endogenous FLAG ChIP
upstream promoter and downstream terminator sequences. The ura4-GFP reporter consists of thefull-length ura4+ gene fused at the C terminuswith a monomeric yeast codon–optimized GFP using Gibson assembly (This construct was epe1∆, red isolate
subsequently cloned downstream of the 10 tetO sites and appended with a 114-bp ura4 promoterelement and the corresponding endogenous ura4 downstream terminator sequence. The plasmid containing TetR-clr4-I was constructed by modify-ing a pFA6a-natMX6-P H3K9me2 ChIP
nmt1 plasmid. The promoter elements in the original plasmid were replacedwith the endogenous clr4+ promoter (using BglIIand PacI restriction sites). The TetR construct con- sists of an N-terminal SV40 nuclear localization sequence followed immediately by a 2X-FLAG epe1∆, white isolate
tag. The clr4+ chromodomain deletion construct consists of a clr4 allele lacking amino acids 7 to59. The synthesis of the TetR-clr4-I fusion with the upstream endogenous clr4 promoter elements was achieved by Gibson assembly. The deletion Fig. 5. Silencing and H3K9me are maintained after deletion of the of the TetR DNA binding element was achieved TetR DNA binding domain. (A) Diagram showing the experimental after modifying a pFA6a-hphMX6-P ,
scheme for conversion of TetR-clr4-I to clr4-ID, which lacks recruitment by insertion of the endogenous clr4+ promoter activity because it can no longer bind to tetO sites. The isolation of sec- ,
red isolate
and a clr4 allele lacking the chromodomain.
tored and red clr4-I D cells demonstrates maintenance in the complete absence of the TetR DNA binding domain (B), whereas white clr4-ID colonies indicate irreversible loss of silencing (C). (D) ChIP-qPCR ex- A strain containing the 10 tetO sites was first periments show that FLAG-tagged TetR-Clr4 signal could be detected made by insertion of the reporter gene at the at tetO sites in clr4-ID cells. (E) ChIP-qPCR experiments show elevated ura4+ locus. The subsequent introduction of the levels of H3K9me in silent clr4-ID cells (red isolates) but background TetR-Clr4-I fusion protein was achieved with the use levels of methylation in ade6+-expressing (white) clr4-ID cells. Error of a PCR-based gene-targeting approach ().
bars in (D) and (E) represent SD.
Strains with the designation TetR-Clr4-I are thosein which the endogenous copy of clr4 is replacedwith the TetR-Clr4-I fusion, making it the only Fig. 6. Inheritance of initiator- source of Clr4 expression in the cells. In strains independent silencing through meiotic where the WT copy of clr4+ is intact (i.e., TetR- cell divisions. (A) Scheme for mating of clr4-I,clr4+), the fusion protein is inserted at the silent (red) ade6+ haploid cells of the trp1+ locus. The deletions of the various RNAi and indicated genotypes in which the TetR- chromatin components were achieved either by Clr4-I was deleted. After sporulation of PCR-based gene-targeting approaches or by a the resulting diploid cells, tetrads were cross followed by random spore analysis and dissected, and the haploid meiotic prog- PCR-based screening to select for colonies that eny were plated on low-adenine medium harbored the reporter gene, the TetR fusion pro- (B). Results of four tetrad dissections are tein, and the appropriate deletion. Strains con- presented and show inheritance and taining deletions of the TetR DNA binding domain variegation of the silent state.
(clr4-ID) were constructed by both PCR-based tar-geting approaches and crosses followed by ran-dom spore analysis. The resulting colonies weretested using allele specific primers. To isolate redcolonies that harbor a deletion of the TetR DNAbinding domain, sectored colonies, which testedpositive for the deletion in the allele-specific PCRscreen, were replated to isolate single red colonieson plates containing limiting adenine. All strainsused in this study are listed in table S1.
Crosses were performed between red isolates of haploid cells of opposite mating type thatharbored a deletion of the TetR DNA binding 3 APRIL 2015 • VOL 348 ISSUE 6230 SCIENCE RESEARCH RESEARCH ARTICLE module. The resulting diploid, which lacks any 30 min at room temperature before quenching sequence-specific establishment factors, was then Cells were grown to a density of 2.5 × 107 cells/ml at with 125 mM glycine for 5 min. The subsequent allowed to sporulate. After tetrad dissection, spores 32°C in yeast extract supplemented with adenine steps for sample processing were performed as were plated on low-adenine medium and allowed (YEA) or YEA containing tetracycline (2.5 mg/ml).
previously described (). Immunoprecipitation to grow at 32°C for 3 days.
Cells were cross-linked with 1% formaldehyde for was performed using the following antibodies:2.5 ml a-H3K9me2 (ab1220, Abcam) for quanti-fying H3K9me2 levels, 2 mg a-H3K9me3 ) forquantifying H3K9me3 levels, and 2.5 ml a-FLAG(M2, Sigma) for quantifying TetR-Clr4-I occupan-cy at the ectopic locus before and after additionof tetracycline. DNA purified from the ChIP ex-periments was analyzed by quantitative PCR usingan Applied Biosystems 7900HT Fast Real-TimePCR system. See table S2 for primer sequences.
ChIP-seq libraries were constructed, sequencedusing an Illumina HiSeq platform, and processedas described previously ).
Strains containing the ade6+ reporter constructwere grown overnight, after which fivefold dilu-tions of each culture were spotted on plates con-taining only yeast extract and glucose withoutany additional adenine supplements with (+tet)or without (–tet) tetracycline (2.5 mg/ml). Eachsilencing assay also included centromeric silenc-ing reporter strains that are unresponsive to tetra-cycline (otr1R:ade6+ and ura4::10XtetO- ade6+) ascontrols to ensure that the addition of tetracyclinedoes not induce any changes in reporter geneexpression.
Cells containing TetR-clr4-I and 10XtetO- ura4-GFP reporter were maintained in log phase ( 2.5 ×107 cells/ml) through the course of sample prep-aration at various time points after addition oftetracycline (2.5mg/ml). Approximately 2.5 × 107cells were harvested and fixed by addition of 70%ethanol for 20 min. The cells were then washedtwice with 1X tris-buffered saline (TBS) (200 mMTris pH 7.5, 150 mM NaCl) and resuspendedin 1 ml of 1X TBS in a FACS tube (BD Falcon).
GFP fluorescence was then measured using aFACScalibur instrument (Becton Dickinson), andexcitation was achieved by using an argon laseremission of 488 nm. Data collection was performedusing Cellquest software (Becton Dickinson), anda primary gate based on physical parameters(forward and side light scatter) was set to ex-clude dead cells or debris. Typically, 20,000 cellswere analyzed for each sample and time point.
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David carmichael

What GlaxoSmithKline Does Not Seem to Want Anyone to Know About Paxil Individuals of al ages should be closely monitored for suicidal and homicidal thoughts and behaviours for at least 1-month after they start taking Paxil and after they increase their dosage. They should also be closely monitored after they stop taking Paxil. By David Carmichael

Les anticancéreux oraux Un regard différent N°8 Septembre 2014 Avec ce nouveau numéro d'Onco News, toute la CAHPP se joint à moi pour vous souhaiter une excellente rentrée 2014. Dans cette lettre en Cancérologie, la CAHPP réaffirme son rôle de centrale de conseil et de référencement souhaitant apporter son expertise en matière d'optimisation des achats et des