supplemental methods - genes &...
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SUPPLEMENTAL METHODS
Recombinant proteins
Recombinant protamines A and B were purified using IMPACT kit (NEB) and concentrated
by cation exchange chromatography (Source 15S). Recombinant protamine chaperones were
purified by Ni-NTA chromatography as described (Fyodorov and Kadonaga 2003). To remove
contaminating nuclease activities, the proteins were additionally purified by size exclusion
(Superose 6) and anion exchange (Sourse 15Q) chromatography. Concentrations were
determined by SDS-PAGE and Coomassie staining along with BSA standards. Details of
cloning, expression conditions and purification protocols are available upon request.
Reconstitution and analyses of model sperm chromatin (MSC) substrate
To prepare MSC by salt dialysis, 100 µg each of the protamines A and B were mixed with
500 µg supercoiled plasmid pGIE-0 (~3.2 kbp) in HEG buffer (25 mM HEPES, pH 7.6, 0.1 mM
EDTA, pH 8.0, 10% glycerol, 1 mM DTT) containing 2 M KCl. The mixture (~0.8 ml) was
dialyzed at 4°C overnight against 4 L HEG buffer containing 200 mM KCl. Substrates that
contained mixtures of tagged and untagged protamines (A-V5 + B-V5, A-V5 + B and A + B-V5)
were prepared. The optimal molecular ratio of protamines to DNA was determined in a series of
empirical titration experiments. When concentration of protamines is increased ~23% (to ~52 bp
DNA per protamine polypeptide), the resulting MSC substrate is no longer functional in
chromatin assembly assays with the S-190 extract (as in Fig. 1C). The empirically optimized
MSC substrate encompasses approximately 1.6 bp DNA for each positively charged amino acid
(Arg, Lys or His) in protamines.
In vitro ChIP was performed as follows. 0.2 pmol/400 ng plasmid DNA, equivalent amount
of oligonucleosomes reconstituted by salt dialysis (Emelyanov et al. 2010) or MSC substrate (A-
V5 + B-V5), was incubated at 27°C for 20 min with 1.5 pmol/50 ng GAL4-VP16 (a gift of Jim
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Kadonaga, UCSD) and cross-linked by addition of formaldehyde to 1%. The template was
diluted 25-fold in 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl and
immunoprecipitated with 1 µg antibody (mouse monoclonal anti-V5, Sigma, or rabbit polyclonal
anti-GAL4, Abcam). Normal mouse or rabbit sera was used for background correction. After
cross-link reversal, relative occupancy was determined by real-time PCR (ViiA 7).
Enzymatic reactions were performed in a 20-µl volume at 37°C for 10 min with 0.1
pmol/200 ng DNA (or equivalend amount of MSC) and: 1 unit Hae III (NEB) in 20 mM Tris-
acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT; 0.002 units
MNase (Sigma) in 10 mM HEPES, pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 2 mM CaCl2, 0.5 mM
EGTA, 1 mM DTT, 10% glycerol; 0.2 units DNAse I (NEB) in 10 mM Tris-HCl, pH 7.6, 2.5
mM MgCl2, 0.5 mM CaCl2, 1 mM DTT; or 0.1 pmol/6 ng (1 pmol/60 ng in MSC-containing
reactions) topoisomerase I fragment ND423 (Fyodorov and Kadonaga 2003) in 50 mM Tris-HCl,
pH 7.5, 0.2 mM EDTA, 10 mM MgCl2, 1 mM DTT, 50 µg/ml BSA. DNA was de-proteinated
with proteinase K, ethanol precipitated, resolved on TBE-agarose gel and stained with ethidium.
ATP-dependent nucleosome assembly
Nucleosome arrays were assembled using purified recombinant ATP-dependent system in
70-µl reactions that contained 2.3 nM DNA (pGIE-0 plasmid, ~3.2 kDa), 80 nM each core
histones, 650 nM NAP-1 and 10 nM CHD1, 5 nM ACF or 25 nM ISWI (Fyodorov and
Kadonaga 2003; Lusser et al. 2005). In some reactions, DNA was substituted with equivalent
amount of MSC, and NAP-1 was substituted or supplemented with equimolar amounts of
TAP/p32, NLP or Nph. Alternatively, oligonucleosomes were assembled with 56 µl S-190
extract in similar conditions (Fyodorov and Levenstein 2002) without supplementation with
exogenous core histones. Chromatin was analyzed by partial micrococcal nuclease (MNase)
digestion with two enzyme dilutions.
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Protamine eviction by S-190 extract
MSC substrate (equivalent to 20 µg plasmid DNA) was mixed with 4 ml S-190 extract with
or without ATP and incubated for 2 hours at 27°C, The reactions were loaded on 5-30% sucrose
gradients in 25 mM HEPES, pH 7.6, 200 mM KCl , 0.1 mM EDTA, pH 8.0, 1 mM DTT and
centrifuged in Beckman SW-41 rotor for 18 hours at 41,000 rpm. The gradients were cut into 12
fractions and analyzed by western for presence of V5-tagged protamines. The following
antibodies were used: mouse monoclonal anti-V5 (Sigma, 1:5,000) and secondary HRP-
conjugated donkey anti-mouse (Jackson ImmunoResearch, 1:5,000).
Protamine eviction by recombinant protamine chaperones
Protamine eviction from MSC was assayed in vitro in 50-µl reactions that contained 0.47
pmol substrate (1 µg DNA, 11.5 pmol protamines A and B each) in buffer R (10 mM HEPES,
pH 7.6, 10 mM KCl, 0.5 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, 10 mM β-glycerophosphate,
10% glycerol) with 200 mM KCl and 0.1 mg/ml BSA with or without 3 mM ATP. Standard
reactions (1x) contained 115 pmol each NAP-1, NLP, Nph and TAP/p32 (5.0, 3.3, 2.0 and 2.1
µg, respectively). After incubation for 1 hr at 27°C, reactions were fractionated on sucrose
gradients (see above) or gravity-flow size exclusion columns (Sephacryl S-500, 2 ml bed
volume) equilibrated to Buffer R + 200 mM KCl. Certain reactions contained different
combinations and/or concentrations (from 0.2x to 4x) of protamine chaperones and were allowed
to proceed from 15 min to 4 hr, as indicated in Figure Legends. Protamines were detected by
anti-V5 western.
Purification of putative protamine chaperones
S-190-mediated protamine eviction reactions were fractionated on sucrose gradient, and
fractions 3-5 that contained V5-immunoreactive material were pooled and immunoprecipitated
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with 50 µl anti-V5-agarose (Sigma). Immunoprecipitated material was eluted in 100 µl by (Fig.
2B) low pH (glycine, pH 2.0) or (Fig. 2C) 10 mg/ml V5 peptide (Sigma) in HEG (Fig. 2C) and
analyzed by SDS-PAGE and silver or Coomassie staining. Major bands were excised from
Coomassie-stained gels, and protein identities were determined by mass-spectrometry. Control
IP reactions contained S-190 extract (not subjected to sucrose gradient fractionation) and no
MSC (Fig. 2B) or fractions 3-5 from sucrose gradient of S-190 extract and no MSC (Fig. 2C).
Subcellular localization analyses
5 g of 0–12 hr embryos were collected, dechorionized, resuspended and homogenized in
TES buffer (10 mM Tris-HCl, pH 7.4, 5 mM CaCl2, 1 mM EDTA, pH 8.0, 0.25 M sucrose, 5
mg/ml APMSF, 5 mg/ml pepstatin, 5 mg/ml leupeptin and 2 mg/ml aprotinin) with 0.25 M
sucrose. The homogenate was centrifuged at 600x g for 10 min, and pellet (crude nuclear
fraction) was resuspended in TES and centrifuged again at 600x g to minimize contamination
with mitochondria. The post-nuclear supernatant was centrifuged at 7,000x g for 10 min. The
supernatant (cytosolic and microsomal fraction) was boiled in SDS-PAGE loading buffer. The
pellet (crude mitochondrial fraction) was resuspended in TES and layered on a discontinuous
gradient of 1.5 M and 1.0 M sucrose in TES. The crude nuclear fraction was homogenized in
TES buffer and layered on discontinuous gradient made by successive layering of 2.8 M, 2.15 M
and 1.85 M sucrose in TES plus 1 mM MgCl2. The gradients were centrifuged in SW-41 rotor at
80,000x g for 1 h. After centrifugation, the phases between the layers of 1.5 and 1.0 M sucrose
and between 2.8 and 2.15 M sucrose were collected as the mitochondrial and nuclear fraction,
respectively. All phases were washed three times with TES and boiled in SDS-PAGE loading
buffer.
Samples were loaded on SDS-PAGE and stained with Coomassie to estimate protein content.
They were further analyzed by western blot, while loading approximately equal total protein
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amount for each sample. The following antibody dilutions were used: rabbit polyclonal anti-
NAP-1 and anti-NLP (a gift of Jim Kadonaga, UC San Diego), 1:10,000; Guinea pig polyclonal
anti-Nph and anti-TAP/p32 (see Materials and Methods), 1:10,000. Rabbit polyclonal anti-
Hsp60 (Cell Signaling Technology) and mouse monoclonal anti-HP1 (Developmental Studies
Hybridoma Bank) were used at 1:1,000; HRP-conjugated secondary donkey anti-Guinea pig,
goat anti-rabbit and goat anti-mouse (all Jackson ImmunoResearch) were used at 1:5,000.
Yeast genetics and Synthetic Genetic Array (SGA) screen
Constructs for genomic deletion of MAM33 and SHE9 were assembled by PCR
megapriming, transformed to a strain (KFY1309) compatible with Synthetic Genetic Array -
Technology (SGA-T), and homologous recombination confirmed by PCR (Keogh et al. 2002;
Janke et al. 2004; Silva et al. 2012).
Growth curves were monitored with a Bioscreen C (Oy Growth Curves). Seed cultures were
grown to mid-log in non-selective media and diluted to OD600 ≤ 0.1 in the appropriate medium
additionally containing 0.2% NP-40: YPD(extrose, 2%, +/- 0.05% MMS), YPE(thanol, 2%) or
YPG(lycerol, 2%). All analyses were performed in triplicate and OD600 curves (30°C, constant
agitation, 15-min time points) monitored for 96 hrs.
Genetic interactions were determined by partially automated Synthetic Genetic Array (SGA)
technology (Tong and Boone 2006). For this study mam33Δ::NatMX was mated in quadruplicate
to a library of ~ 4800 non-essential genes individually deleted with KanMX (Winzeler et al.
1999). Strains were arrayed at 1536-density per 12.5 x 8.5cm plate and replica-plated with a
Singer RoToR. The growth of all double-mutant haploid daughters was compared to the
respective single-mutant parents to identify and quantify positive (i.e. epistasis, suppression) or
negative (SS/SL) genetic interactions. Negative interaction scores (Suppl. Table S2) are a
product of the average growth of each replicate of each double mutant on a five point scale: -4
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(dead) to 0 (no change), measured by two independent observers. “Interactions” with deletions of
factors required for mating, metabolic pathways used in the screening protocol, or within the
MAM33 linkage group (+/- 50 kB) were excluded from consideration. Any enrichment of
specific Gene Ontology terms in the list of 83 negative genetic interactors (<–1.0) was
determined with GOrilla (http://cbl-gorilla.cs.technion.ac.il) (Eden et al. 2009).
Fly genetics
Null P32 alleles were prepared by a modified protocol for ends-out homologous
recombination (Huang et al. 2008). The targeting event was designed to eliminate 899 bp of P32
transcription unit, including the coding sequence from Arg5 to Lys263 at the C-terminus. 5’ and
3’ flanking homology arms spanning 5,080 and 3,937 bp, respectively, were cloned in pRK1 (a
gift of Yang Hong, University of Pittsburg). After establishing 3 independent P insertion alleles,
homologous recombination was induced by heat-shock to excise linear donor DNA fragments
with FLPase and I-SceI enzymes. One white+ targeted progenitor was selected from crosses with
each original insertion and resultant alleles were designated P32[1], P32[2] and P32[3].
hsp70::white+ marker was excised from P32[1] and P32[2] by crossing to y, w, P{Crey}1b;
Sco/CyO allele and heat-shock induction of Cre recombinase to generate white– knock-out alleles
P32[4] and P32[5], respectively (Suppl. Fig. S4A). All targeting/excision events were
confirmed by PCR with primers outside of the homology regions that were used in the knockout
construct (Suppl. Fig. S4B). Construct and primer sequences are available upon request.
Df(3R)Nph[Nlp] was generated by imprecise excision of P{EPgy2}EY21985 P-element as
described (Fyodorov et al. 2004) and balanced by TM6B, Tb. Breakpoints of the deficiency were
determined by PCR and sequencing (all sequences are available upon request). A total of 110
individual chromosomes were analyzed by PCR. The excision event resulted in deletion of 924
bp (coordinates 3R:25830437-25831360 in D. melanogaster Gene Models Database, version
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R5.53) in the regulatory regions of CG7911/Nph and Nlp that extended into the coding sequence
of Nlp. To examine expression of NLP and Nph in vivo, whole wild-type or homozygous mutant
L3 larvae were grinded and boiled in SDS-PAGE loading buffer. The Nph[Nlp] allele was
confirmed to contain null mutations of both Nph and Nlp by western analyses of the
homogenates (Suppl. Fig. S4D). Equal protein loading was confirmed by SDS-PAGE and
Coomassie staining of equivalent amounts of larval lysates.
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SUPPLEMENTAL REFERENCES
Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z. 2009. GOrilla: A Tool For Discovery And
Visualization of Enriched GO Terms in Ranked Gene Lists. BMC Bioinformatics 10: 48.
Emelyanov AV, Konev AY, Vershilova E, Fyodorov DV. 2010. Protein complex of Drosophila
ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin
in vivo. J Biol Chem 285: 15027-15037.
Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A,
Doenges G, Schwob E, Schiebel E et al. 2004. A versatile toolbox for PCR-based tagging
of yeast genes: new fluorescent proteins, more markers and promoter substitution
cassettes. Yeast 21: 947-962.
Keogh MC, Cho EJ, Podolny V, Buratowski S. 2002. Kin28 is found within TFIIH and a Kin28-
Ccl1-Tfb3 trimer complex with differential sensitivities to T-loop phosphorylation. Mol
Cell Biol 22: 1288-1297.
Silva AC, Xu X, Kim HS, Fillingham J, Kislinger T, Mennella TA, Keogh MC. 2012. The
replication-independent histone H3-H4 chaperones HIR, ASF1, and RTT106 co-operate
to maintain promoter fidelity. J Biol Chem 287: 1709-1718.
Tong AH, Boone C. 2006. Synthetic genetic array analysis in Saccharomyces cerevisiae.
Methods Mol Biol 313: 171-192.
Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R,
Benito R, Boeke JD, Bussey H et al. 1999. Functional characterization of the S.
cerevisiae genome by gene deletion and parallel analysis. Science 285: 901-906.
SUPPLEMENTAL TABLES
Supplemental Table S1. Mass-spec data of protamine-chaperone complexes.
Protein bands of interest were manually excised from a Coomassie-stained SDS-PAGE gel (Fig.
2B) and submitted for MALDI-TOF MS sequencing of tryptic digestion peptides to the
Proteomics Resource Center at Rockefeller University. For protein identification, the measured
tryptic peptide masses were batch processed and searched against a non-redundant NCBI
database using the software MASCOT 2.1.
Accession number
Protein name
Expected MW, Da
Peptide matches
Specific hits
Sequence coverage
MASCOT score
p-value
gi|28574150 CG31731 176,571 137 6 <1% 39 ~0.1
gi|17137142 NAP-1 42,637 40 37 46% 1,251 <10-8 gi|20130085 CG6459 28,845 13 7 22% 200 <10-5 gi|24651247 CG7911 17,238 10 7 28% 88 <10-3 gi|17738283 NLP 16,865 7 6 31% 128 <10-4
Supplemental Table S2. Negative genetic interactors of Sc mam33.
Summary of negative genetic interactions between mam33Δ allele and a collection of deletion alleles for ~4,800 non-essential S. cerevisiae genes.
Interactions were scored as described in Materials and Methods. Statistically significant interactions from –1 to –4 (on a 5-point scale) are shown
and arranged in blocks of genes conforming to specific GO terms.
GENE ORF PLATE ROW COLUMN NOTE SCORE ANNOTATION GROUP
ARP8 YOR141C 6 15 6 -3.4 Nuclear actin-related protein involved in chromatin remodeling; component of chromatin-remodeling enzyme complexes; has mRNA binding activity CHROMATIN
IOC4 YMR044W 5 3 4 -2.5Member of a complex (Isw1b) with Isw1p and Ioc2p; interacts directly with H3K36me3 nucleosomes through its PWWP domain to recruit the Isw1b complex to open reading frames in a Set2p-dependent manner; Isw1b exhibits nucleosome-stimulated ATPase activity and acts within coding regions to coordinate transcription elongation with termination and processing
CHROMATIN
ARP6 YLR085C 5 29 3 -1.8 SWR-C: Actin-related protein that binds nucleosomes; a component of the SWR1 complex, which exchanges histone variant H2AZ (Htz1p) for chromatin-bound histone H2A CHROMATIN
CDC73 YLR418C 5 5 36 -1.8 PAF complex subunit CHROMATIN
VID21 YDR359C 2 15 37 -1.6 Component of the NuA4 histone acetyltransferase complex; acts as a platform for assembly of NuA4 subunits into the native complex CHROMATIN
RSC2 YLR357W 5 11 9 -1.3 Component of the RSC chromatin remodeling complex CHROMATIN
SNF5 YBR289W 1 21 20 -1.1 Subunit of the SWI/SNF chromatin remodeling complex; involved in transcriptional regulation CHROMATIN
YDR532C 2 29 40 -1.0 Subunit of a kinetochore-microtubule binding complex; complex bridges centromeric heterochromatin and kinetochore MAPs and motors; required for sister chromatid bi-orientation and kinetochore binding of SAC components; CHROMATIN
MPH1 YIR002C 4 29 3 -3.8 DNA REPAIR: 3'-5' DNA helicase involved in error-free bypass of DNA lesions DNA DAMAGE
DDI1 YER143W 2 3 46 -3.0 DNA damage-inducible v-SNARE binding protein; role in suppression of protein secretion; may play a role in S-phase checkpoint control; has ubiquitin-associated (UBA), ubiquitin-like (UBL), and retroviral-like proteinase (RVP) domains DNA DAMAGE
MMS22 YLR320W 5 3 33 -1.8Subunit of E3 ubiquitin ligase complex involved in replication repair; stabilizes protein components of the replication fork, such as the fork-pausing complex and leading strand polymerase, preventing fork collapse and promoting efficient recovery during replication stress; required for accurate meiotic chromosome segregation
DNA DAMAGE
RAD52 YML032C 5 25 12 -1.3 DNA repair. Stimulates strand exchange; stimulates strand exchange by facilitating Rad51p binding to single-stranded DNA; anneals complementary single-stranded DNA; involved in the repair of double-strand breaks in DNA during vegetative growth and meiosis DNA DAMAGE
BRE1 YDL074C 1 27 12 -1.1 E3 ubiquitin ligase; forms heterodimer with Rad6p to monoubiquinate histone H2B-K123, which is required for the subsequent methylation of histone H3-K4 and H3-K79; required for DSBR, transcription, silencing, and checkpoint control DNA DAMAGE
SRB2 YHR041C 3 29 14 -2.2 RNApII mediator complex MEDIATOR
SIN4 YNL236W 6 27 35 -1.2 Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme MEDIATOR
SRB8 YCR081W 1 7 36 -1.2 Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme MEDIATOR
KGD1 YIL125W 7 7 24 -4.0 Subunit of the mitochondrial alpha-ketoglutarate dehydrogenase complex; catalyzes a key step in the tricarboxylic acid (TCA) cycle, the oxidative decarboxylation of alpha-ketoglutarate to form succinyl-CoA MITO
MDM31 YHR194W 3 7 22 petite -2.0 Mitochondrial protein that may have a role in phospholipid metabolism; inner membrane protein with similarity to Mdm32p; required for normal mitochondrial morphology and inheritance; interacts genetically with MMM1, MMM2, MDM10, MDM12, and MDM34 MITO
SHE9 YDR393W 2 23 29 -1.8 Protein required for normal mitochondrial morphology; mitochondrial inner membrane protein; may be involved in fission of the inner membrane; forms a homo-oligomeric complex MITO
MMR1 YLR190W 5 29 25 -1.7 Phosphorylated protein of the mitochondrial outer membrane; localizes only to mitochondria of the bud; interacts with Myo2p to mediate mitochondrial distribution to buds; mRNA is targeted to the bud via the transport system involving She2p MITO
GON5 YPL183W-A 7 11 45 petite -1.6 Protein involved in translation; mutants have defects in biogenesis of nuclear ribosomes; sequence similar to prokaryotic ribosomal protein L36, may be a mitochondrial ribosomal protein MITO
YME1 YPR024W 7 13 16 petite -1.3 Catalytic subunit of the i-AAA protease complex; complex is located in the mitochondrial inner membrane MITO
YLR193C 5 29 13 -1.0Phosphatidic acid transfer protein; plays a role in phospholipid metabolism by transporting phosphatidic acid from the outer to the inner mitochondrial membrane; localizes to the mitochondrial intermembrane space; null mutant has altered cardiolipin and phosphatidic acid levels; ortholog of human PRELI
MITO
YJL046W 4 21 13 petite -1.0 Putative lipoate-protein ligase; required along with Lip2 and Lip5 for lipoylation of Lat1p and Kgd2p; similar to E. coli LplA; null mutant displays reduced frequency of mitochondrial genome loss MITO
RIM13 YMR154C 5 3 22 -1.5 RIM101: Calpain-like cysteine protease; involved in proteolytic activation of Rim101p in response to alkaline pH RIM101
RIM8 YGL045W 3 29 39 -1.3 RIM101: involved in proteolytic activation of Rim101p; part of response to alkaline pH RIM101
RIM21 YNL294C 6 11 33 -1.3 RIM101: pH sensor molecule, component of the RIM101 pathway RIM101
RIM101 YHL027W 3 13 22 -1.2 RIM101: Cys2His2 zinc-finger transcriptional repressor; involved in alkaline responsive gene repression as part of adaptation to a alkaline conditions RIM101
RIM9 YMR063W 5 7 4 -1.0 Plasma membrane protein of unknown function; involved in the proteolytic activation of Rim101p in response to alkaline pH; interacts with Rim21p and Dfg16p to form a pH-sensing complex in the Rim101 pathway and is required to maintain Rim21p levels RIM101
RIM20 YOR275C 7 21 35 -1.0 Protein involved in proteolytic activation of Rim101p; part of response to alkaline pH; PalA/AIP1/Alix family member; interaction with the ESCRT-III subunit Snf7p suggests a relationship between pH response and multivesicular body formation RIM101
SPE2 YOL052C 6 5 22 -2.3 S-adenosylmethionine decarboxylase; required for the biosynthesis of spermidine and spermine; cells lacking Spe2p require spermine or spermidine for growth in the presence of oxygen but not when grown anaerobically SPE
SPE1 YKL184W 4 3 12 -2.3 Required for the biosynthesis of spermidine and spermine SPE
SPE3 YPR069C 7 25 20 -1.7 Required for the biosynthesis of spermidine and spermine SPE
YJL028W 4 17 5 -3.7 Unknown function; may interact with ribosomes, based on co-purification experiments RIBO
RIC1 YLR039C 5 17 3 -1.7 involved in retrograde transport to the cis-Golgi network; forms heterodimer with Rgp1p that acts as a GTP exchange factor for Ypt6p; involved in transcription of rRNA and ribosomal protein genes RIBO
RPS0B YLR048W 5 21 15 -1.4 component of the small (40S) ribosomal subunit RIBO
RPS1B YML063W 5 9 46 -1.3 Ribosomal protein 10 (rp10) of the small (40S) subunit; homologous to mammalian ribosomal protein S3A, no bacterial homolog; RPS1B has a paralog, RPS1A, that arose from the whole genome duplication RIBO
BUD21 YOR078W 6 27 28 petite -1.3 Component of small ribosomal subunit (SSU) processosome RIBO
RPP1A YDL081C 1 3 34 -1.3 Ribosomal stalk protein P1 alpha; involved in the interaction between translational elongation factors and the ribosome; RIBO
ASC1 YMR116C 5 19 8 -1.3 G-protein beta subunit and guanine dissociation inhibitor for Gpa2p; ortholog of RACK1 that inhibits translation; core component of the small (40S) ribosomal subunit RIBO
RPL22A YLR061W 5 25 19 petite -1.2 Ribosomal 60S subunit protein L22A RIBO
GZF3 YJL110C 4 3 3 -3.5GATA zinc finger protein; negatively regulates nitrogen catabolic gene expression by competing with Gat1p for GATA site binding; function requires a repressive carbon source; dimerizes with Dal80p and binds to Tor1p; GZF3 has a paralog, DAL80, that arose from the whole genome duplication
TRANSCRIPTION
STB5 YHR178W 3 3 18 -2.7 Transcription factor; involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitro TRANSCRIPTION
INO4 YOL108C 6 21 26 -1.6 Transcription factor involved in phospholipid synthesis TRANSCRIPTION
RRN10 YBL025W 1 5 25 -1.0 Protein involved in promoting high level transcription of rDNA; subunit of UAF (upstream activation factor) for RNA polymerase I TRANSCRIPTION
BGL2 YGR282C 3 29 4 -3.8 Endo-beta-1,3-glucanase; major protein of the cell wall, involved in cell wall maintenance
TRF4 YOL115W 6 25 42 -3.8 Non-canonical poly(A) polymerase; involved in nuclear RNA degradation as a component of TRAMP; catalyzes polyadenylation of hypomodified tRNAs, and snoRNA and rRNA precursors
TIR3 YIL011W 3 15 30 -3.7 Cell wall mannoprotein; member of Srp1p/Tip1p family of serine-alanine-rich proteins; expressed under anaerobic conditions and required for anaerobic growth; TIR3 has a paralog, TIR2, that arose from the whole genome duplication
FYV4 YHR059W 3 3 12 -3.3 Unknown function; required for survival upon exposure to K1 killer toxin
RCY1 YJL204C 4 3 45 -3.2 F-box protein involved in recycling endocytosed proteins; involved in recycling plasma membrane proteins internalized by endocytosis; localized to sites of polarized growth
YGL024W 3 25 43 -2.8 Dubious open reading frame; unlikely to encode a functional protein, based on available experimental and comparative sequence data; partially/completely overlaps the verified ORF PGD1/YGL025C
SUR2 YDR297W 2 27 3 -2.7 Sphinganine C4-hydroxylase; catalyses the conversion of sphinganine to phytosphingosine in sphingolipid biosyntheis
YER119C-A 2 23 20 -2.7Dubious open reading frame; unlikely to encode a functional protein, based on available experimental and comparative sequence data; not conserved in closely related Saccharomyces species; deletion mutation blocks replication of Brome mosaic virus in S. cerevisiae, but this is likely due to effects on the overlapping gene SCS2
TCO89 YPL180W 7 7 17 -2.7 Subunit of TORC1 (Tor1p or Tor2p-Kog1p-Lst8p-Tco89p); TORC1 complex regulates growth in response to nutrient availability; cooperates with Ssd1p in the maintenance of cellular integrity; deletion strains are hypersensitive to rapamycin
YLR261C 5 15 19 -2.5Dubious open reading frame; unlikely to encode a functional protein, based on available experimental and comparative sequence data; not conserved in closely related Saccharomyces species; 98% of ORF overlaps the verified gene YPT6; deletion causes a vacuolar protein sorting defect
YLL044W 4 27 10 -2.3 Dubious open reading frame; unlikely to encode a functional protein, based on available experimental and comparative sequence data; transcription of both YLL044W and the overlapping gene RPL8B is reduced in the gcr1 null mutant
YPT6 YLR262C 5 15 15 -2.3 Rab family GTPase; Ras-like GTP binding protein involved in the secretory pathway, required for fusion of endosome-derived vesicles with the late Golgi, maturation of the vacuolar carboxypeptidase Y; has similarity to the human GTPase, Rab6
ARC1 YGL105W 3 9 13 -2.3 binds tRNA and methionyl- and glutamyl-tRNA synthetases; involved in tRNA delivery, stimulating catalysis, and ensuring localization
GSH1 YJL101C 4 3 15 -2.3 Gamma glutamylcysteine synthetase; catalyzes the first step in glutathione (GSH) biosynthesis; expression induced by oxidants, cadmium, and mercury; protein abundance increases in response to DNA replication stress
BTS1 YPL069C 7 7 19 -2.1 Geranylgeranyl diphosphate synthase; increases the intracellular pool of geranylgeranyl diphosphate, suppressor of bet2 mutation that causes defective geranylgeranylation of small GTP-binding proteins that mediate vesicular traffic
VRP1 YLR337C 5 7 29 petite -1.7 Proline-rich actin-associated protein; involved in cytoskeletal organization and cytokinesis; related to mammalian Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP)
YML013C-A 5 21 24 -1.7 Dubious open reading frame; unlikely to encode a functional protein, based on available experimental and comparative sequence data; partially overlaps the verified gene SEL1
SSE1 YPL106C 7 15 11 -1.7ATPase component of heat shock protein Hsp90 chaperone complex; plays a role in determining prion variants; binds unfolded proteins; member of the heat shock protein 70 (HSP70) family; localized to the cytoplasm; SSE1 has a paralog, SSE2, that arose from the whole genome duplication
BUD31 YCR063W 1 3 40 -1.6 Component of the SF3b subcomplex of the U2 snRNP; diploid mutants display a random budding pattern instead of the wild-type bipolar pattern; facilitates passage through G1/S Start, but is not required for G2/M transition or exit from mitosis
DEG1 YFL001W 7 3 34 -1.5 tRNA:pseudouridine synthase; introduces pseudouridines at position 38 or 39 in tRNA, important for maintenance of translation efficiency and normal cell growth, localizes to both the nucleus and cytoplasm
BEM4 YPL161C 7 3 37 -1.5 Involved in establishment of cell polarity and bud emergence; interacts with the Rho1p small GTP-binding protein and with the Rho-type GTPase Cdc42p; involved in maintenance of proper telomere length
LEA1 YPL213W 7 19 41 -1.5 Component of U2 snRNP complex; disruption causes reduced U2 snRNP levels; physically interacts with Msl1p; putative homolog of human U2A' snRNP protein
YLR402W 5 27 41 -1.4 Dubious open reading frame
YKE2 YLR200W 5 3 43 -1.3 Subunit of the heterohexameric Gim/prefoldin protein complex; involved in the folding of alpha-tubulin, beta-tubulin, and actin; prefoldin complex also localizes to chromatin of actively transcribed genes in the nucleus and facilitates transcriptional elongation
GRR1 YJR090C 4 27 41 very slow growing -1.3 F-box protein component of an SCF ubiquitin-ligase complex
YLR338W 5 7 25 petite -1.2 Dubious open reading frame; partially overlaps the verified ORF VRP1/YLR337C
YNL080C 6 17 9 -1.2 Involved in N-glycosylation; deletion mutation confers sensitivity to exidative stress and shows synthetic lethality with mutations in the spindle checkpoint genes BUB3 and MAD1
UBP3 YER151C 2 3 26 -1.2 Ubiquitin-specific protease involved in transport and osmotic response; interacts with Bre5p to co-regulate anterograde and retrograde transport between the ER and Golgi
PHO5 YBR093C 1 23 35 -1.1 Repressible acid phosphatase; 1 of 3 repressible acid phosphatases that also mediates extracellular nucleotide-derived phosphate hydrolysis
YNL198C 6 15 15 -1.1 Dubious open reading frame
CBC2 YPL178W 7 7 25 -1.1 Small subunit of the heterodimeric cap binding complex with Sto1p; interacts with Npl3p, possibly to package mRNA for export from the nucleus
PEX10 YDR265W 2 23 43 -1.0 Peroxisomal membrane E3 ubiquitin ligase; required for for Ubc4p-dependent Pex5p ubiquitination and peroxisomal matrix protein import
SLX8 YER116C 2 23 36 -1.0 Subunit of Slx5-Slx8 SUMO-targeted ubiquitin ligase (STUbL) complex; stimulated by prior attachment of SUMO to the substrate; contains a C-terminal RING domain; forms nuclear foci upon DNA replication stress
GCN1 YGL195W 3 29 21 -1.0 Positive regulator of the Gcn2p kinase activity; forms a complex with Gcn20p; proposed to stimulate Gcn2p activation by an uncharged tRNA
GTR2 YGR163W 3 5 44 -1.0 Putative GTP binding protein; negatively regulates Ran/Tc4 GTPase cycle; activates transcription
VPS51 YKR020W 4 19 16 -1.0 Component of the GARP (Golgi-associated retrograde protein) complex; this complex is required for the recycling of proteins from endosomes to the late Golgi
INP52 YNL106C 6 25 17 -1.0 Polyphosphatidylinositol phosphatase; dephosphorylates a number of phosphatidylinositol phosphates (PtdInsPs, PIPs) to PI; involved in endocytosis
Supplemental Table S3. Developmental progression in P32 mutant embryos.
Drosophila embryos from wild-type or P322/P324 mutant mothers were collected 0–2 or 0–4
hours after egg deposition (AED) and stained with propidium iodide (PI). The number of nuclei
and phenotypic appearance were used as measures of progression of development. The sample
size was 500 stained embryos for each genotype and collection time.
Development stage
Typical timing in wild type
% embryos wt, 0-2 h AED
% embryos P32, 0-2 h AED
% embryos wt, 0-4 h AED
% embryos P32, 0-4 h AED
degraded nuclei unknown 0.4% 1.6% 2.4% 2.0%
1-4 nuclei/ pronuclei 0-30 min 25.6% 35.6% 10.8% 33.4%
division cycles 3-13 0.5-2.5 hr 70.6% 43.6% 57.4% 31.4%
cellular blastoderm + >2.5 hr 3.4% 19.2% 29.4% 33.2%
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure S1. Drosophila protamines and model sperm chromatin (MSC)
substrate.
A. Alignment of amino acid sequences of D. melanogaster Mst35Ba (protamine A) and
Mst35Bb (protamine B). Sequences of predicted proteins encoded by Mst35Ba (146 residues)
and Mst35Bb (144 residues) are aligned. Vertical lines indicate identical amino acids.
B. Purified recombinant protamines A and B. Recombinant chimeric V5-tagged and untagged
proteins fused to intein-chitin binding domain were expressed in E. coli and purified by affinity
chromatography on chitin resin. After elution by intein self-cleavage, the proteins were
concentrated by Source 15S chromatography. Purified proteins were examined by SDS-PAGE
and Coomassie staining. Molecular masses (kDa) and positions of protein marker bands are
shown on the left.
C. In vitro ChIP analyses of MSC. Naked plasmid DNA (blue bars), reconstituted
oligonucleosomes (red bars) or MSC containing V5-tagged protamines A and B (green bars) was
incubated with purified recombinant GAL4-VP16, crosslinked and analyzed by in vitro qChIP
with V5 and GAL4 antibodies. The ordinate indicates the amounts of qChIP DNA samples
relative to input DNA. All experiments were performed in triplicate. Error bars, standard
deviation.
D. Nuclease sensistivity of MSC. Naked plasmid DNA or MSC, undigested (–) or treated with
indicated nucleases (+) was deproteinated, and resulting DNA fragments were resolved on an
ethidium-stained agarose gel. Marker, 123 bp DNA ladder.
E. Relaxation of DNA supercoiling in MSC substrate by topoisomerase I. Naked plasmid DNA
or MSC, undigested (–) or treated with Drosophila topoisomerase I (+/++) was deproteinated,
and resulting DNA fragments were resolved on an ethidium-stained agarose gel. Note that the
MSC-containing reaction (++) was performed with 10 times greater amount of the enzyme as
compared to the DNA-containing reaction (+). Marker, 123 bp DNA ladder.
Supplemental Figure S2. Analyses of Drosophila protamine chaperones.
A. Purified recombinant NAP-1, NLP, Nph and TAP/p32. Recombinant His-tagged proteins
were expressed in E. coli and purified by a succession of Ni-NTA, Superose 6 and Source 15Q
chromatography. Purified proteins were examined by SDS-PAGE and Coomassie staining.
Molecular masses (kDa) and positions of protein marker bands are shown on the left. Note that
recombinant NLP is resolved as oligomers (apparent molecular masses of 100-150 kDa) despite
having been boiled for over 20 min in a buffer containing 2% SDS and 2.5% β-mercaptoethanol.
B. Overlap in physical interaction networks of NLP, Nph and TAP/p32. Substantial overlap
among candidate protein interaction partners of NLP, Nph and TAP/p32 from DPiM, Drosophila
Protein interaction Map (Guruharsha et al. 2011), is presented in a Venn diagram. For
comparison, no significant overlap is observed between their interaction partners and those of
Hsp60 (mitochondrial protein).
C. Subcellular distribution of protamine chaperones. Drosophila embryos (0–12 hr AED) were
fractionated into nuclear, mitochondrial and cytosolic/microsomal fractions, and protein
subcellular distribution was examined by western blot. Antibodies to Hsp60 and heterochromatin
protein 1 (HP1) were used as mitochondrial and nuclear markers, respectively. The lanes were
loaded with approximately equivalent amounts of total protein based on SDS-PAGE of samples
and Coomassie staining of the gel.
D. MSC remodeling by combinations of protamine chaperones. MSC substrates were
reconstituted with both V5-tagged protamines, remodeled in reactions containing 2.3 µM
TAP/p32 supplemented with various combinations of other protamine chaperones (2.3 µM each)
and analyzed as in Fig. 3C.
Supplemental Figure S3. Intra- and extra-mitochondrial roles for budding yeast Mam33p.
A. Mam33p is required for growth on non-fermentable carbon sources. Growth of mam33Δ cells
in YPD(extrose, 2%), YPE(thanol, 2%) or YPG(lycerol, 2%) was analyzed in a Bioscreen C, and
estimated by culture density over time (red line; see Materials and Methods). Deletion of the
mitochondrial morphology regulator She9p (she9Δ) acts as a positive control (green line). WT,
wild-type (blue line).
B. Mam33 (but not She9) is required for resistance to the alkylating genotoxin MMS. Growth in
YPD +/- 0.05% MMS was analyzed and presented as in (A).
C. SGA technology was used to place mam33Δ in the context of ~ 4,800 non-essential gene
deletions, and genetic interactions revealed by comparing the growth rate of each double deletion
haploid daughter to that of their single deletion haploid parents. The identified 83 negative
genetic interactors (Suppl. Table S2) include a significant over-representation of genes that are
involved in recombinatorial DNA repair [RAD52, a recombinase central to DNA double-strand
break (DSB) repair; MMS22, a subunit of an E3 ubiquitin ligase involved in replication repair;
BRE1, an E3 ubiquitin ligase required for DSB repair; and MPH1, a 3’-5’ DNA helicase
involved in the error-free bypass of DNA lesion], encode subunits of SWI/SNF family chromatin
remodeling complexes [ARP8 (Ino80); IOC4 (Isw1b); ARP6 (SWR); RSC2 (RSC); SNF5 (SWI-
SNF)] or regulate biosynthesis of polyamines spermine and spermidine [SPE1; SPE2; and
SPE3].
.
Supplemental Figure S4. Mutant alleles of Drosophila Nlp, Nph and P32.
A. Targeting of P32 by ends-out homologous recombination. The targeting construct was
prepared in pRK backbone (Huang et al. 2008) with ~5 kbp and ~4 kbp homologous arms
encompassing adjacent genes, eIF3-S8, CG30108, CG30109 and Sema-1b. Positions of loxP
sites and hsp70-white+ marker are indicated as light ovals and a dark arrow, respectively. Closed
and open rectangles designate protein coding sequences and mRNA untranslated regions,
respectively. Thin lines, introns and intergenic regions; black arrows, transcription start sites; red
arrowheads, PCR primers used for allele genotyping (B); scale bar, 1,000 bp. In P32[1], P32[2]
and P32[3] alleles, a genomic interval of 899 bp that encompasses most of the coding sequence
of P32 is replaced with 2,189-bp hsp70-white+ transgene. In P32[4] and P32[5] alleles, the
white marker is further excised by Cre recombinase.
B. Sequence analyses of null mutant alleles of P32. Genomic DNA was prepared from
homozygous males of indicated genotypes and analyzed by PCR to the left (primers L+ to L–) or
to the right (primers R+ to R–) of the targeted locus.
C. Imprecise excision of P{EPgy2}EY21985. The P-element insertion is positioned in the
vicinity of overlapping regulatory regions of Nph/CG7911 and Nlp. Closed rectangles, protein
coding sequences; open rectangles, mRNA untranslated regions; thin lines, introns and intergenic
regions; black arrows, transcription start sites; scale bar, 1,000 bp. Italicized numbers next to the
ends of DNA diagrams represent genomic coordinates according to D. melanogaster Gene
Models Database, version R5.53. P{EPgy2}EY21985 was excised in a series of genetic crosses
as described in the Materials and Methods. In one of the excision alleles, Nph[Nlp], a deficiency
of 924 bp was introduced (examined by PCR and sequencing).
D. Df(3R)Nph[Nlp] is a null mutant allele of CG7911/Nph and Nlp. The expression of Nph and
NLP proteins was examined by western blot in whole L3 larvae (wild-type, homozygous original
EY21985 insertion or homozygous Nph[Nlp] excision alleles). Equal protein loading was
verified by Coomassie staining of equivalent SDS-PAGE gels and/or western of NAP-1 (not
shown). Positions of molecular mass markers (kDa) are shown on the left.
Mst35Ba 1 MSSNNVNECKSLWNGIISISAKDESPKGLTEMCNHPIRRAPQKCKPMKSCAKPRRKAACAKATRPKVKCAPRQ 73 |||||||||||||||||||||||||||||||||||| |||| ||||||||||||||||||||||||||||| |Mst35Bb 1 MSSNNVNECKSLWNGIISISAKDESPKGLTEMCNHPKRRAPPKCKPMKSCAKPRRKAACAKATRPKVKCAPSQ 73
Mst35Ba 74 KCSKQGPVTNNAYLNFVRSFRKKHCNLKPRELIAKAAKAWARLSENRKDRYRRMACKVTTSERHKRRRICQQY 146 |||||||||||||||||| |||||| ||| |||| |||||| | ||||||||||||||||||||||||||Mst35Bb 74 KCSKQGPVTNNAYLNFVRFFRKKHCDLKPQELIAEAAKAWAELPEHRKDRYRRMACKVTTSERHKRRRICK 144
0
20
40
60
80
100
120
DN
AN
ucl
MSC
anti-V5 anti-GAL4
DN
AN
ucl
MSC
A
B C
D
DNA MSC
Hae III– + – +
MNAse– + – +
DNAse I– + – +
DNA MSC DNA MSC
E
DNA MSC
Topo I– + – ++
Agarose GelEtBr-Stained
Mst
35Bb
Mst
35Bb
-V5
Mst
35Ba
-V5
Mst
35Ba
SDS-PAGE, Coomassie-Stained
15
20
25
In v
itro
ChI
P, R
elat
ive
Occ
upan
cy, %
Emelyanov_FigS1
Emelyanov_FigS2
A
CG79
11/N
phCG
6459
/TAP
/p32
NLP
NAP-
1
SDS-PAGE, Coomassie-Stained
37
2025
50
75100150250
Protein-protein interaction network
164
11035
19
1
1
5
810
16
Nph
NLP
TAP/p32
Hsp60
B
D
MSC + chaperones, 30 minGel filtration, V5 western
F B F B F B FB B F B F B F
NLP
Nph
NA
P-1
NLP
NA
P-1
NA
P-1
Nph
NLP
Nph
–
A-V5B-V5
TAP/p32C
NAP-1
NLP
Nph
TAP/p32
HP1
Hsp60
mito
chon
dria
cyto
sol
nucl
ei
whol
e
Western blot
Emelyanov_FigS3
EtOH
50 60 70 80 90 100
0.00
0.25
0.50
0.75
1.00
Time (hrs)
OD
600
WTmam33mdm33
YPD
0 5 10 15 20 25 30
0.00
0.25
0.50
0.75
1.00
Time (hrs)
OD60
0
WTmam33mdm33
WT mam33Δ she9Δ
0
0.5
1.0 0.75
0.25
0 5 10 15 20 25 30
OD
600
YPD(extrose)
YP Glycerol
50 60 70 80 90 100
0.00
0.25
0.50
0.75
1.00
Time (hrs)
OD
600
WTmam33mdm33
0
0.5
1.0 0.75
0.25
0
0.5
1.0 0.75
0.25
OD
600
OD
600
0 10 20 30 40 50
0 10 20 30 40 50 Time (hrs)
YPE(thanol)
YPG(lycerol)
A MMS (0.05%)
0 5 10 15 20 25 30
0.00
0.05
0.10
0.15
0.20
0.25
Time (hrs)
OD
600
WTmam33mdm33
B
0.05
0.15
0.25 0.2
0.1
0
0 5 10 15 20 25 Time (hrs)
30
MMS
WT mam33Δ she9Δ
OD
600
MAM33 C
GO: 0000725 Recombinatorial
repair
p = 0.0000158
RAD52, SNF5, MMS22, BRE1, RSC2, MPH1
GO: 0070603 SWI/SNF-type
complex
p = 0.000212
ARP6, ARP8, IOC4, SNF5,
RSC2
GO: 0006596 Polyamine
biosynthesis
p = 0.0000289
SPE1, SPE2, SPE3
Emelyanov_FigS4
3R:25829300
3R:25836200
P{EPgy2}EY21985
Df(3R)Nph[Nlp] (924 bp)
Nlp
Nph/CG7911 CG7912
1,000 bpC
899 bp
2,189 bp5,080 bp 3,937 bp
P32 Sema-1bCG30109CG30108eIF3-S8
1,000 bp
hsp70::w+loxP loxP
pRK1
P32[1], P32[2] and P32[3]: white+
P32[1] P32[4] and P32[2] P32[5]: white–
A
Nph[
Nlp]
EY21
985
wild
typeD
Western, anti-NLP
37
2025
50100
15
Western, anti-Nph
37
2025
50100
15
L+ R+ L– R– wild
type
P32[
4]
P32[
1]
PCR: L+ to L–
Agarose gel, EtBr-stained
wild
type
P32[
4]
P32[
1]
PCR: R+ to R–
B