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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Struct Mol Biol. Author manuscript; available in PMC Jun 1, 2010.
Published in final edited form as:
PMCID: PMC2788674
NIHMSID: NIHMS145198

An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe

Summary

Histone variant H2A.Z has a conserved role in genome stability, although it remains unclear how this is mediated. Here we demonstrate in fission yeast that the Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or unacetylatable mutation of Pht1 leads to genome instability, primarily caused by chromosome entanglement/breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat length are unaffected by the absence of Pht1. Strikingly the chromosome entanglement in pht1Δ anaphase cells can be rescued by forcing chromosome condensation prior to anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes.

Keywords: Chromosome architecture, condensin, H2A.Z, KAT5, RCA, S. pombe

Introduction

The basic repeating unit of chromatin is the nucleosome core particle: 146 bp of DNA wrapped around a core histone octamer composed of a (H3-H4)2 tetramer and two H2A-H2B dimers. One means of nucleosome-specialization is the replacement of a major histone with a specific variant. These single-copy, non-allelic isoforms are generally expressed throughout the cell-cycle, and many enter nucleosomes through the action of specific deposition machineries 1. Both major and variant histones can be further distinguished by the addition of small chemical moieties, including phosphorylation, acetylation, methylation and ubiquitylation 2,3.

H2A.Z is one of the most studied histone variants. In metazoans it is essential 4, regulating chromosome stability 5, gene activation 6,7 and spermatogenesis 8. The histone is subject to multiple N-terminal acetylations in Saccharomyces cerevisiae (Sc) 911, Tetrahymena thermophila 12, and metazoans 13,14. Though the role(s) of these modifications remains uncertain, defects in heterochomatin restriction 11 and chromosome stability 9 are observed when unacetylatable H2A.Z alleles are expressed as the sole source of the histone in budding yeast.

A phenotype common to all H2A.Z-deficient species is genomic instability. This manifests in mammalian cells as chromatin bridges in anaphase 5. Deletion of Sc H2A.Z (htz1Δ) results in an increased rate of chromosome loss, sensitivity to microtubule destabilizing agents, and synthetic genetic interactions with components of the kinetochore and spindle checkpoint machineries 15. Many of these phenotypes are shared by an unacetylable htz1 mutant, suggesting a role for acetylation in genomic stability 9. However we still have little mechanistic understanding of what leads to the chromosome segregation defect in these cells, though centromere organization, kinetochore attachment, cohesin recruitment and arm sister chromatid cohesion appear normal 9.

To explain the role of H2A.Z in chromosome stability we turned to the fission yeast Schizosaccharomyces pombe (Sp) as its three large chromosomes (Sc, 16 × 230kb – 1.3 Mb; Sp, 3 × 3.5 – 5.7 Mb) can be more easily monitored during mitosis. We show that Sp H2A.Z (Pht1; Pseudo-histone 1) co-purifies with a complex almost identical in composition to the Sc SWR-C, with this complex required for the insertion of Pht1 into chromatin. Chromatin-associated Pht1 is acetylated on its N-terminus by the KAT5 acetyltransferase (Sp Mst1). This modification is essential for Pht1 function, with unacetylatable mutants phenocopying complete deletion of the histone variant in all analyses, including genome-scale genetic-interaction and gene-expression studies. In addition we show that chromosome loss in pht1 mutants is primarily caused by broad architectural defects and can be suppressed by improving chromosome condensation. Consistent with this we show by Chromatin Immunoprecipitation that the condensin complex is prematurely released from chromatin at anaphase in pht1Δ cells.

Results

Pht1 is acetylated as a component of chromatin

Sp Pht1 has four potential acetylatable lysines (K) on its N-terminus: K5, 7, 12 and 16 (Fig. 1a). We synthesized a peptide containing all four acetyl-lysines and immunized rabbits to create polyclonal anti-Pht1Ac. On immunoblots of Sp whole cell extracts (WCEs) this recognized a protein of the appropriate size for Pht1. This signal disappears in WCEs from pht1Δ, −NΔ (deletion of the N-terminus), or mutants where all four lysines were mutated to unacetylatable arginine (R) or glutamine (Q) (Fig. 1b). Thus the Pht1 N-terminus is acetylated and we have a specific reagent for the modification.

Figure 1
Chromatin-associated Pht1 is acetylated by Mst1

The primary acetyltransferase for Sc Htz1 is Esa1, the catalytic subunit of the NuA4 complex 911. Esa1 is a member of the KAT5 family, with the most likely Sp homolog the essential protein Mst1 16. We created a temperature sensitive (ts) allele of Mst1 (mst1-L271P), that was slow at the permissive temperature (25°C) and lethal >34°C (Fig. 1c). WCEs from mst1-L271P cells showed no appreciable change in total Pht1 levels but a profound reduction in Pht1Ac (Fig. 1d), indicating that Pht1 acetylation is Mst1-dependent. Cell fractionation showed that Pht1Ac is chromatin-associated, though acetylation is not required for entry to this cellular compartment (Figs. 1e–f).

Sc Htz1 associates with the SWR-Complex, containing the Swr1-ATPase, and incorporation of the histone into chromatin is markedly reduced in swr1Δ cells 1719. We subjected WCEs from Pht1.TAP to sequential affinity purification and identified the associated proteins by Mass Spectrometry (Supplementary Table 1). Reciprocal tagging and purification of these factors delineates the Sp SWR-C. This is almost identical in composition to the Sc SWR-C, even to the level where subunits shared between the Sp SWR and Mst1-acetyltransferase complexes are those shared by their Sc counterparts, the SWR-C and NuA4 (Fig. 2a). Western analysis with anti-Pht1Ac distinguished those subunits of the Sp SWR-C required for the efficient acetylation of the histone (Fig. 2b), most likely because of inefficient assembly of the variant into chromatin in each background (Fig. 2c). Thus a pathway first identified in Sc also operates in Sp: SWR-C inserts Pht1 into chromatin, where it is acetylated by Mst1.

Figure 2
Pht1 is inserted into chromatin by the SWR-C

Acetylation is integral to Pht1 function

To determine the relevance of N-terminal acetylation to Pht1 function, we compared the gene expression and genetic interaction profiles of various unacetylatable mutant alleles (pht1- NΔ,4KR, and −4KQ) to pht1Δ.

The transcription profiles of the unacetylatable mutants were almost identical to pht1Δ (e.g. relative P-value: Δ to −4KR = 2e−195; Fig. 3a). A striking observation was that almost all genes were modulated to some degree in pht1 mutants (Fig. 3b), suggesting that the histone acts as a general transcriptional regulator. By comparison swr1Δ showed weaker changes in gene expression (Fig. 3b). Since loss of the ATPase strongly reduces, but does not completely ablate chromatin-associated Pht1 (Fig. 2c), it is likely that the remainder is sufficient to mask most H2A.Z-dependent phenotypes.

Figure 3
Unacetylatable pht1 mutants phenocopy pht1Δ

We then used the Pombe Epistatic Mapper 2 (PEM-2) system to quantitatively analyze genetic interaction patterns 20,21. To this end we individually crossed pht1Δ, −NΔ, −4KR, −4KQ and swr1Δ strains to a library of 2161 non-essential Sp deletions (the Bioneer collection), and derived scores covering each negative (e.g. synthetic sick/lethal) and positive (e.g. suppression) genetic interaction using colony size as a quantitative read-out 20,2224. Positive interactions enrich for factors that are co-complexed or function in the same pathway 20,22,23. Consistent with this, pht1Δ, unacetylatable pht1, or swr1Δ each gave rise to positive genetic interactions in combination with deletions of all non-essential subunits of the SWR-C (20 and Fig. 3c). This implies that preventing Pht1 acetylation disables the primary function of the SWR-C/Pht1 pathway in fission yeast.

The genetic interaction profile of a particular mutant can be used as a high-resolution phenotype. Comparison of this profile to those generated from other mutants can identify functionally related factors 20,22,23. When compared to data from >100 genetic screens (not shown), pht1Δ and swr1Δ were highly correlated (Fig. 3d), confirming that they function in the same pathway. Indeed, of all mutants analyzed, the most highly correlated to pht1Δ were −NΔ, −4KR and −4KQ, suggesting that N-terminal acetylation is critical for Pht1 function (Fig. 3e). Inspection of individual interactions showed that all unacetylatable pht1 alleles were synthetic with deletions of factors involved in chromatin modification/remodeling (e.g. COMPASS, RSC, SET3-C), transcription (e.g. Mediator) and chromosome segregation/cytokinesis (e.g. cut8, DASH complex) (e.g. Fig. 3f). These are reminiscent of the synthetic interactions displayed by Sc htz1Δ 15,17, further suggesting strong conservation of H2A.Z function in each organism.

Chromosome loss in pht1Δ is caused by entanglement at anaphase

Knockout or depletion of H2A.Z in Sc 15 or mammalian cells 5 leads to increased rates of chromosome loss. This phenotype was also observed if any component of the Sp Pht1Ac pathway is disrupted, including mutants in swr1 (and msc1), pht1 (pht1Δ, −4KR or −4KQ), or mst1 (Supplementary Table 2 and 16,25,26). One possible explanation for chromosome instability is disruption of the centromere 27. However as in htz1Δ cells 9, centromere structure and function appeared normal in pht1 cells (Supplementary Fig. 1).

On cytological analysis of individual Sp cells we observed a >8-fold increase (relative to WT) in the number of pht1Δ cells with anaphase chromosome segregation defects (Fig. 4a). Three specific categories of anaphase defects were distinguished using the kinetochore marker Nuf2-GFP: (i) lagging chromosomes, where the mis-segregating chromatin contains at least one kinetochore, (ii) chromosome entanglement, where the mis-segregating chromatin is stretched between the spindle poles but contains no kinetochore, and (iii) entanglement leading to breakage, where broken pieces of chromatin with no kinetochore lag on the spindle (Fig. 4b). The primary segregation defect in pht1Δ cells was entanglement: >80% of defective anaphases. This was clearly distinct from the primary anaphase segregation defect in heterochromatin-deficient clr4Δ cells: >90% lagging chromosomes (Fig. 4c). Lagging chromosomes result from merotely, a defect where a single kinetochore attaches to microtubules emanating from opposite spindle poles 28. Thus, to a large extent, kinetochore - microtubule attachments appear normal in pht1Δ cells, supporting our ChIP observation of a WT-like centromere in pht1 mutants (Supplementary Fig. 1c). Consistent with this result, pht1Δ cells did not activate the spindle checkpoint, nor did they rely on spindle checkpoint genes for survival (not shown).

Figure 4
Lack of Pht1Ac leads to chromosome segregation defects in anaphase

Chromosome entanglement in pht1Δ cells was particularly obvious when we followed telomere segregation at anaphase. To do so, we used the telomere-binding protein Taz1-GFP as a marker. In a normal mitosis, six Taz1-GFP spots (identifying the left and right telomeres on the three Sp chromosomes) migrate to opposite poles with the bulk of DNA. However in pht1Δ cells we often observed two or more Taz1-GFP foci entangled in the centre, leading to stretched chromosome arms (Fig. 4d). One possible explanation for this entanglement is a defect in telomere function comparable to that observed in cells lacking the protection factor Taz1 29. However many of the phenotypes of taz1Δ were not shared by pht1Δ: e.g. cold-sensitivity, size of the chromatin bridge, presence of Rad22 foci, rqh1-SM suppression (Supplementary Fig. 2). In addition, and most striking, while telomere-repeat length was dramatically increased in taz1Δ, it was comparable to WT in pht1Δ (Fig. 4e).

To quantify how frequently the entanglement in pht1Δ cells leads to chromosome loss, we employed a series of strains containing: (i) lacOperator (lacO) repeats integrated at different chromosomal locations, and (ii) LacI-GFP, the fluorophore marked lacO binding protein 30. Surprisingly, marker loss in mitotic pht1Δ cells increased with distance from the centromere, suggesting that they generally lost broken pieces of chromosomes rather than whole chromatids (Fig. 4g–h). This is in sharp contrast to sgo2Δ cells, which are unable to correct chromosome bi-orientation defects 31,32. In sgo2Δ cells marker loss was similar irrespective of chromosome position, suggesting the loss of whole chromatids (Fig. 4h). Together these observations suggest that the primary role of Pht1 in chromosome transmission is to maintain overall chromosome architecture rather than to regulate the function of a specific region (such as the centromere or telomere).

Pht1 plays a role in chromosome architecture/compaction

Chromosomes condense to a folded rod-shaped structure upon mitotic entry. As the chromatids are pulled to the spindle poles in early anaphase, the chromosome arms trail behind the centromeres, forming a distinctive “arrowhead” structure. This was lost in >50% of cells lacking pht1 (Fig. 5a). Such a generalized defect further supports the idea that Pht1 regulates overall chromosome architecture rather than that of specific loci.

Figure 5
Pht1 plays a role in chromosome architecture/compaction

The primary chromosome segregation defect in pht1Δ cells (i.e. chromosome entanglement despite accurate centromere segregation; Fig. 4) is reminiscent of that observed in mutants of condensin, a five-subunit complex instrumental to the architecture and segregation of chromosomes in mitosis 3336 (Supplementary Fig. 3a). We thus speculated that pht1 mutants may mis-regulate chromosome condensation during anaphase. If so, it may be possible to suppress their defect by forcing chromosome hyper-condensation. To this end we used the cold-sensitive tubulin mutant, nda3-KM311, which fails to assemble microtubules at the restrictive temperature (20°C), leading to a spindle-dependent checkpoint arrest 37. This prolonged early mitotic arrest induces chromosome hyper-condensation 37, and when shifted to the permissive temperature (32°C) these cells enter a synchronous anaphase 31,37. Strikingly, this prolonged mitotic arrest specifically rescued the chromosome segregation defect of pht1Δ but not taz1 cells (Fig. 5b). Arresting pht1Δ cells in S-phase with hydroxyurea (HU, depletes nucleotide pools) had no effect on the chromosome segregation defect, showing that merely prolonging the cell-cycle was not sufficient to rescue pht1Δ defects (Fig. 5b).

A functional relationship would be indicated by genetic interaction, so we tested that between pht1 and condensin. Unacetylatable pht1 or pht1Δ each raised the restrictive temperature of ts alleles of three complex subunits: cut3-477 (smc4-S1147P), cut14-208 (smc2-S861P) and cnd2-1 (cnd2-A114T) (Fig. 5c and Supplementary Fig. 3b). This partial rescue was specific, as pht1Δ was synthetic with rad21-K1, a mutant in the condensin-related complex cohesin, which holds sister-chromatids together prior to anaphase onset. This suppression is unlikely to be mediated through indirect transcriptional effects (e.g. increased expression of condensin), since the mRNA levels (by gene expression microarray) of all tested complex subunits and known regulators (e.g. Cut17, Ark1, Top2, Fin1, Pim1, Pic1, Acr1, Nuc1) were comparable to WT in pht1Δ and unacetylatable pht1 cells (Supplementary Fig. 3c and not shown). In addition the partial rescue was not observed on deletion of two other transcriptional regulators: the Set1 methyltransferase or Gcn5 acetyltransferase (Fig. 5c).

Our results are consistent with a model where an acetylated form of Pht1 regulates condensin loading and/or localization in mitosis. To test this, we arrested WT or pht1Δ cells expressing C-terminally tagged Cut3 (Cut3.HA3) at the G2/M boundary, and then monitored condensin occupancy kinetics through a synchronous mitosis (Fig. 5d). To first ensure that mitotic progression in WT and pht1Δ cells followed similar kinetics, we examined the peak septation index and H3-K9Me2 in each population. A newly formed septum indicates the completion of mitosis and can be visualized by specific staining with Calcofluor (Supplementary Methods). H3-K9Me2 is markedly reduced at centromeric repeats (otr) during mitosis, increasing as cells enter G1/S 38. Each pattern was indistinguishable between WT and pht1Δ cells (Fig. 5d). When we examined condensin recruitment, Cut3.HA3 initially (≤75 mins) followed similar kinetics at the centromere (cnt, inr and otr) and rDNA of WT and pht1Δ cells (Fig. 5d and Supplementary Fig. 3d). However significant differences were then observed: Cut3.HA3 levels continued to increase in WT cells, peaking at 100 mins before dropping at 125 mins (the latter corresponding to the peak septation index time point; Fig. 5d). However in pht1Δ condesin delocalization began by 100 mins, indicating the premature dissociation of the complex from chromatin. This likely explains many of our observations in these cells: loss of the anaphase arrowhead structure leading to chromosome entanglement and the loss of telomere-proximal regions (Fig 5).

Discussion

In this work we identify and characterize a previously unknown role for the histone variant H2A.Z (Sp Pht1) in chromosome architecture at anaphase. In the absence of Pht1, chromosomes frequently entangle in anaphase, which can lead to breakage and loss, particularly of telomere-proximal regions. We provide evidence that chromosome entanglement in pht1Δ is most likely due to premature dissociation of the condensin complex in anaphase. We also demonstrate that the factors involved in the chromatin-loading and acetylation of H2A.Z are highly conserved in Sc and Sp. The last common predecessor of these yeasts was ~ 380 million years ago (mya): by comparison that of the entire mammalian class existed ~ 165 mya 39, while the primate line split to humans and gorillas ~ 8 mya 40. We propose that since the pathways regulating H2A.z localization, modification, and function are so well conserved, the role of the histone variant in chromosome architecture will be equally penetrant.

Extensive conservation of the Swr1-C

Comprehensive proteomic analyses identify two subunits in the Sp SWR-C not seen in its Sc counterpart: Msc1 and SPAC4H3.02c (Fig. 2a, Supplementary Table 1, and 41). Both deletions are epistatic with pht1 (Fig. 3c and 20), further suggesting a functional relationship. Msc1 was originally identified as a multi-copy suppressor of cells defective for checkpoint kinase Chk1 (Rad27) function 42. The Walworth lab have demonstrated an epistatic chromosome loss phenotype in pht1Δ and msc1Δ, and suggested that Msc1 was upstream of Pht1 in this pathway 26, a relationship our data supports (e.g. Fig. 2c). Msc1 was recently shown to interact with the Mst1 acetyltransferase by yeast-2-hybrid analysis 16. Despite this, any physical interaction between Mst1 and Msc1 is likely transient, as Msc1.TAP purification did not identify Mst1 (or any non-shared subunit of the Swr1 and Mst1 Complexes: Supplementary Table 1), and unique members of the Mst1-C do not co-purify Msc1 (not shown). However this is an interesting link between the Swr1 and Mst1 complexes, and may suggest that Msc1 directly recruits Mst1 to sites of Pht1 integration.

N-terminal acetylation is integral to Pht1 function

The Swr-C is required for the assembly of Pht1 into chromatin, where it is acetylated by Mst1 (Figs. 1 and and2).2). This relationship between the ATPase (Swr1), histone variant (H2A.Z) and KAT5-family acetyltransferase (Mst1) appears to be widely conserved 9,15,4346. Unacetylatable pht1 phenocopies pht1Δ throughout this work, but is perhaps most striking in large-scale gene expression and genetic analyses (Fig. 3). This strongly suggests that acetylation of the histone is integral to its function. Based on Sc Htz1, it is likely that all four lysine residues in the Pht1 N-terminus are modified 10,11. However our pht1Ac antibody was raised to a tetra-acetylated peptide (Fig. 1a) and the unacetylatable pht1 used throughout this work has mutations at all four N-terminal lysines, so this is as yet unconfirmed. mst1-L271P reduces Pht1Ac below the threshold of detection (Fig. 1d), suggesting we have identified the major enzyme for this modification. In this regard, we note that mst1 mutants also show increased rates of chromosome loss 16, likely due in part to reduced Pht1Ac.

Pht1 regulates chromosome architecture at anaphase

Knockout or depletion of H2A.Z in Sc 15, Sp (this work and 25,26), or mammalian cells 5 leads to increased rates of chromosome loss. An acetylated form of H2A.Z mediates the chromosome stability role in both Sc 9 and Sp (this work), strongly suggesting that this will prove to be the case in other organisms. Directly monitoring chromosome segregation in individual Sp cells allowed us to show that lack of Pht1 induces chromosome arm entanglements in anaphase that can lead to chromosome breaks (Fig. 4). Furthermore, Pht1 is required for the stable association of condensin with chromatin through anaphase (Fig. 5d), and the chromosome entanglement in pht1Δ cells can be rescued by a pre-anaphase arrest where chromosomes hyper-condense (Fig. 5b). Thus the lack of Pht1 interferes with chromosome architecture in anaphase. However it also improves the viability of mutants that regulate this process, such as those in condensin (cut3-477, cut14-208, cnd2-1) or topoisomerase II (top2-191) (Fig. 5c and Supplementary Fig. 4). To resolve this apparent contradiction, we propose that Pht1 actually plays a dual role in mitotic chromosome architecture (see below).

Mitotic defects in a chicken condensin mutant primarily occur in anaphase, such that chromosomes prematurely lose their compact organization as they move to the poles 47. Thereafter individual chromatids can no longer be distinguished and prominent chromatin bridges are visible in >90% of cells (e.g. Fig. 6b and Supplementary Fig. 3a). These phenotypes can be overcome if Repo-Man, a targeting subunit for protein phosphatase 1 (PP1), is unable to direct the phosphatase to chromosomes in anaphase 47. In WT cells Repo-Man is subject to CDK-dependent phosphorylation(s) to abrogate chromosome binding at early mitosis when CDK levels are high. Chromosome compaction at this stage is only slightly affected in condensin mutants, prompting the Earnshaw lab to propose that an as-yet-uncharacterized activity, Regulator of Chromosome Architecture (RCA), drives initial condensation. RCA is then inhibited in a Repo-Man/PP1-dependent manner after CDK levels fall at anaphase onset and condensin steps in to stabilize chromosome architecture until mitosis completes 47 (all modeled in Fig. 6d).

Figure 6
H2A.Z plays a role in higher order chromosome architecture

The above model suggests that there are at least two steps in chromosome condensation. The first is RCA-dependent compaction to the characteristic X-shaped mitotic chromosome. The second is the condensin-dependent maintenance of a robust architecture that can withstand the pulling forces of microtubules in anaphase. Our data suggests that Pht1 regulates both steps: promoting the inhibition of RCA, and stabilizing the association of condensin. As above, artificially maintaining RCA activity in anaphase by preventing the loading of PP1 to chromatin partly rescues the chromosome segregation defects of chicken smc2 mutants 47. This is reminiscent of the genetic interactions between pht1 and condensin (e.g. Fig. 5c). It is highly likely that RCA is conserved in Sp as deletion of PP1Dis2, the fission yeast PP1 ortholog that localizes on chromatin, also partly rescues cut3-477 (Supplementary Fig. 4b). Finally we have recently shown that Sc Bud14, a regulator of Sc PP1 (Glc7), is required for the efficient loading of Htz1 onto chromatin (and its subsequent acetylation) 48. This suggests that H2A.Z could act downstream of PP1 to inhibit RCA in anaphase (Fig. 6d). Further studies are underway to resolve this model.

Supplementary Material

Acknowledgments

We thank Robin Allshire (University of Edinburgh), Marc Bühler (Friedrich Miescher Institute for Biomedical Research), Julie Cooper (Cancer Research UK), Dan Finley (Harvard Medical School), Susan Forsburg (University of Southern California, Los Angeles), Keith Gull (University of Oxford), Charles Hoffmann (Boston College), Junko Kanoh (Kyoto University), Rich Maraia (NIH), Danesh Moazed (Harvard Medical School), Toru Nakamura (University of Illinois at Chicago), Frank Neumann (Rockefeller University), Paul Nurse (Rockefeller University), Matthew O’Connell (Mount Sinai School of Medicine), Janet Partridge (St. Judes Childrens’s Research Hospital), Mitsuhiro Yanagida (Kyoto University) and the Yeast Genome Resource Center (Osaka City University) for the generous supply of antibodies and yeast strains (detailed in Supplementary Tables 3 – 4). We also thank Guoquing Zhong, Shamanta Chandran, Thanuja Punna (all University of Toronto), and Mike Shales (UCSF) for technical support. Finally we are grateful to Gwyneth Ingram (University of Edinburgh) for expertise with the Telomere Repeat length assay. Work in the J.B. lab is funded by Cancer Research UK, T.K. lab by a start-up grant from the OCU, K.G.H. lab by a program grant from the Wellcome Trust, and M-C.K. lab by the Speaker’s Fund for Biomedical Research: Toward the science of Patient Care, awarded by the City of New York.

Footnotes

Author Contributions

H-S.K. and V.V. contributed equally to this work. H-S.K. was responsible for the data in Fig. 1 (with A.T. contributing Fig. 1c), Fig. 2 (with Mass Spectrometry help from J.F., T.K., A.E. and J.F.G.), Fig. 5c–d and Supplementary Figs 1 and 3. V.V. (with help from K.G.H.) was responsible for the data in Fig. 4, Fig. 5a–c and Supplementary Figs 2, 3a and 4. S.W. and J.B. performed and analyzed the microarrays in Fig. 3a–b; A.R. and N.J.K. performed and analyzed the genetic screens in Fig. 3c–f. L.R.C and C.S.B. created the anti-Pht1 antibodies used in Fig. 1. H-S.K., V.V. and M-C.K. planned experiments, analyzed the data and wrote the manuscript.

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