Comprehensive Proteomic Analysis of PGC7-Interacting Proteins

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Comprehensive Proteomic Analysis of PGC7-Interacting Proteins Liu Hongliang, Zhang Lei, Wei Qing, Shi Zhaopeng, Shi Xiaoyan, Du Juan, Huang Chenyang, Zhang Yong, and Guo Zekun J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00883 • Publication Date (Web): 16 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Journal of Proteome Research

TITLE: Comprehensive Proteomic Analysis of PGC7-Interacting Proteins AUTHOR: Liu Hongliang†,‡, Zhang Lei†,‡, Wei Qing†,‡, Shi Zhaopeng†,‡, Shi Xiaoyan‡,§, Du Juan‡,∥, Huang Chenyang†,‡, Zhang Yong†,‡, Guo Zekun*†,‡ AUTHOR ADDRESS †

College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi

712100, China ‡

Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F

University, Yangling, Shaanxi 712100, China §

Medical Experiment Center of Shaanxi University of Chinese Medicine, Xianyang,

Shaanxi, 712000, China ∥

Medicine School of Yan‘an University, Yan’an, Shaanxi, 716000, China

ABSTRACT Primordial germ cell 7 (PGC7), a maternal factor essential for early development, plays a critical role in the regulation of DNA methylation, transcriptional repression, chromatin condensation, and cell division and the maintenance of cell pluripotentiality. Despite the fundamental roles of PGC7 in these cellular processes, only a few molecular and functional interactions of PGC7 have been reported. Here, a streptavidin–biotin affinity purification technique combined with LC−MS/MS was used to analyze potential proteins that interact with PGC7. In total, 291 potential PGC7-interacting proteins were identified. Through an in-depth bioinformatic 1

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analysis of potential interactors, we linked PGC7 to critical cellular processes including translation, RNA processing, cell cycle, and regulation of heterochromatin structure. To better understand the functional interactions of PGC7 with its potential interactors, we constructed a protein–protein interaction network using the STRING database. In addition, we discussed in detail the interactions between PGC7 and some of its newly validated partners. The identification of these potential interactors of PGC7 expands our knowledge on the PGC7 interactome and provides a valuable resource for understanding the diverse functions of this protein. KEYWORDS: PGC7, unfolded protein, LC−MS/MS, protein−protein interaction network, RNA processing, cell cycle, heterochromatin structure

INTRODUCTION The maternal factor primordial germ cell 7 (PGC7), also known as Dppa3 and Stella, was initially identified as a gene preferentially expressed in primordial germ cells (PGCs)1. In mice, PGC7 expression begins during the process of PGC specification at embryonic day 7.25 (E7.25) and continues until E15.5. Subsequently, PGC7 expression resumes in the oocytes and is maintained in preimplantation embryos after fertilization1-3. PGC7 deficiency in mice leads to aberrant epigenetic modifications, abnormality in oocytogenesis and severe early embryogenesis defects3-6. In addition, the transition from non-surrounded nucleolus-type to surrounded nucleolus-type oocytes is significantly impaired in PGC7-null oocytes5. The examination of PGC7-null embryos revealed abnormal cleavage at the 2–4-cell stages4,7,8. 2


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To date, multiple studies have shown that PGC7 protects the DNA methylation status in the maternal genome and at certain imprinted loci in the paternal genome3,4,9,10. The underlying molecular mechanisms have been clarified: PGC7 suppresses the enzymatic activity of TET2 and TET3, protects DNA methylation from Tet-mediated 5-mC to 5-hmC conversion11, and selectively binds to chromatin containing dimethylated histone 3 lysine 9 (H3K9me2), thereby blocking the activity of the TET3 methylcytosine oxidase in these loci9. Conversely, PGC7 overexpression in NIH3T3 cells has been shown to induce global DNA demethylation12,13 instead of maintaining DNA methylation, suggesting that PGC7 plays a critical role in regulating DNA methylation status. In addition, PGC7 is involved in transcriptional repression, chromatin condensation, cell division, and cell pluripotentiality maintenance 3,6,9,14. Despite our understanding of the fundamental roles of PGC7 in these cellular processes, only a few molecular and functional interactions of PGC7 have been reported2,3,14. Here, the STRING online tool (http://string-db.org/) was used to investigate the known and predicted protein–protein interactions of PGC7, and the only protein identified in humans was IPO5. To identify additional PGC7-interacting proteins, we performed the first proteomic characterization of the protein–protein interaction network of PGC7 and found 290 previously unknown PGC7-interacting proteins. The discovery of these PGC7 interactors considerably expands our knowledge on the PGC7 interactome, and their analysis also clarifies previously unknown molecular functions of PGC7 in RNA processing, cell cycle, and regulation of heterochromatin structure. Additionally, knowledge of these new PGC7-interacting 3



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proteins will likely explain the findings of previous studies on PGC7 and provide a substantial resource for subsequent in-depth studies on PGC7 functions.

MATERIALS AND METHODS Plasmid Construction The coding sequence of biotin holoenzyme synthetase (BirA) gene was isolated by polymerase chain reaction (PCR) using the DNA extract of Escherichia coli strain BL21 as template. The full-length coding region of the BirA gene was cloned in-frame with a linker encoding a Myc-tag in a pCDH-MCS-T2A-PURO-MSCV expression vector (System Biosciences, Palo Alto, CA, USA ), which was named pCDH-Myc-BirA. The cDNA for the house mouse PGC7 (ID no. AF490347; GenBank) was PCR-amplified with a mouse embryonic stem cell cDNA library and specific primers. PGC7 amplicon harboring a FLAG epitope tag and a BirA recognition/substrate peptide sequence (MSGLNDIFEAQKIEWHEGAPSSR) at its N-terminal was also cloned into the pCDH-MCS-T2A-PURO-MSCV expression vector. The vector was named pCDH-FlagBio-PGC7. To co-express BirA and FlagBio-PGC7, Myc-tagged BirA cDNA with a linker encoding P2A peptide sequence15 at its C-terminus was cloned from the pCDH-Myc-BirA vector using PCR and specific primers, and then inserted into the pCDH-FlagBio-PGC7 vector. The resultant

recombinant

plasmid

was

pCDH-Myc-BirA-P2A-FlagBio-PGC7-T2A-PURO-MSCV. This vector was named pCDH-BirA-FlagBio-PGC7. 4


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Cell Culture HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), penicillin (100 units/mL), and streptomycin (100 mg/mL). The cultures were maintained at 37°C in 5% CO2. Western blot and Immunofluorescence Assay HEK293T

cells

were

transfected

with

plasmids

pCDH-Myc-BirA,

pCDH-FlagBio-PGC7 and pCDH-BirA-FlagBio-PGC7 for 48 h, and then the whole cell lysates were collected via the lysis of RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM NaF, 5 mM EDTA, 1 mM sodium or thovanadate, and complete protease inhibitor mixture) for 1h on ice and clarified via centrifugation. The supernatants assayed by western blotting (Figure S1). Mouse monoclonal anti-MYC tag antibody (9E10, Sigma-Aldrich) and anti-FLAG antibody (M2, Sigma-Aldrich) were used to detect the expression of Myc-BirA and FlagBio-PGC7. The streptavidin-HRP (Amersham, RPN1231) was used to detect the biotinylated PGC7. 24-well

plates

of

HEK293T

cells

were

transfected

with

plasmids

pCDH-FlagBio-PGC7, pCDH-BirA-FlagBio-PGC7 and pCDH-Flag-PGC7 for 48 h, and then the cells were fixed with cold 4% paraformaldehyde. The cellular localization of tagged PGC7 were analyzed by immunofluorescence (Figure S2) using antibodies against FLAG (M2, Sigma-Aldrich). The nuclei were stained with 0.01% 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). The fluorescent images were 5



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examined under an inverted fluorescence microscope (Nikon, Tokyo, Japan). Protein Sample Preparation and One-step Streptavidin Purification of Protein Complexes Twenty

15-cm

plates

of

HEK293T

cells

were

transfected

with

pCDH-BirA-FlagBio-PGC7 (co-expressing BirA and biotinylated PGC7); equal amounts of HEK293T cells were transfected with pCDH-Myc-BirA (only expressing BirA control); Forty eight hours after transfection, the cells were harvested for protein extraction. The nuclear extracts (NEs) of the BirA and BirA + biotinylated PGC7 samples were prepared according to the method described by Wang et al16. The protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein assay kit. Equal amounts of NEs (as determined by BCA protein assay) from the BirA (control) and BirA + biotinylated PGC7 samples were used to perform the follow-up experiment. The NE sample was precleared with protein G-agarose (100 µl of partial slurry per 10 mg of protein) for 2 h at 4°C with continuous mixing. The samples were then spun at 300 × g for 5 min at 4°C, and the precleared NE samples were transferred to new tubes. Subsequently, PGC7-associated and control protein complexes were purified from the precleared BirA + biotinylated PGC7 and BirA samples, respectively, using the method one-step streptavidin purification of protein complexes described by Wang et al16. The protein concentration of the purified complexes was determined by BCA protein assay. Samples were quick-frozen in liquid nitrogen and stored at −80°C. SDS-PAGE and In-Gel Digestion 6


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Large sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were prepared using 5% stacking gels and 12% resolving gels. The enriched protein complexes samples were separated on the 12% resolving gel and visualized by Coomassie blue staining. Each gel lane was divided into five slices and cut into small pieces. The gel pieces were destained in 50 mM NH4HCO3/50% acetonitrile at 37°C until depigmentation. Subsequently, 500 µl of 100% acetonitrile was added, and the samples were incubated at room temperature with occasional vortexing until the gel pieces became white and shrunk. Dried gel pieces were incubated with enough trypsin buffer (0.01 µg/µl) in 40 mM NH4HCO3 overnight at 37°C. The resulting tryptic peptides were extracted for mass spectrometry analysis. LC-ESI-MS/MS analysis based on Triple TOF 5600 Each sample was resuspended in 20 µl buffer A [5% acetonitrile (ACN), 0.1% formic acid (FA)] and centrifuged at 20,000 × g for 10 min. Approximately 5 µl supernatant (approximately 2.5 µg protein) was loaded on a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) by the auto sampler onto a 2 cm C18 trap column (inner diameter 200 µm, Waters). The peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 µm, Waters) packed in-house. The samples were loaded at 8 µl/min for 4 min, and the 35 min gradient was run at 300 nl/min starting from 5% and increasing to 35% buffer B (95% ACN, 0.1% FA), followed by a 5 min linear gradient to 60%, followed by a 2 min linear gradient to 80%, maintenance at 80% buffer B for 2 min, and finally a return to 5% for 1 min. According to a modified method described by Andrews et al17, data acquisition 7



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was performed with a TripleTOF 5600 System (AB SCIEX, Concord, Canada) fitted with a Nanospray III source (AB SCIEX) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA, USA). Data were acquired using an ion spray voltage of 2.5 kV, a curtain gas of 30 psi, a nebulizer gas of 15 psi, and an interface heater temperature of 150°C. The mass spectrometer (MS) was operated with a resolving power (RP) of greater than or equal to 30,000 fwhm for time-of-flight (TOF) MS scans. For information-dependent acquisition (IDA), survey scans were acquired in 100 ms, and as many as 40 product ion scans were collected if exceeding a threshold of 150 counts per second (counts/s) was exceeded with a 2+ to 5+ charge-state. Total cycle time was fixed to 2.8 s. Four time bins were summed for each scan at a pulser frequency value of 11 kHz through monitoring of the 40 GHz multichannel time-to-digital converter (TDC) detector with four-anode/channel detection. A sweeping collision energy setting of 35 ±15 eV was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (∼15 s) and then the precursor was refreshed off the exclusion list. MS Data Analysis and Protein Identification The acquired peak-lists of all tandem MS (MS/MS) spectra were combined into one Mascot generic format (MGF) file and searched with Mascot software (version 2.3.02) against the International Protein Index (IPI) human sequence database (version 3.87, 91,464 sequences). The search parameters were set as follows: MS/MS Ion search; trypsin as the enzyme, with one missed cleavage allowed; mass values of Monoisotopic; mass tolerance of 0.05 Da for peptides and 0.1 Da for fragmentations; 8


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fixed

modifications

of

Carbamidomethyl

(C);

variable

modifications

of

Gln->pyro-Glu (N-term Q), Oxidation (M), and Deamidated (NQ). The Mascot instrument setting parameter was set to ESI-QUAD-TOF. Mascot ‘identity score’ provides an acceptance threshold with false identification probability at a confidence level of 0.05. To reduce the probability of false peptide identification, only peptides with significance scores greater than “identity score” were counted as identified, and each confident protein identification involves at least one unique peptide. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomeexchange.org) via the PRIDE18 partner repository (http://www.ebi.ac.uk/pride) with the dataset identifier PXD006362. Bioinformatic Analysis To examine the biological and functional properties of the identified proteins, Gene Ontology (GO) annotation was conducted by searching the GO website (http://www.geneontology.org). Functional category analysis was performed with protein2go and go2protein. The Clusters of Orthologous Groups of proteins (COGs) annotation was performed using Blast software by searching the COG databases (http://www.ncbi.nlm.nih.gov/COG/). The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 was used for functional enrichment analysis of GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A false discovery rate (FDR) of 4 and nodes > 10. For example, the top cluster we identified (cluster 1) comprised ribosome-associated proteins. In addition, many RNA processing-associated proteins were connected in a dense protein–protein interaction network that formed cluster 2. This analysis is consistent with our functional annotation analyses. Co-immunoprecipitation Validation Of the 291 PGC7-interacting proteins identified from human 293T cells, most were not previously known to be interacting partners of PGC7. To validate whether PGC7 interacts with these orthologous proteins in mice, we selected 28 murine proteins (which were orthologous proteins of human) from the 291 PGC7 interactors (Table 2). The sequence similarity of the 28 proteins with orthologous proteins in human were calculated and are shown in Table 2. The criteria for choosing these proteins were as follows. Firstly, in order to further understand the roles of PGC7 in above-mentioned (Table 1) GO biological processes, six proteins, YBX1, HNRNPR, U2AF2, PPP2R1A, FBL, and NOP2, were selected from RNA processing (GO: 14


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0006396) term, and nine proteins, including UHRF1, HSPA2, CDK2, CDK5, RBBP4, SMC2, SMC3, MCM6, and AKAP8L, were chosen from cell cycle (GO: 0007049) term. Secondly, six proteins were selected based on their function, although some of them exhibiting low coverage and scores. Such as, NUPL1, KPNA1, KPNB1, and CSE1L may be involved in the transport of PGC7 between the cytoplasm and nucleus22,23; DMAP1, a DNMT1 interacting protein, is known to play an important role in epigenetic reprogramming during preimplantation development24; and HP1BP3 is essential for postnatal viability and growth in mice25. Finally, the remaining seven proteins, including MAGED2, GNB2L1, STAT1, RPL14, CANX, NME1, and YWHAG, were selected randomly. In total, 28 proteins, approximately 10% of those identified, were chosen for validation. Next, we attempted to validate the interaction of PGC7 with the selected proteins. To do so, all the genes were cloned into vectors containing an N-HA tag. Co-immunoprecipitations were performed from whole cell extracts using HA-tag antibodies and matched normal IgG as a negative control. We examined a total of 28 selected proteins and were able to confirm binding of 23 to PGC7 (Figure 3). These data provide a level of confidence for the many other PGC7-interactors identified from the mass spectrometry screen.

DISCUSSION PGC7 was initially identified as a gene expressed specifically in preimplantation embryos and germ cells1. After that, a series of studies showed that PGC7 plays 15



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pivotal roles in oocytogenesis and early embryogenesis3,5,8. However, the molecular mechanism that regulates these biological processes was not clear, and none of PGC7 interacting proteins were reported. Later, Nakamura et al7 reported that PGC7 protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. In this article, Ran binding protein 5 (RanBP5, also known as IPO5) were identified binding to PGC7, and plays a critical role in the nuclear localization of PGC7. To further elucidate the molecular mechanism of PGC7 protects DNA methylation, Nakamura et al9 found that PGC7 binds histone H3K9me2 to protect 5mC from Tet3-mediated conversion to 5hmC, and Bian et al11 further demonstrated that PGC7 interacts with TET2 and TET3 to suppress the enzymatic activity of TET2 and TET3. In addition, Funaki et al12 found that PGC7 interacting with UHRF1, and the binding of PGC7 to UHRF1 induced global DNA demethylation in NIH3T3 cells. To date, only the five above-mentioned proteins have been reported to interact with PGC7. To gain a better understanding of the molecular and functional interactions of PGC7, we attempted to identify a comprehensive set of proteins that interact with PGC7 in 293T cell nuclear extracts. We identified 291 potential PGC7-interacting proteins using a streptavidin–biotin affinity purification method. Of the previously known PGC7-interacting proteins, only UHRF1 was detected in our screen. Thus, 290 novel PGC7-interacting proteins were identified in 293T cells. PGC7 is a small protein (150 amino acids) in the mouse. Thus, we are surprised why we discover so many PGC7 interacting proteins. To investigate its reason, we found that the sequence of PGC7 protein contains a high proportion of particular polar 16


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and charged amino acids (63.3%). Then, the amino acid sequences of PGC7 protein were

submitted

to

the

fold

index

software

(FoldIndex,

http://bioportal.weizmann.ac.il/fldbin/findex)26 to analyze the amino acid sequence that provide the maximum favored condition for folding of each domains27,28. As shown in Figure 4, PGC7 is an intrinsically unfolded protein (also known as intrinsically disordered proteins). Unfolded proteins with high degrees of structural ﬂexibility can more easily adapt to a variety of binding partners to perform their function. This structural ﬂexibility can also help play a role in proteins associated with protein-protein binding, protein-DNA binding, protein-RNA binding, cell-cycle control, cell signaling and longevity of the proteins29-33. Therefore, it is reasonable that we found so many PGC7 interacting proteins. The unfolded structure also revealed that PGC7 has a variety of specific physiological functions, which is consistent with our subsequent analysis. Based on the functional annotations, PGC7-interacting proteins were found to possess many molecular functions, such as binding, catalytic activity, structural molecule activity, and transporter activity (Figure 1A). Further functional enrichment analysis of the PGC7-associated proteins revealed enrichment for distinct functional GO categories. The top-ranked GO molecular function categories included nucleotide binding and RNA binding, consistent with previously characterized functions of PGC77,10. For GO biological processes, these proteins were significantly enriched in 12 GO terms. However, there was no evidence for a role of PGC7 in these GO biological process terms. Thus, our study uncovered previously unknown associations 17



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of PGC7 with proteins involved in translation, RNA processing, cell cycle, ribonucleoprotein complex biogenesis, and RNA splicing. To further elucidate the functional relationships between the PGC7-interacting proteins and identify specific functional complexes, we mapped PGC7 and its interactors using the STRING interaction database. As shown in Figure 2A, a PPI network of PGC7 interactome was generated with a confidence score of >0.4 and then visualized by Cytoscape. Of the 291 PGC7-interacting proteins, 243 (83.5%) were mapped to the interaction network. However, based on the latest version of the STRING database, there were no functional relationships between PGC7 and its interactors. Thus, our study considerably expands the knowledge on the PGC7 interactome and suggests testable hypotheses for elucidating of novel PGC7 functions. The interaction network was further analyzed for highly connected clusters using the MCODE program. Interestingly, the top three highly connected clusters identified included translation, RNA processing, and transportation cluster (Figures 2B–2D). This analysis aligned with our GO functional annotation analyses. The PPI network analysis also revealed multiple PGC7-associated complexes that suggest previously unknown functional settings for PGC7. This study identified proteins from 293T cell nuclear extracts, most of which were not previously known to be interacting partners of PGC7. We validated the interaction of PGC7 with these newly identified proteins. According to the criteria described above, 28 murine proteins, approximately 10% of the identified proteins, were chosen for further experimental validation. Of the 28 proteins examined 23 18


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(82.14%) were successfully validated using co-immunoprecipitation (Figure 3). This high validation percentage provides a certain degree of confidence for the many other PGC7-interacting proteins revealed by the mass spectrometry analysis. Importantly, these 23 novel validated PGC7-interacting partners could contribute to the elucidation of novel PGC7 functions. Although motif analysis of PGC7 suggested that it functions in RNA processing34, none of the RNA processing-associated proteins were previously reported to interact with PGC7. Here, of the 34 proteins identified, most are known to be involved in RNA splicing. These include, YBX1, an RNA-binding protein that is required for regulating the splicing of certain mRNAs35-38; HNRNPR, a member of the spliceosome C complex that functions in pre-mRNA processing39; and U2AF2, the U2 auxiliary factor (U2AF) large subunit that plays a critical role in 3′ splice site selection40-42. In this study, a total of six RNA processing-associated proteins were validated to interact with PGC7 (Figure 3, Figure 5). Thus, PGC7 may play a previously unknown role in RNA processing. Additional studies are necessary to clarify these molecular mechanisms. Previous studies have reported that PGC7 was essential for early development. Oocytes from PGC7-deficient mice exhibit early embryogenesis arrest at the 4-cell stage following fertilization4,7,8. Although Nakamura et al9 determined that the molecular mechanism of early embryogenesis arrest involved abnormal DNA demethylation, we hypothesized that PGC7 also plays a role in driving progression through phases of the cell cycle. Here, our functional enrichment analysis showed that 19



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34 PGC7-interacting proteins are involved in the cell cycle. In addition, the binding of several of these proteins, including CDK2, UHRF1, HSPA2, AKAP8L, RBBP4, and MCM6, to PGC7 was validated. Cyclin dependent kinases (CDKs) are known to regulate progression through cell cycle phases, and CDK2 plays a critical role in G1-to–S-phase transition43. UHRF1 also plays a major role in the G1-to-S-phase transition by regulating topoisomerase II alpha44-46. Both HSPA2 and AKAP8L are required for the G2-to-M-phase transition47,48. RBBP4 is involved in regulating oocyte meiotic progression49, and MCM6 is a highly conserved mini-chromosome maintenance protein (MCM) that is essential for the initiation of eukaryotic genome replication. The interactions between PGC7 and these cell cycle-associated proteins further strengthen the potential roles of PGC7 in the cell cycle. In addition to this, PGC7 plays a role in chromosomal organization. Previous gene deletion analyses have implicated PGC7 in chromatin condensation5, maternal chromosome integrity4 and chromatin reorganization50. However, to date, no PGC7 interactors involved in chromatin organization have been reported. Here, three chromatin organization-associated proteins were identified: RBBP4, DMAP1, and HP1BP3. RBBP4 is a ubiquitously expressed nuclear protein that belongs to a subfamily of WD-repeat proteins51. It was found to be a component of two protein complexs that have been implicated in chromatin organization, the Mi-2 chromatin-remodeling complex52 and the chromatin assembly factor 1 complex53. In addition, RBBP4 was shown to be required for proper chromosome segregation during MI in mouse oocytes49. Given its interaction with RBBP4, PGC7 may play a 20


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more active role in chromatin regulation than previously thought. DMAP1, one of the core components of the Swr1 chromatin-remodeling complex54, was originally identified as a DNMT1-interacting protein55. Subsequently, the DMAP1–DNMT1 complex was found to interact with the p33ING1–Sin3–HDAC complex, and both were shown to be indispensable for the maintenance of pericentric heterochromatin structure throughout cell division56. Additionally, DMAP1 plays a crucial role in the maintenance of chromosomal integrity through the DNA damage repair pathway57,58. Interestingly, based on previous studies, we noticed that DMAP1 interacted with DAXX59. We had previously found that PGC7 was essential for chromatin reorganization through the regulation of DAXX expression in early embryos50. In this study, the interaction between PGC7 and DMAP1 was validated as well. Taken together, these findings strongly indicate that DMAP1 forms a complex with PGC7 and DAXX and that this novel complex may play a role in chromatin reorganization. HP1BP3 (also known as HP1-BP74), a component of heterochromatin, was initially discovered as a heterochromatin protein (HP1)-binding protein60. HP1BP3 is enriched in the heterochromatin in vivo and plays a key role in mediating heterochromatin formation and propagation25,61. Recent studies have revealed that HP1BP3 depletion disrupts the heterochromatin structure because of chromatin unpacking25,62, suggesting that HP1BP3 is required for maintaining heterochromatin integrity. In this study, because we observed an interaction between PGC7 and HP1BP3, we further analyzed the interaction between PGC7 and the heterochromatin 21



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marker HP1α (also known as CBX5) using co-immunoprecipitation. Interestingly, PGC7 interacted with HP1α as well (data not shown). This discovery prompted us to speculate that PGC7 may play a role in the regulation of heterochromatin structure by forming a complex with HP1α and HP1BP3. Regarding the molecular mechanism of PGC7 in protecting DNA methylation, we will examine in the future whether PGC7 inhibits the access of TET3 to heterochromatin in association with HP1α and HP1BP3. Among the PGC7-interacting proteins, four proteins (KPNB1, KPNA1, YWHAG, and NUPL1) involved in protein transportation were identified. KPNB1 and KPNA1 are nucleocytoplasmic transport proteins that participate in protein transport between the nucleus and cytoplasm63. It is known that PGC7 protects the maternal genome from demethylation only after localizing to the nucleus. Here, we found that PGC7 could directly bind to KPNB1 and KPNA1. Although RanBP5 has been reported to interact with PGC7 and facilitate its nuclear import, KPNB1 and KPNA1 may play a role in the nuclear translocation of PGC7 as well7. However, further studies are needed to validate this possibility. CONCLUSION Our proteomic analysis has provided a wealth of new information regarding PGC7 protein interactions in the cell. In this study, 290 previously unknown PGC7-interacting proteins were identified. An in-depth bioinformatic analysis placed PGC7 in new pathways governing critical cellular processes, including translation, RNA processing, and cell cycle. In addition, we validated numerous potential PGC7 22


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interactors that participate in diverse and functionally important cellular processes. As shown in Figure 5, PGC7 may be involved in RNA processing, cell cycle, and regulation of heterochromatin structure, roles that were not previously linked to PGC7. In addition, a network was generated to summarize the interaction of PGC7 with its partners. ASSOCIATED CONTENT Supporting Information Figure S1：Validation of biotinylation of PGC7 by transient transfection. Figure S2：Detection of cellular localization of tagged PGC7. Figure S3：Separation of PGC7 interacting proteins in 293T cells by SDS-PAGE. Table S1: The complete list of 291 identified interacting proteins of PGC7. Table S2: GO and KEGG enrichment analysis of PGC7-interacting proteins. Table S3: The COG function classification of PGC7-interacting proteins. Table S4: Protein network analysis of PGC7-interacting proteins by using STRING database by MCODE. Table S5 ： Detailed peptide data for PGC7 interacting proteins identified by LC-MSMS. Supplemental Data S1: The result files of protein identification using Mascot software. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author 23



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*Corresponding author: Guo Zekun Tel.: (86)29-87080092; Fax: (86)29-87080085. E-mail: [email protected] Notes The authors declare no competing financial interest. Funding Sources This work was supported by the National Natural Science Foundation of China (No. 31172279 to ZG, No. 31572405 to ZG), and Key Science and Technology Innovation Team in Shaanxi Province (No. 2014KCT-26 to ZG). ACKNOWLEDGMENTS We thank Yongyan Wu and Zhiying Ai for advice, reagents and help with experiments. We also thank Haoya Chang for proofreading.

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Tables Table 1. GO terms enriched in the PGC7 interactome. The top three GO biological process, molecular function, and cellular component terms enriched in the PGC7 interactome are listed. Category

Term

Description

Biological process

GO:0006412 translation GO:0006396 RNA processing GO:0007049 cell cycle Molecular function GO:0000166 nucleotide binding GO:0003723 RNA binding GO:0005198 structural molecule activity Cellular component GO:0043232 intracellular non-membrane-bounded organelle GO:0043228 non-membrane-bounded organelle GO:0043233 organelle lumen * Number of PGC7 interacting proteins

31


Counts*

P-Value

FDR

43 34 34 69 58 48 107 107 75

5.61E-24 2.50E-10 1.36E-06 1.06E-07 2.39E-23 5.64E-19 2.61E-18 2.61E-18 2.13E-11

9.28E-21 4.14E-07 0.002248 0.000149 3.37E-20 7.93E-16 3.53E-15 3.53E-15 2.88E-08


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Table 2. List of the 28 selected murine proteins. The interactions of these proteins with PGC7 was examined by co-immunoprecipitation in this study. Protein name

Symbols

Accession number

Protein similarity *

E3 ubiquitin-protein ligase UHRF1 isoform A melanoma-associated antigen D2 importin subunit beta-1 exportin-2 structural maintenance of chromosomes protein 2 guanine nucleotide-binding protein subunit beta-2-like 1 rRNA 2'-O-methyltransferase fibrillarin signal transducer and activator of transcription 1 isoform 1 cyclin-dependent kinase 2 isoform 2 calnexin precursor heterogeneous nuclear ribonucleoprotein R isoform a structural maintenance of chromosomes protein 3 nuclease-sensitive element-binding protein 1 heat shock-related 70 kDa protein 2 cyclin-dependent kinase 5 histone-binding protein RBBP4 A-kinase anchor protein 8-like serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform probable 28S rRNA (cytosine-C(5))-methyltransferase DNA replication licensing factor MCM6 isoform 1 60S ribosomal protein L14 14-3-3 protein gamma splicing factor U2AF 65 kDa subunit isoform 1 heterochromatin protein 1-binding protein 3 isoform 1 DNA methyltransferase 1-associated protein 1 nucleoporin p58/p45 importin subunit alpha-5 nucleoside diphosphate kinase A *Protein similarity show the sequence similarity of these human.

UHRF1 MAGED2 KPNB1 CSE1L SMC2 GNB2L1 FBL STAT1 CDK2 CANX HNRNPR SMC3 YBX1 HSPA2 CDK5 RBBP4 AKAP8L

NP_001104548.1 NP_001186175.1 NP_032405.3 NP_076054.1 NP_001288341.1 NP_032169.1 NP_032017.2 NP_001192242.1 NP_058036.1 NP_001103969.1 NP_001264050.1 NP_031816.2 NP_035862.2 NP_001002012.1 NP_031694.1 NP_033056.2 NP_059504.2

72.76% 86.20% 99.20% 76.31% 91.65% 100.00% 95.43% 92.72% 98.99% 92.92% 99.53% 99.92% 98.46% 98.44% 99.66% 100.00% 91.81%

PPP2R1A

NP_058587.1

76.23%

NOP2 NP_620086.2 75.06% MCM6 NP_032593.1 95.86% RPL14 NP_080250.1 85.91% YWHAG NP_061359.2 100.00% U2AF2 NP_001192160.1 99.79% HP1BP3 NP_001116369.1 89.93% DMAP1 NP_075667.1 97.86% NUPL1 NP_733479.1 90.82% KPNA1 NP_032491.2 97.77% NME1 NP_032730.1 94.08% murine proteins with orthologous proteins in

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Figure legends Figure 1. GO classifications of the PGC7-interacting proteins. (A) GO molecular function analysis. Binding, catalytic activity and structural molecule activity are the top three listed functions. (B) GO biological process analysis. Cellular process, metabolic process, and single-organism process are top three listed biological process. (C) GO cellular component analysis. The top-ranked categories include cell, cell part, and organelle.

Figure 2. Protein–protein interaction network of the PGC7 interactome. (A) The complete PGC7 interaction network obtained from the STRING database with a confidence score of >0.4. The network contains 243 nodes and 1,676 edges. (B)–(D) The three top-ranked and tightly connected network clusters obtained with MCODE are color-coded and rendered as separate modules. Detailed cluster information is listed in supplemental Table S4.

Figure 3. Validation of the selected PGC7-interacting proteins. PGC7 and N-HA tagged interactors were transiently co-expressed in 293T cells. Total cell lysates were used to validate the protein–protein interactions by co-immunoprecipitation. HA-tagged antibodies were used to capture N-HA-tagged protein–PGC7 complexes, and normal mouse IgG served as a negative control. In western blot analysis, PGC7-specific antibodies were used to detect PGC7. Of the 28 selected proteins, 23 were successfully validated.

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Figure 4. Bioinformatics analysis for folding of PGC7 using Fold index software. Figure 5. The interaction network of PGC7 with its partners. In this network, 24 newly validated and four previously reported PGC7-interacting proteins were included. The STRING online tool was used to determine the known interactions between PGC7 and its partners. We further visualized the network in Cytoscape and grafted our identified interactions onto it.

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For TOC only

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Figure 1. GO classifications of the PGC7-interacting proteins. 180x322mm (150 x 150 DPI)


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Figure 2. Protein–protein interaction network of the PGC7 interactome. 165x112mm (300 x 300 DPI)



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Figure 3. Validation of the selected PGC7-interacting proteins. 141x85mm (300 x 300 DPI)


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Figure 4. Bioinformatics analysis for folding of PGC7 using Fold index software. 132x184mm (300 x 300 DPI)



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Figure 5. The interaction network of PGC7 with its partners. 152x99mm (300 x 300 DPI)


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Comprehensive Proteomic Analysis of PGC7-Interacting Proteins

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