1

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

2

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.

3

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

4

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

5

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

6

(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

7

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.

8

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