Design of a versatile episomal/integratable synthetic histone cassette
To develop a maximally useful and flexible resource, we designed a synthetic cassette with unique features allowing 1) use of a wide range of selectable markers, 2) delivery either as a replication-competent episome or integrated at a native histone locus with high fidelity, and 3) use in complex phenotypes analyses too labor intensive for individual mutants (overall design; ).
Fig. 1 Features of synthetic histone cassette. A. Schematic representation of histone H3/H4 cassette in pRS414. The two selectable markers, TRP1 and URA3, can be ued to select an episomal copy or an integrated cassette respectively. B. Cassettes contain synthetic (more ...)
There are two selectable markers in the construct, TRP1
is designed for use in introducing the mutations as part of a CEN (episomal single copy) vector (). URA3
and flanking DNA segments HHT2L
are used to integrate the cassette at HHT2-HHF2
(replacing one of two loci at which H3/H4 genes normally reside; ). URA3
is itself flanked by loxP
sites, facilitating URA3
removal in E. coli
or yeast (Sauer, 1987
), or replacement of URA3
by any other loxP
flanked selectable marker (Güldener et al, 2002
). This design allows one mutant library to be used in a wide variety of genetic contexts.
To be useful for targeted integration, efficiently targeting of the resident HHT2-HHF2
locus and replacement with the mutant copy on the plasmid is needed, gene-targeting was precise (supplemental methods
). Tests of simple phenotypes can be scaled up and performed in the traditional manner on the surface of agar plates by replicating devices. However some phenotypes are more complex, and/or require plating on a series of different media before phenotypic scoring. Molecular barcodes or TAGs have been used for such applications, and to study genetic interactions. Pairs of TAGs were assigned to each mutant; a subset of the ~12,000 molecular barcodes used in the yeast knockout collection (Winzeler et al, 1999
) was used to tag the mutants (Experimental Procedures).
Library of Histone H3 and H4 Mutants
Gene synthesis was employed to create a library of 486 bar-coded histone H3 and H4 mutants containing a number of systematic amino acid substitutions (Supplemental methods
; ). For example, in addition to a complete alanine scan, all lysine residues were additionally mutated to arginine and glutamine to potentially mimic constitutively de-acetylated/acetylated states. The collection also contains sets of 52 and 27 systematic deletion alleles of the N-termini of H3 and H4, respectively, ranging in size from 4 to 36 residue deletions. These deletion series extend from the N termini to residues H3 36 and H4 24, the positions at which the tails emerge from the nucleosome core; endpoints are placed every 4 residues; deletions made and studied previously by other investigators were added.
All 486 mutations were integrated at HHT2-HHF2 in two distinct strain backgrounds, GRF167 and S288C, in the presence of wild-type histone proteins encoded by shuffle plasmid pJP11; viable mutants were also banked after shuffling.
Histone mutation database
A histone mutation database documenting the validated phenotypes of all mutants described here and in other systematic mutagenesis studies (Hyland et al, 2005
; Matsubara et al, 2007
; Park et al, 2002
) was established that allows searching by residue, phenotypes, and other parameters. It displays phenotypic data, performs clustering and other analyses, and allows visualization. It was essential for interpreting comprehensive screens. The database, available at www.histonehits.org
, will be described in detail elsewhere (H.H., E.M.H., J.D., A. Norris, P. Lee, J.D.B. and J.S.B., in preparation).
Complex lethality profiles of H3 and H4 mutants
The ability of each mutant to survive in the absence of wild-type was initially determined by a plasmid shuffle technique. Mutants were denoted lethal if integrated copies failed to produce plasmid-free segregants after extended subculturing on nonselective medium. In total, 47 of 407 point mutants, encompassing 40 residues, failed to support viability in both strain backgrounds (). It is remarkable how many residues in these highly conserved proteins can be mutated and retain basic nucleosomal function.
Fig. 2 Analysis of lethal histone alleles. A. Substitution mutations above the sequence are lethal in the S288C strain background (blue); those below are lethal in GRF167 (green). Arrows indicate substitution mutations with strain-specific lethality. Clusters (more ...)
Mapping the residues affected by lethal substitution mutations to the nucleosome structure revealed two nucleosome surface locations highly sensitive to amino acid changes, the nucleosome entry/exit site and the nucleosome dyad axis (). The surface residues required for viability were largely restricted to those interacting directly with DNA (). A second set of lethal mutants was in the hydrophobic core. Nearly all of these fell in alpha helices and tended to be more highly conserved residues (), suggesting that the mutations interfered with nucleosome assembly or histone stability.
Amino acid substitutions that increase net negative surface charge are prominent among the lethal mutants. These fall into two classes; one class is on the DNA binding (lateral) surface and consists of positively charged residues that when changed to neutral residues is lethal but when changed to other positive residues is not. The second class includes semi-buried uncharged residues near the end of alpha helices in the core; when these residues are changed to acidic residues (but not other neutral residues) it can lead to lethality. Additionally, 9 of 79 N-terminal tail deletions resulted in lethality in both strains (). The pattern of lethality in histone tails is complex. In histone H3, the two longest tail deletions are inviable, and only in one of two strain backgrounds tested. A single much shorter deletion is inviable in both backgrounds; this protein accumulates at near normal levels, thus lethality presumably results from a toxic junction sequence. In histone H4, the pattern also defies simple explanation but many of the longer deletions are inviable in both backgrounds; the data suggest deletion of 4−5 lysine residues is incompatible with viability.
Strain differences matter
For the most part, the list of inviable mutations agreed in the two strain backgrounds, with some notable exceptions (). In a few cases, a lethal mutant in one strain background was “sick” in the other, but in most cases, mutations causing lethality in one background supported near normal growth in the other. These discrepant results between strain backgrounds help explain earlier reports of phenotypic discrepancies in the literature, for example discordant results with histone tail multipoint K to R mutants (Megee et al. 1990; Johnson et al. 1990
; Park and Szostak 1990
One especially interesting cluster of residues near the C-terminus of histone H4 falls into this category, along with some residues essential (or conferring slow growth) in both backgrounds. These include Y88, L90 and Y98. These residues appear to form an important surface inside
the nucleosome core. Viewing the nucleosome structure from the disk face with the dyad at 12 o'clock, the upper half of the nucleosome is packed with the H3 H4 tetramer and the H2A C-termini and largely solvent-free. The lower half of the nucleosome consist of two separate half-disks separated by a water-filled but somewhat tortuous cleft in a castanet-like structure (Fig. S1
). The residues noted above are part of a surface that forms the base of this cleft; the residues are interwoven with portions of histones H3 and H2B to form this surface. We suggest that these residues could serve as a molecular spring that maintains tensile strength in the lower half of the nucleosome. Tyrosine 88 of H4 stacks on Tyrosine 86 of H2B in the structure, forming a spring-like structure (Fig. S1B
). Whereas mutation to phenylalanine supports viability, a more severe substitution to glutamate does not.
Episome remediality and protein stability of lethal mutants
While the number of lethal mutations is small, the number of essential residues detected here is somewhat higher than reported in other studies. The lethal mutants are likely to be lethal for a variety of reasons.
We hypothesized that the slightly higher frequency of lethal mutants observed in this study relates to the fact that the alleles were integrated in single copy as opposed to other studies in which they were maintained episomally; unstable plasmid copy number could potentially mitigate the deleterious effects of a mutated histone, presumably by increased expression as a result of selection for mildly increased copy number. We systematically tested episome remediality of lethal mutations by introducing them on a centromeric plasmid. We found that 25% (14/55) of the point mutants that were lethal in strain JDY23 were episome-remedial (Table S1
We tested protein stability of the lethal mutant histones in the presence of a tagged wild-type histone ( and S2
) and found that 35% (12/34) of the H3 and 38% (8/21) of the H4 lethal point mutants led to a significant decrease in protein abundance. Decreased RNA abundance did not explain reduced protein abundance in these mutants (Fig. S3
). Intriguingly, several of these mutants appear instead to overproduce
histone mRNA, suggesting regulatory compensation for a histone deficit. All lethal tail deletions showed only minor effects on protein stability. However, many of the lethal mutants produced high levels of protein, and protein was detectable in all of the mutants excepting H3 D123A/N.
High-throughput phenotyping of individual H3 and H4 Mutants
Phenotypes associated with each of the viable histone mutants in the GRF167 background were determined in thirteen separate assays grouped into five phenotypes: temperature sensitivity, DNA damage response, transcriptional elongation, transcriptional silencing, and response to microtubule disruption. Cold-sensitive (Cs) alleles were scored at 16°C; temperature-sensitive alleles (Ts) were scored at both 37°C and 39°C. The DNA damage response was tested in the presence of hydroxyurea (HU), camptothecin (CPT), methyl methanesulfonate (MMS), or UV irradiation. Mutants with potential defects in transcriptional elongation or in their response to microtubule disruption were identified by their sensitivities to 6-azauracil (6AU) or to benomyl, respectively. The role of specific residues in transcriptional silencing was assessed using reporter genes inserted at all three transcriptionally silent regions in yeast, namely the rDNA, the telomeres and HMR
loci, as well as by mating competency. Data for the individual mutants in each of these assays are available on the Histone Mutation Database (www.histonehits.org
) and are visually represented on the nucleosome surface in Fig. S3
. Most of the histone point mutants had phenotypes in 0−1 classes (defined in Experimental Procedures); few mutants had phenotypes of >three classes (). Overall agreement with data from previous studies (Hyland et al., 2004; Matsubara et al, 2007
) was excellent (Fig. S5
Fig. 3 High-throughput phenotypic analysis. A. Pie chart shows % of mutants with pleiotropy values of 0−5, defined as the number of phenotype classes with at least one non-wild-type phenotype. B. Distinct but overlapping substitution mutations affect (more ...)
Histone substitutions had the greatest effect on transcriptional silencing with a total of 183 (38%) and 148 (30%) of the alleles displaying altered levels of reporter gene expression from rDNA and telomeric heterochromatic loci respectively. The assays permitted detection of both increased silencing and loss of silencing and the results indicate that approximately 80% and 75% of these alleles have a negative influence on rDNA and telomeric silencing respectively.
illustrates the nucleosomal position of the mutations affecting silencing with a screenshot from www.histonehits.org
. Histone substitutions on the nucleosome face were significantly enriched for both loss of telomeric silencing (LTS) and loss of HM
silencing (see HMR
silencing results, Fig. S3 I
). These residues cluster around the previously identified LRS surface (Park et al, 2002
), encircling the H3 K79 methylation site, known to play a role in demarcating euchromatin and heterochromatin (van Leeuwen et al, 2002
). At the rDNA, nucleosome surface mutations predominantly lead to an increased rDNA silencing (IRS) phenotype. This is perhaps not surprising given the mechanistic differences underlying rDNA silencing and silencing at the telomeres and HM
loci. It is striking, however, that this distinction can be manifested in histone mutations at similar nucleosome positions and suggests that heterochromatin structure at these different silent loci varies dramatically. Mutations on the lateral surface show similarly discordant results at telomeric and rDNA loci. These substitutions are statistically enriched for LTS, in addition to IRS phenotypes. Within chromatin, this surface is inaccessible as it lies beneath the DNA helix and presumably anchors histone-DNA interactions. However the different classes of phenotypes arising from alterations in the amino acid composition at this interface would suggest that these residues play more complex roles in formation and maintenance of locus-specific heterochromatin.
The tail deletions showed striking silencing phenotype patterns. 77% of H3 tail deletions have an LRS phenotype whereas only 33% and 8% showed an effect at telomeres and HMR respectively, indicating the main role of the H3 tail in silencing is at the rDNA. The converse pattern of silencing phenotypes was noted in strains expressing deletions in the H4 tail. Nearly 80% of viable H4 tail deletions lost the ability to silence the reporter at both telomeres and at HMR, whereas only 17% affected rDNA silencing. These data further extend the discordance between rDNA and telomeric/HMR silencing mechanisms and support the hypothesis that they converge on distinct nucleosome domains.
To investigate whether pleiotropy correlates with evolutionary conservation, we used Consurf (Glaser et al, 2003
; Landau et al, 2005
) to calculate evolutionary conservation scores for each residue, assigning discrete scores from 1 (most variable) to 9 (most conserved). As expected, residues with higher pleiotropy values were significantly more conserved, (; p = 0.005, one-sided Kendall's rank correlation test).
We explored whether certain phenotypic classes were overrepresented geographically on the nucleosome (Table S2; supplemental methods
). The frequencies of phenotypic values for different domains were significantly different (; p = 0.001, Kruskal-Wallis rank sum test). Tail residue mutations are significantly less pleiotropic than mutations in buried regions (p = 0.004) or the disk face (p = 0.004 and p = 0.0012 respectively, two-sided Wilcoxon signed-rank test corrected for multiple comparisons). 6-AU sensitive mutants were overrepresented on the lateral surface and were absent from the disk face (histonehits.org
). Temperature sensitive mutants were overrepresented on the disk face and absent from the tails (Table S2
). shows the correlation between phenotypic value frequencies and change in side chain pKa
values (supplemental methods
); different pKa
classes had significantly different numbers of phenotype classes (p = 0.003, Kruskal-Wallis rank sum test). Post hoc paired comparisons demonstrated that acid-to-neutral and basic-to-neutral changes were significantly more likely to produce more severe phenotypes than basic-to-basic changes (p = 0.04 and 0.03 respectively, two-sided Wilcoxon tests corrected for multiple comparison). Tail deletions demonstrated a significant correlation between deletion length and pleiotropy (; p = 2 × 10−12
, one-sided Kendall's rank correlation test).
Effect of tail deletions on nucleosome core modifications
Interdependencies of histone modification sites have been noted previously (Kouzarides, 2007
). In parallel work, Nakanishi et al. (A. Shilatifard, pers. comm.) systematically mapped interdependencies of histone modifications using an alanine scanning substitution series. To test the utility of our resource for discovering interdependencies we probed N-terminal tail requirements for histone H3 acetylation at K56 and methylation at residues K4 and K79.
Immunoblotting was performed on whole cell extracts from yeast strains harboring viable deletions of histones H3 or H4 (). Every histone H4 tail deletion lacking residues 17 to 23 completely blocked dimethylation of H3 K79. Loss of K79 dimethylation is specific to H4 tail deletions, as none of the H3 tail deletions had this effect (data not shown). The effect of H4 tail deletion on K79 dimethylation is specific; tail deletions affected neither K56 acetylation nor K4 methylation (data not shown). To pinpoint critical residues within H4 important for K79 dimethylation, we exploited the available single point mutations (); mutants R17A, H18A, and R19A completely inhibit K79 dimethylation. Additionally the I21A mutant dramatically, if not completely blocked modification and L22A had a minor inhibitory effect. These results extend recent reports that basic patch residues (residue 17−20) on histone H4 regulate H3 K79 methylation (Altaf et al, 2007
; Fingerman et al, 2007
). Our results show clearly that at least one residue (I21) beyond the basic patch residues identified previously is also critical for H3 K79 dimethylation.
Fig. 4 Tail deletions affect K79 methylation. A. Immunoblots of extracts of cells expressing wild-type or indicated H4 tail-deletion mutants using antibodies against dimethylated K79 (diMeK79). Antibodies against histone H3 and H4 were used as loading controls; (more ...)
Survival of the fittest – behavior of the mutant pool in a chemostat
The high level of sequence conservation of histone proteins across phyla suggests a fitness advantage of these particular amino acid sequences during evolution. Yet comprehensive analysis indicates that many histone mutations have no recognized phenotype. To see if some mutants cause subtle fitness reductions, we compared growth over several generations, measuring relative mutant fitness under steady-state growth conditions. We cultured the pool of viable histone mutants in glucose-limiting medium in parallel chemostats, maintaining the population in steady state exponential growth (Novick and Szilard, 1950
). It has previously been shown that glucose-limited chemostat experiments extending >20 generations select for fitter genetic variants, providing a convenient model for adaptive evolution by natural selection (Horiuchi et al, 1962
; Paquin and Adams, 1983
). After 10 days' growth, (~30 generations), populations were sampled, and amplified TAGs were analyzed. depicts the TAG array intensity ratios on the chemostat samples from days one and ten relative to the original inoculum; red spots indicate reduced hybridization on day ten. Data indicate that ~40% of the viable histone mutants were reduced or eliminated in the pool (log2
ratio of signal intensity of day 10 versus inoculum <−1.5). Surprisingly, a subset of 27 histone mutants show a higher intensity after growth (log2
ratio >+1.5) suggesting they are collectively fitter and maintain a selective advantage under glucose limitation. illustrates high concordance between two independently grown chemostats at day 10. We grew the chemostat 10 more days and analyzed this population. show allele distributions in chemostats on days 10 and 20, respectively. Mutants were grouped and colored based on their day 10abundance. shows that most mutants are reduced in the population after 20 days; most of those that thrive after 20 days were already abundant at day 10 (Table S3
); thus these strains exhibit a fitness advantage over the other mutants. Indeed 8 out of the 27 day 10 “winners” still dominate the population at day 20.
Fig. 5 Chemostat growth profiles reveal subset of mutants that outgrow wild-type. Cells were cultured in a glucose-limited chemostat at 30°C and sampled as indicated. A. Microarray ratio images. Red features represent mutants depleted after chemostat. (more ...)
To determine whether the “winner” histone mutations accounted for their success in the chemostat we cultured the un-evolved day 10 “winning” histone mutants in the presence of wild-type (WT) for 30 generations. Each histone mutant was represented at the same level as WT in the initial inoculum. indicates that the winner histone mutants had outcompeted WT by day 10, equating to ~6% faster growth under these conditions.
Analysis of double mutants
SLAM (Synthetic Lethality Analyzed by Microarray) is a high-throughput technology for identifying genetic interactions. SLAM was used to probe synthetic gene interactions between the histone mutant library and two genes encoding key regulators of chromatin biology, UBP8
. These regulators control the levels of H2B K123 ubiquitylation (K123Ub) and H3 K36 methylation, respectively. We hypothesized that the ability of a cell to respond to perturbed modification, through the deletion of these loci, may depend on processes involving other key histone residues. To probe this, the histone mutant library was transformed with specific kanMX
targeting constructs, and histone tags were amplified and analyzed. The intensities of each histone mutant TAG on the array were compared with those amplified from a control histone library with a deletion at a “neutral” locus, HO
. plots log ratios of the TAGs in ubp8
relative to ho
, for viable mutants. Two synthetic fitness interactions with ubp8
Δ were validated, H3 K56Q and H4 K91A (). These double mutant combinations resulted in a synthetic fitness (SF) interaction; the double mutant grew but exceedingly poorly. Direct testing revealed three additional interactions with the H3 K56R, H3 K56A and H4 K91R alleles (not shown). It is striking to note that both H3 K56 and H4 K91 are potentially acetylated residues situated within the nucleosome globular core (Masumoto et al, 2005
; Xu et al, 2005
; Ye et al, 2005
), suggesting a functional interaction between these histone marks and Ubp8p. Since deubiquitylation of Histone H2B K123 by Ubp8p is a key regulatory step in the switch from transcription initiation to elongation (Wyce et al, 2007
, Henry et al, 2003
), K56 and/or K91 modification status may affect the transcription cycle.
Fig. 6 Assaying complex phenotypes using TAG arrays. A. Double mutant analysis of histone library with ubp8Δ. Scatter plot depicting log2 ratio of abundance of each mutant in a ubp8Δ strain. Gray vertical lines (alleles 235−288 and 461−488) (more ...)
We also identified three histone alleles, H3 T107A, H3 K9A, and H3 Del [9−20], that reproducibly gave rise to synthetic fitness defects in a set2Δ background (). H3 T107 is buried within the core of the nucleosome close to the dyad axis and has no known biological role to date. Both the H3 K9A and H3 Del [9−20] alleles eliminate H3 K9 acetylation, a mark required for transcriptional activation. Eliminating both these marks is detrimental and may suggest a role for H3 K9 acetylation in normal chromatin function in the absence of Set2p methylation.
H3 tail and NHEJ
The DNA double-strand break (DSB) is a toxic DNA lesion resulting from environmental stress, chemicals insult or stalled replication forks. To probe histone requirements for DSB repair, we applied our histone mutant pool to study the nonhomologous end joining (NHEJ) pathway using a transformation-based plasmid repair assay (Ooi et al, 2001
). A number of mutants showed apparent NHEJ defects, including several N-terminal tail mutants of histone H3 ( and Table S4
). Six mutants significantly defective in NHEJ were individually confirmed; NHEJ efficiency in these was ~1/3 that of WT, and about 4-fold higher than a lig4
control strain (). One striking finding is that most of the H3 tail deletions appear defective in NHEJ repair. In contrast, only a small number of tail deletions on histone H4 have this effect, suggesting a specific role for the H3 tail in NHEJ. Other mutations affecting NHEJ include H3 K56, an important site of acetylation. Although K56R only modestly affects NHEJ (Masumoto et al, 2005
), a recent report demonstrated that loss of Rtt109p, the K56 acetyltransferase, impairs NHEJ (Jessulat et al, 2008
), consistent with our results. Other histone mutants significantly affecting NHEJ are listed in Table S4
. It has been reported that lysine 16 on histone H4 is deacetylated in a SIN3
-dependent manner in the vicinity of DNA DSBs. Deletion of SIN3
confers an NHEJ defect, presumably due to the inability to remove H4 K16 acetylation (Jazayeri et al, 2004
). Consistent with this, K16Q, which mimics acetylation (but not K16R), is also defective in NHEJ.