Fam208a orchestrates interaction protein network essential for early embryonic development and cell division

Experimental Cell Research 382 (2019) 111437

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Fam208a orchestrates interaction protein network essential for early embryonic development and cell division

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Veronika Gresakovaa,c, Vendula Novosadovab, Michaela Prochazkovab, Shohag Bhargavaa, Irena Jenickovab, Jan Prochazkaa,b,∗∗, Radislav Sedlaceka,b,∗ a

Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i, Průmyslova 595, 252 50, Vestec, Czech Republic Czech Centre of Phenogenomics, Institute of Molecular Genetics, ASCR v.v.i, Průmyslova 595, 252 50, Vestec, Czech Republic c Palacky University in Olomouc, Faculty of Medicine and Dentistry, Hněvotínská 3775 15, Olomouc, Czech Republic b

ARTICLE INFO

ABSTRACT

Keywords: Genome stability Fam208a Multipolar spindle apparatus HUSH

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining the genome stability during the cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 leads to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by qRT-PCR profiling and in situ hybridisation [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is multi-interacting protein affecting genome stability on the level of cell division at the earliest stages of development and also by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an additional role in zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp.

1. Introduction Genome stability can be impaired by variable processes including erroneous DNA1 replication or unequal sister chromatid segregation [2]. Moreover, natural decay or exogenous genotoxic agents such as ultraviolet light, oxidative stress, chemical mutagens, and radiation are

constantly affecting the stability of the DNA. To compensate this instability, multiple mechanisms were developed to prevent accumulation of these changes. The DNA repair system is a complex machinery involving a system of checkpoints, homologous recombination or nonhomologous end-joining process, posttranscriptional RNA2 modifications (m6A, alternative splicing) and posttranslational modifications of

Abbreviations: MPP8, Mphosph8; FAM, Fam208a; DAPI, 4′,6-diamidino-2-phenylindole hydrochloride; GFP, green fluorescent protein; A488, Alexa 488; IMPC, The International Mouse Phenotyping Consortium; ENU, N-ethyl-N-nitrosourea; KO, knock out; EPLIN, epithelial protein lost in neoplasm; Y2H, yeast two hybrid ∗ Corresponding author. Laboratory of Transgenic Models of Diseases, Institute of molecular genetics of the ASCR, v.v.i, Průmyslova 595, 252 50, Vestec, Czech Republic. ∗∗ Co-corresponding author. Laboratory of Transgenic Models of Diseases, Institute of molecular genetics of the ASCR, v.v.i, Průmyslova 595, 252 50, Vestec, Czech Republic. E-mail addresses: [email protected] (V. Gresakova), [email protected] (V. Novosadova), [email protected] (M. Prochazkova), [email protected] (S. Bhargava), [email protected] (I. Jenickova), [email protected] (J. Prochazka), [email protected] (R. Sedlacek). 1 Deoxynucleotide acid. 2 Ribonucleic acid. https://doi.org/10.1016/j.yexcr.2019.05.018 Received 2 April 2019; Received in revised form 11 May 2019; Accepted 14 May 2019 Available online 18 May 2019 0014-4827/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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proteins (histone methylation, phosphorylation etc.) [3,4]. Methylation processes are also involved in maintaining genome stability and are driven by several signalling pathways, one of which includes the HUSH (Human silencing hub) complex with FAM208a in its core [5]. Fam208a is a large protein, which was originally designated as RAP140a and described as an interaction partner for human partial retinoblastoma protein, Rb [6]. The first suggestions about its role were linked to intracellular translocation of Rb and general involvement in cell-cycle control, gene expression, and tumorigenesis. Subsequently, Fam208a was identified as a direct transcriptional target of Oct4, critical for cell pluripotency, differentiation activation, and gene repression [7]. In 2013, Fam208a was described as a potential suppressor of variegation in ENU3 mutagenesis screen with integrated multi-copy green fluorescent protein (GFP) transgene under the control of the haemoglobin promoter [8]. Two independent lines with induced mutations in Fam208a, MommeD6 (L130P) and MommeD20 (introduction of a stop codon in the intron 1 region) were identified and described as embryonically lethal [9]. Studies on Fam208a-L130P suggested its involvement early embryonal development [10]. The usage of near-haploid KBM7 cells in forward genetic screen identified a complex of four proteins, FAM208a, MPHOSPH8, SETDB1, and PPHLN1, designated as HUSH (human silencing hub) complex [11]. HUSH complex was shown to be involved in regulation of silencing integrated retroviruses as well as endogenous regions by recruiting methyltransferase SETDB1 to H3K9 methylation sites. Recently, it was proved that FAM208a in HUSH complex bind to sequences of endogenous retroviruses (ERVs) and long interspersed nuclear elements (LINE-1s/L1s). Moreover, HUSH complex was also shown that together with TRIM28, they are responsible for co-repression of young retrotransposons and new genes promoted by noncoding DNA silencing [12]. Our recent study shows that Fam208a, known to be part of HUSH complex, can also play an important role in spindle pole assembly and sister chromatids segregation in the initial steps of embryonic development. We identified several new putative interaction partners of Fam208a such as SVIL, INTB3BP, GPSM2, EML1 and AMN1, which are known to play a role in cell division and mitotic spindle assembly. Moreover, knocking down endogenous Fam208a by RNA interference in murine zygotes leads to formation of multipolar spindles and increased ratio of arrested or incorrectly developed embryos, suggesting that the early embryonic lethal phenotype might be associated with improper regulation of cell division resulting in chromosomal aberrations. These findings suggest a new role of Fam208a in very early events of embryonic development and in adult somatic cells, where its major function is associated mostly with function in HUSH complex.

extension at 72 °C for 5 min at the end. PCR products were separated in 1% agarose gels. The animals used in the study came from at least 3 back-crosses of heterozygotes with wild type C57Bl/6NCrl mice. 2.2. Mutant Hek293t cell lines CRISPR/Cas9 targeting sequence for both genes, FAM208a and MPHOSPH8, were cloned into pX330 Venus vector (Suppl. Table 2). All constructs were verified by sequencing. Hek293t cells were incubated with transfection mixture consisted of 30 μg of vector DNA and 90 μl of X-treme GENE HP DNA Transfection Reagent (Roche, 06 366 236 001) and DMEM (D6429). After 24 h incubation, cells were sorted by FACS for GFP positive cells and 10000 cells were plated for further cultivation. 48 h later, GFP negative sorting took place, followed by single cell plating. This sorting was further incubated and single cell colonies were formed and analysed by PCR followed by sequencing. Finally, 6 stable lines were selected for next experimental plans: HekMT (no mutation); Fam-a1 (−39bp, exon 4/11); Fam-a2 (−34bp, exon 4/11); Mpp8-a (−19bp, exon 8) and Mpp8-b (−46bp, exon 8). Both transcription variants of FAM208a should be effected, in FAM208a-202 exon 4, in FAM208a-209 exon11. In MPHOSPH8 were CRISPRS targeted to Ankyrin rich domain, as this was identified by Y2H system as interaction domain with Fam208a, but the mutation lead to complete protein ablation. 2.3. E9.5 LC-MS analysis We collected 4 littermate embryos at the stage of E9.5, and performed separation from yolk sack and maternal residues. Two wild types, one heterozygote and one homozygote embryo were lysed in 100 mM TEAB containing 2% SDC and boiled at 95 °C for 5 min. Protein concentration was determined using BCA protein assay kit (Thermo) and 20 μg of protein per sample was used for MS sample preparation in technical triplicates. Cysteines were reduced with 5 mM final concentration of TCEP (60 °C for 60 min) and blocked with 10 mM final concentration of MMTS (10 min at Room Temperature). Samples were digested with trypsin (trypsin/protein ratio 1/20) at 37 °C overnight. After digestion, samples were acidified with TFA to 1% final concentration. SDC was removed by extraction to ethyl acetate [1,11] and peptides were desalted on Michrom C18 column (Michrom BioResources Inc.). 2.4. Hek293t mutant cell LC-MS analysis

2. Material and methods

Mutant Hek293t cells were grown in 6-well plate till confluence. Cell pellets were lysed in 100 mM TEAB containing 2% SDC and boiled at 95 °C for 5 min and the subsequent steps are identical to previously described method (E9.5 LC-MS analysis).

2.1. Fam208a KO murine strain

2.5. nLC-MS 2 analysis

The knock out (KO) mouse for Fam208a (first exon deleted, see Suppl. Table 2) was generated with CRISPR/Cas9 technology. The active RNA for CRISPR/Cas9 complex was microinjected into C57BL/ 6NCrl zygotes with two pronuclei on Leica micromanipulator equipped with Eppendorf FemtoJet. The microinjected zygotes were transferred to ICR recipient in the same day. The screening of mutant mice was performed with polymerase chain reaction (PCR). The Dream Taq (ThermoFisher, K1081) genotyping PCR was run with DNA prepared from tail tips (Quick extract solution, Lucigen, QE09050), using F, R1 and R2 primers (for sequence see Suppl. Table 2), under the following conditions: 95 °C for 5 min, 40 cycles of melting at 95 °C for 30 s, annealing at 64 °C for 40 s, and extension at 72 °C for 30 s, with additional

Nano Reversed phase columns (EASY-Spray column, 50 cm × 75 μm ID, PepMap C 18,2 μm particles, 100 Å pore size) were used for LC/MS analysis. Mobile phase buffer A was composed of water, 2% acetonitrile and 0.1% formic acid. Mobile phase B was composed of 80% acetonitrile, 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5 μm, 300 Å Wide Pore, 300 μm × 5 mm, 5 Cartridges) for 4 min at 15 μl/min loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. Peptides were eluted with Mobile phase B gradient from 2% to 40% B in 120 min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analysed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1,5Th with the quadrupole, HCD fragmentation with normalized collision energy of 30,

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N-ethyl-N-nitrosourea. 2

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and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS 2. The dynamic exclusion duration was set to 45s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2s cycles [1].

supplier and mRNA from murine D3 cell line was used. Final product of cDNA was diluted into final concentration of 100 ng/μl. Both products were cloned into pGBKT7 vector (Clontech, 630489) by using Phusion PCR reaction and primers with integrated XmaI/SalI or EcoRI/SalI restriction sites. Set of constructs was prepared for Y2H screen: C′ terminal construct (536-1640aa) was used as construct C, construct A (536947aa) was prepared from construct C digested with SmaI and blunted, construct B (536-1160aa) was C construct digested with PstI with spliced out cassette exon 17 (60bp), α (536-1498aa) was C construct without exon 17 and 274bp deletion in exon 24 and β (536-1640aa) was C construct without exon 17 and 179bp deletion in exon 22. Further steps were following producer's manual for Matchmaker Gold Yeast two hybrid system (Cat. No. 630489) with murine embryonal library of 11 days of age (Clontech, 630478). Finally, library plasmids were isolated with lysis buffer (50 mM Tris-HCL pH8; 10 mM EDTA + 20 mg/ml RNaseA) and purified with incubation with 200 mM NaOH, 1% SDS and after 5 min with addition of 3 M Sodium acetate, pH4.8. Overnight precipitation with isopropanol was followed by standard ethanol washes. Identified vectors were used for transformation into DH5α E. coli cells to increase a yield and later were re-purified and sent for sequencing to analyse putative partners.

2.6. Data analysis All data were analysed and quantified with the MaxQuant software (version 1.5.3.8) (Cox, Hein et al., 2014). The false discovery rate (FDR) was set to 1% for both proteins and peptides and we specified a minimum peptide length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search against the Human database (downloaded from Uniprot on September 2015, containing 147 934 entries). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Dithiomethylation of cysteine was selected as fixed modification and N- terminal protein acetylation and methionine oxidation as variable modifications. The ‘match between runs’ feature of MaxQuant was used to transfer identifications to other LC-MS/MS runs based on their masses and retention time (maximum deviation 0.7 min) and this was also used in quantification experiments. Quantifications were performed with the label-free algorithms described recently. Data analysis was performed using Perseus 1.5.2.4 software [13–15].

2.9. BIOMARK and qRT-PCR Gene expression analysis was performed using the BioMark high throughput microfluidic qPCR platform (Fluidigm, San Francisco, CA). Prior to the qPCR the samples were pre-amplified as follows: 2 μl of cDNA (10 ng RNA/μl) was mixed with 1.25 μl of 200 nM primer mix (all primers together at a final concentration of each primer of 25 nM), 5 μl of iQ Supermix (BioRad, Prague, Czech Republic) and 1.75 μl of RNase/DNase-free water (ThermoFisher Scientific). The mixture was first incubated for 3 min at 95 °C, then 18 cycles of 15 s at 95 °C, and finally 4 min at 59 °C. Pre-amplified cDNA was diluted 40 × . qPCR was carried out in the GE Dynamic array 48.48 in a BioMark HD System (Fluidigm, San Francisco, CA). 5 μl of Fluidigm sample premix consisted of 1 μl of 40 × diluted pre-amplified cDNA, 0.25 μl of 20 × DNA Binding Dye Sample Loading Reagent (Fluidigm), 2.5 μl of Sso Fast EvaGreen Supermix (Bio-Rad, Czech Republic), 0.1 μl of 4 × diluted ROX (Invitrogen, USA) and 1.15 μl of RNase/DNase-free water. Each 5 μl assay premix consisted of 2 μl of 10 μM primers (forward and reversed primer each at a final concentration of 400 nM), 2.5 μl of DA Assay Loading Reagent (Fluidigm, USA) and 0.5 μl of RNase/DNasefree water. Thermal qPCR protocol was: 50 °C for 5 s and 98 °C for 3 min, 40 cycles of 98 °C for 5 s, and 60 °C for 5 s. The data were collected with the BioMark 4.2.2. Data Collection software and analysed with the BioMark Real-Time PCR Analysis Software 4.1.3. (Fluidigm, USA).

2.7. Immunofluorescence and RNAi in early embryos C57Bl/6NCrl females at the age of 4–6weeks were stimulated for superovulation with injection of PMSG at 2:00 pm of day one. Next day, HCG was applied and males were added to the cages. Third day morning, females were screened for vaginal plugs and fertilized zygotes were isolated and incubated in M2medium (Sigma, M7167). Downregulation of desired proteins was mediated by ON-target smart pool siRNA from Dharmacon (Fam208a- L-047440-00-0005; GAPDHD-001830-20-05; Non Targeting pool- D-001810-10-05). RNA was dissolved in PCR Ultra H2O from Top-Bio (P340) for final concentration 5,8 μM. Electroporation (EP) was performed by NEPA21 electroporator (Nepagene) with impedance set to range 0.18–0.22Ω and transfer pulse set to 5 V for 50msec with 5 pulses and 5 zygotes per run. After EP, cells were incubated at 37 °C with 5% CO2 in M2media. 24, 48 & 72 h later, zygotes were fixed with 4% PFA for 45 min. Three sets of washes in PBS/FBS 5% were performed followed by permeabilization in 0.5% PBST for 60 min. Blocking step was performed with solution containing 5% NGS, 0.3 M glycine and 0.1% Triton X in PBS for at least 2 h. Primary antibody incubation took overnight at 4 °C with 1% NGS and β-tubulin antibody (Cell signalling, #2146) with dilution 1:50. Another set of washing took place next morning followed by incubation with secondary antibody donkey anti-Rabbit AlexaA488 (Invitrogen, A21206) with dilution 1:1000 for 90 min. Last set of washes followed by nuclear staining with DAPI and glycerol series with mounting in 90% glycerol with 5% NPG took place. Cells were visualized by Dragonfly spinning disc microscope with 40× objective.

3. Results 3.1. Full ablation of Fam208a leads to embryonic lethality at early somite stage Since previously published data were generated using mutant lines generated with ENU mutagenesis (MommeD6 and MommeD20) [9], where a random mutation does not necessarily result in a complete loss of function alleles, we prepared a full Fam208a knock out (KO) mouse line, where the whole critical exon 1 was deleted by CRISPR/Cas9 according to the IMPC standard [16]. Guide RNAs (G1 and G2) were designed to target an intronic regions before or after the exon 1 (Fig. 1A) resulting in the deletion of 866 nucleotides. Quantitative polymerase chain reaction (qPCR) verified decreased levels of Fam208a mRNA in homozygote E9.5 embryos (Fig. 1B) and slight increase of its expression in heterozygotes, which might be caused by compensatory mechanisms as mutated mRNA is decayed and cells drive higher expression from wt allele. In breeding using heterozygous mice, we

2.8. Yeast two hybrid system Screening for identification of Fam208a interaction partners divided this large protein into two overlapping parts N’ (3-741aa) and C’ (5361640aa) terminal part. To create cloning sites for Y2H vector (pGBKT7, Clontech, 630489), unique primers were designed (Suppl. Table 3) so the PCR product kept reading frame after subcloning into vector sequence. Restriction sites XmaI/SalI for N′ terminal and EcoRI/SalI for C′ terminal part were used. Standard protocol for Phusion PCR reaction (NEB, M0532S) was used. Template cDNA was prepared by M-MLV reverse transcription (Promega, M170A) with original protocol by 3

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Fig. 1. Downregulation of Fam208a in zygotes leads to embryonic lethality. A) Mutagenesis design shows location of genotyping primers (F’; R1′ and R2′) and guide RNAs (G1 and G2) with labeled PAM sequence, yellow. B) Quantitative analyses of mRNA levels shows a slight increase of transcription levels of Fam208a in heterozygous and reduction of RNA in homozygous mutants. C) Within five litters, no viable homozygote is observed. D) Bright-field microscopy shows morphological differences between the development of Fam208a +/+; Fam208a −/+ and Fam208a −/− embryos at embryonic day 9.5. E) As no mutants were born, we examined embryos at stages E8.5 and E9.5. In 43 dissected embryos (E8.5 + E9.5), 12 were full mutants with Fam208a −/− genotype, 13 embryos were wild types and 18 embryos were heterozygous for deletion in Fam208a. F) Graph representing the number of somites at embryonic stage E9.5 with milder differences observed between wild types and heterozygotes and rapid elimination of the number of somites in Fam208a −/− embryos. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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observed fully penetrant pre-weaning lethal phenotype, consistent with previously published data. Instead of viable homozygotes, five litters with a total number of 32 pups provided 23 heterozygotes and 9 wild type animals (Fig. 1C). In order to investigate the cause of this lethal phenotype, we analysed embryonic development of Fam208a KO mutants with the aim to identify the critical developmental period of Fam208a malfunction. We followed the IMPC embryonic lethal screen guidelines [17] and started at embryonic stage E12.5. As expected, no viable homozygous embryos were observed at this stage (Fig. 1C). The heterozygote littermates were of similar size and weight, and the somite number was the same as in the wild-type (wt) embryos (Suppl. Fig. S1). Next, we examined embryos at E8.5 and E9.5 stage but we did not observe any loss of Fam208a null embryos before the placentation as live null embryos harvested at E8.5 and E9.5 were comparable to the wild type littermates (Fig. 1D). Nevertheless, Fam208a null embryos went through gastrulation process (Fig. 1E); however, they reached the maximal number of 4 somites at E9.5 compared to the wild type embryos which averaged 28 somites. Interestingly, the delayed development was also observed in heterozygote embryos with reproducible difference in formation of up to 6 somites less than in wild type littermate embryos at E9.5 (Fig. 1F). In E8.5 and E9.5 embryos, we observed the same genotype distribution (similar number of wt and homozygotes and higher numbers of heterozygotes, wt:het:homo; 13:18:12). Based on these findings, we conclude that Fam208a ablation causes embryonic lethality with robust developmentally delayed phenotype observed at E8.5, progressing through E9.5 with full lethality by E12.5. Remarkably, at earlier stages of development, the dose dependent effect of mutated allele is visible in delayed developmental progress of heterozygous embryos; however, this effect is fully compensated for in later developmental stages and results in fully viable and fertile mice. There are obvious compensatory mechanisms which assist heterozygotes to proceed with the development. Based on provided clues and knowledge, it might be driven by increased expression (higher mRNA levels) or by alternative signalling pathways (Kap1, HUSH2).

3.3. Downregulation of Fam208a in zygotes leads to immediate cell division phenotype To further investigate the role of Fam208a in embryonic development preceding post-gastrulation and pre-placentation lethal period (E8.5-E12.5), we systematically focused on the earlier events in embryonic development, employing microarray profiling of whole zygotic transcriptome at several stages. To explore the effects of waves of zygotic transcription activation, fully grown GV oocytes, metaphase IIarrested eggs, 1-cell zygotes, 2-cell zygotes, 4-cell zygotes, morula, and blastocyst datasets were analysed and compared [18,19]. RNA levels of Fam208a showed clustering with genes with lower expression in the first three stages i.e. GV, MII oocytes, and 1-cell zygote. However, the expression increases after two-cell stage transcription activation and stays relatively high during subsequent stages, i.e. 4-cell stages, morula and blastocyst (Fig. 3A). Increased expression is represented by more frequent and higher peaks located in intronic regions (exons are represented by blue boxes in the gene scheme in the last row of Fig. 3A). Based on the transcriptomics data, we investigated the putative function of Fam208a in the first zygotic divisions. To avoid effects of maternally delivered mRNA (from heterozygous mothers, as homozygotes are not viable) into early zygotes, which can significantly diminish the null phenotype [20], we used siRNA to knockdown Fam208a in the zygote immediately after electroporation. We introduced RNAi by electroporation into 1-cell stage zygote of C57Bl/6NCrl and observed the ability to further develop. The downregulation of Fam208a in zygotes, which overcame the two-cell stage block, leads to the problem with proceeding through typical cell division and multipolar spindles were formed (Fig. 3B). Tri-polar spindle apparatus was formed most frequently (n = 9) in siRNA-knockdown Fam208a cells (Fig. 3C). These abnormal spindles were observed at all selected time points. The most dramatic differences were observed 48 and 72 h after siRNA delivery. All controls (siGAPDH, non-targeting siRNA and water) developed spindles without any observable disturbances (n = 11) (Fig. 3B). Our findings suggest that Fam208a is not limited to epigenetic regulation, but can also play a role also in other processes such as H3K9 methylation. Moreover, Fam208a may directly form a part of the spindle apparatus regulatory pathways with potentially novel spectrum of interacting proteins distinct from the epigenetic modifiers discovered before (HUSH complex). This is also supported by the finding that the methylation state in early zygote is stable, until massive de-methylation at 4-cell stage embryo occurs [21].

3.2. Fam208a mutation massively impacts protein expression profile in homozygous mice In order to reveal the impact of Fam208a depletion in molecular landscape, during early embryonic development of Fam208a null embryos, we performed unbiased differential proteomics by LC-MS analyses in E9.5 embryos. Proteomics data showed apparent divergence between homozygote and wild type embryos. Examination of samples disclosed the fact that while both, wild type and heterozygote, were almost identical, homozygote embryos exhibited completely altered protein expression profile (Fig. 2A). We identified over 4800 proteins, from which 800 showed highly significant differences (Fig. 2B). The most numerous groups with a common function were proteins with nucleic acid binding activity (170) followed by proteins involved in transcription regulations (165). The third group included proteins playing different roles in epigenetic processes (122), crucial for embryonal development (Fig. 2C). Proteins involved in cell cycle control and cell division processes were also highly affected (83 and 103 proteins, respectively). Regarding the expression of previously published interaction partners in HUSH complex [11,12], Pphln1 and Setdb1 showed only mild alteration of the expression in Fam208a null embryos; however, the level of Fam208a itself was decreased, and Mphosph8 (Mpp8) in homozygotes was just at the detection limit of the method (Suppl. Fig. S2). Neither of HUSH partners had severe detectable differences in the expression levels of heterozygote embryos (Fam208a+/-). This proteomic data suggests that there might be other complexes, which include Fam208a, that are influenced by the heterozygous expression or milder ablation of Fam208a protein as well. That would partially explain observed developmental retardation in heterozygotes and no changes in the levels of HUSH proteins in them.

3.4. Y2H screen revealed novel Fam208a interaction network in spindle assembly machinery In order to study Fam208a interactome, we used the Y2H system for unbiased identification of all the potential binding partners. The advantage of the Y2H system is the possibility to search for putative interacting partners, which were not identified by proteomic approaches in differentiated cell cultures. The already known and verified interaction partners of Fam208a from HUSH complex (MPP8, PPHLN1, and SETDB1) served as an internal control. However, none of those has been linked with establishment of spindle poles or direct DNA binding with actin and tubulin fibres of spindle apparatus. Because of its large size, we split Fam208a (1610aa) into two overlapping parts for the purpose of Y2H system. N′ terminal part covered first 740aa. C′ terminal part with 1100 amino acids was further studied by generation of four deletion constructs based on natural variants (alternative reading frame, exon skipping and partial deletions). All constructs were used to closely describe potential protein binding interaction domains (Fig. 4A). Our results confirmed possibility of this in silico prediction as there were no verified interaction partners for N′ terminal part (possible DNA/RNA binding part), while the Y2H screen identified 20 putative interaction partners for C’ terminal part (Ncbp1, Eml2, Hcfc2, S100a10, Inpp5a, Amn1, Parpbp, Psmd8, 5

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Fig. 2. Fam208a mutation massively impacts protein expression profile in homozygous mice. A) Heat map representing overall identified genes for LC-MS data obtained from E9.5 embryos. Wild types and a heterozygote show high similarity in the expression profile, while a homozygous embryo shows a different pattern. B) Detail visualization of affected proteins and distinct expression profiles identified in a homozygous Fam208a KO embryo. C) The affected proteins were grouped according to their ontologies and the chart represents their distribution amongst these groups; numbers in lines represent common ontologies for a protein.

Slc22a3, Capn7, Stk38, Pphln1, Alb, Etfa, Gpsm2, Itgb3bp, Mphosph8, Svil, Cntn1 and Tmem 100). Size and colour intensity of the yeast colonies were used as strength markers for different interaction partners (Fig. 4B). One of the strongest colour signal was observed in colonies with Ankyrin domain of Mphosph8 protein, which confirmed our approach as the direct interaction between Fam208a and Mphosph8 was already described [11]. Reciprocal verification mating ruled out four (Capn7, Stk38, Slc22a3

and Pphln1) of twenty identified constructs (Fig. 4B). As an example, Periphilin1 was firstly identified in 39 out of 65 diploid yeast colonies, all possible transcription variants were pulled, surprisingly none of them was ’in frame’. Nevertheless, our control mating did not confirm this protein as a direct binder to Fam208a. In the screening assay, Fam208a-Mphosph8 interaction was confirmed, as the Ankyrin repeat domain (known protein binding domain) was identified in Mphopsh8 sequences which were pulled by Y2H 6

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Fig. 3. Downregulation of Fam208a in zygotes leads to formation of multipolar spindles. A) RNA-seq results show increased expression of Fam208a after two-cell stage activation. The first line of expression profile in one-cell stage shows a low amount of identified RNA mainly obtained from maternal RNA. In the third line presenting expression after two-cell stage activation (four cells), we see increased levels of both introns and exons, which is evidence of transcription coming from embryonic RNA. In morula, the fourth line, the trend is still detectable, but obviously the highest expression peak for Fam208a is during the first rounds of zygotic division. B) Immunofluorescence staining of zygotes, which are either 42 h or 72 h after siRNA electroporation, shows increased incidence of formation of multipolar spindles in the absence of Fam208a (n = 9/11). Red arrows point to spindle poles of dividing cells. C) Detailed view of the spindle apparatus with a multipolar spindle in Fam208a downregulated cell at different planes (z) to observe all formed spindle centers (left panel) and two different planes of a normal spindle apparatus with two spindle poles in a control cell (right panel).. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 7

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Fig. 4. Y2H screen revealed novel Fam208a interacting network in spindle assembly machinery The yeast two-hybrid screen used several constructs as a bait to pull down interaction partners; a scheme of prepared constructs is shown with an outline of possible interaction domains with competitive binding partners. Constructs α, β and B lack cassette exon 17 (60 bp), the α construct had also an identified alternative reading frame caused by splicing differences, and the β variant had deletion in exon 22 that influences the reading frame in the rest of the protein. B) All identified preys are listed in a table with symbols to identify the observed strength of their interaction based on the size and colour of the yeast diploid colonies after mating. C) Proposed scheme with already identified function of proteins involved in the cell division process. Gpsm2, mitotic spindle pole organisation; Amn1, nuclear orientation checkpoints; Eml1, assembles and organizes microtubules and regulates orientation of the spindle apparatus; Itgb3bp, member of centromere-specific complex, recruits histone H3 to the centromere region; Svil, coordinates actin filaments and myosin II during cell spreading. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

system, and the region between amino acids 600–904 of Fam208a were responsible for their binding. In addition, novel putative interaction partners were identified from the different protein function groups such as calcium regulating proteins (S100a10, Inpp5a), proteins with DNA/ RNA binding (Ncbp1, Hcfc2, Parpbp) and proteins involved in regulation of the cell division (Eml1, Svil, Gpsm2, Itgb3bp, and Amn1) (Table 1). These proteins play an important role in sister chromatid segregation, spindle assembly, and cytokinesis (Fig. 4C). Gpsm2 (LGN) is a protein well known to regulate cell polarity and spindle organisation. RNA interference driven depletion of Gpsm2 in oocytes caused similar effects on spindle poles as in the case of Fam208a, which resulted misplacement and multiple, pole spindle apparatuses [22]. Supervillin (Svil), another key player in cell mitosis and cytokinesis, represents a molecular link between the central spindle and the contractile ring necessary for the ingression of the cleavage furrow. Its depletion in MCF10A cells led to de-organisation of spindles and impairment in final daughter cell separation [23]. Fam208a might be therefore involved in organising the formation of spindle poles and in case of its downregulation, multipolar spindles can occur. Proteomics data support this hypothesis, as there is a selective elimination of all putative interaction partners in KO embryos analysed by LC-MS. Svil, Eml1, Gpsm2, and Itgb3bp were all below the detection limits of the LCMS method in E9.5 homozygous embryos. Although, all these interaction partners exhibited stable and strong expression in wild-type and

Table 1 List of identified and verified putative interaction partners with their ontologies. Gene

Name

Ontology

Eml1

Echinoderm microtubule-associated protein-like Supervillin G-Protein Signalling Modulator 2 Integrin Subunit Beta 3 Binding Protein Antagonist Of Mitotic Exit Network 1 Homolog Contactin 1 Electron Transfer Flavoprotein Alpha Subunit Proteasome 26S Subunit, Non-ATPase 8 Inositol Polyphosphate-5-Phosphatase A Calpactin M-Phase Phosphoprotein 8 Periphillin Transmembrane protein 100 Albumin PARP1 Binding Protein Host Cell Factor C2 Nuclear Cap Binding Protein Subunit 1

cell division, spindle

Svil Gpsm2 Itgb3bp Amn1 Cntn1 Etfa Psmd8 Inpp5a S100a10 Mphosph8 Pphln1 Tmem100 Alb Parpbp Hcfc2 Ncbp1

cell division, adhesion cell division, spindle cell division, kinetochore cell division, spindle adhesion energy proteasome, degradation Ca regulation Ca binding HUSH HUSH differentiation carrier protein DNA/RNA mechanism DNA/RNA mechanism DNA/RNA mechanism

8

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heterozygote samples. Taken together, we found increased incidence of impaired spindle apparatuses following Fam208a downregulation and identified several presumed interaction partners linked to spindles pole establishment and functioning. These findings suggest that Fam208a plays an important role in spindle pole assembly during zygotic division and might be also a part of novel protein complex other than HUSH.

dependence on the tissue type and involved physiological process. Moreover, tissues with higher proliferation levels have higher expression of Fam208a and HUSH partners while gametogenic tissues are richer in partners important for spindle apparatus control. 3.6. Ablation of FAM208a in somatic cells did not impair the cell division processes

3.5. Fam208a has tissues specific subsets of interacting partners

To observe the possible effects of ablation of FAM208a in fully differentiated somatic cells, we used CRISPR/Cas9 to delete exon 4 (exon 10 in alternative splicing variant) in Hek293t cells. In order to study assumed overlapping roles of FAM208a and HUSH complex, we also prepared a MPHOSPH8 deletion mutant by introducing a mutation in exon 7, in which Ankyrin repeat region was identified and thus only the Fam208a function in HUSH complex was targeted. We established three lines for FAM208a (Fam-a1, Fam-a2 and Fam-a3), and two lines for mutated MPHOSPH8 (Mpp8-a; Mpp8-b) protein. To test whether FAM208a deletion in somatic cells also affects cell division, we performed Trypan Blue Viability measurement. Evaluated parameters included concentration of viable cells, their diameter, and total viability. We did not detect any significant differences between mutant variants of FAM208a and control samples (Fig. 6A). Conversely, MPHOPH8 KO lines showed diminished cell viability after 48 h. In addition, we noticed increased cellular diameters in the mutant lines Fam-a2, Fam-a3, and Mpp8-a. These data propose a marginal effect of FAM208a depletion on cell proliferation as well as on the functionality of HUSH complex. To further analyse FAM208a, we performed proteomic analysis of all lines using LC-MS approach, in which more than 4000 proteins were detected and used for differential proteomics. Original Hek293t cells (HekWT) and cells, which underwent the whole mutagenesis procedure but did not have edited genome (HekMT), were used as controls. LC-MS analyses verified absence of FAM208a in lines Fam-a1, Fam-a2, and Fam-a3 and MPHOSPH8 in lines Mpp8-a and Mpp8-b. (Fig. 6B). Evaluating the presence of other members of HUSH complex showed no difference in levels of PERIPHILIN1. On the other hand, SETDB1 could not be detected in any of our mutant lines (Fig. 6B). Approximately 25% of LC-MS detected proteins were differentially expressed among the mutated lines (Fig. 7A). To show the most significantly up- and down-regulated FAM208a-interacting proteins, we filtered out 127 of them using the most stringent analytical criteria. Selection filter was set up at several levels as follows: firstly, the protein had to be detected in either both control samples or in neither of them. Secondly, the levels of these proteins had to be beyond the detection limit in at least two out of three FAM208a KO lines or opposite. As a result, 104 proteins were detected in both controls, HekWT and HekMT, but they were not measured in at least two of FAM208a KO lines (leading to downregulated expression due to FAM208a depletion). Moreover, 23 proteins were upregulated only in mutant Hek293t lines (upregulated expression) (Fig. 7B). The same method was used for MPHOSPH8 cell lines. This gave us the final number of 67 downregulated and 16 upregulated proteins compared to non-mutated HekWT and HekMT (Fig. 7B). Based on the gene ontologies (Table 1) and identified functions, FAM208a-dependent proteins can be clustered into 6 groups: DNA/RNA binding group with 24 downregulated and 7 upregulated proteins, transcription regulation group with 22 downregulated and 2 upregulated proteins, cell cycle regulation group with 15 downregulated and 2 upregulated proteins, cell division-linked proteins with 9 downregulated and 3 upregulated proteins, and proteins connected with cellular apoptosis with 4 downregulated and 2 upregulated identified changes (Fig. 7C). The majority of proteins had more than just one role and can therefore be involved in multiple categories. Proteomics data from somatic cell lines showed largely effected protein levels, allowing us to assume the impact of depletion of FAM208a in transcription regulations, nucleic acid binding, epigenetic regulation without real impairment of cell viability (based on Trypan blue

The Y2H system provided an unbiased view on pleiotropy of putative binding partners of Fam208a. However, identification of biological processes where possible interactions play a role is a challenging question. In order to study interactions between Fam208a and its potential binding partners, we analysed the expression pattern of all identified interacting proteins in adult murine tissues. 20 murine organs were used for the preparation of RNA library and subsequently used for BIOMARK q-RT-PCR screen with 18 gene-specific primers (Suppl. Table 1), from which 16 were designed based on identified Y2H preys. Primers for Pphln1 and Setdb1 from HUSH complex were also included to the screen. Different tissues showed various expression levels of Fam208a as well as of all different binding partners. Fam208a is generally ubiquitously expressed at low levels in the majority of the organs and increased levels were detected in kidneys, spleen, thymus, seminal vesicles, uterus, and ovaries; however, its expression was almost six times higher in male tissues than in females (Fig. 5A). Three other HUSH proteins (Mphosph8, Pphln1, and Setdb1) also exhibited higher expression in kidneys, uterus, seminal vesicles, and testes. Besides that, their expression was higher in the brain and lungs. Genes involved in spindle apparatus assembly and function, and cell division regulation (Amn1, Eml1, Gpsm2, Itgb3bp, and Svil) are commonly highly expressed in seminal vesicles, lungs, duodenum, and brain. Amn1 and Itgb3bp had highest expression in testes. Stomach and lungs exhibited higher level of Eml1 whereas Gpsm2 appears to be predominantly expressed in proximal colon and ileum. Svil is strongly expressed in heart and tongue. Interestingly, Hcfc2 and Ncbp1 together with HUSH proteins, Psmd8 and Parpbp exhibited very high expression in testes (Fig. 5B). To investigate these findings further, we decided to study the expression of Fam208a and other preys at the single cell resolution. All previously examined tissues are composites of several cell types and so it is not possible to state whether expression levels are based on heterogeneity of tissue samples or protein partners. We selected three adult tissues with the highest expression of Fam208a (testes, ovaries, and brain) for in situ hybridisation [1]. Diversity of co-expressed interaction partners with Fam208a based on observed organ sample was obviously supporting contextual nature of Fam208a protein interactions also at the cell resolution level. A closer investigation of tissues from testes (Fig. 5B) showed possible co-localisation of Fam208a and several other partners. Fam208a itself was highly expressed in seminiferous tubules with strong a signal in Sertoli cells, spermatogonia, and spermatocytes. Eml1, Gpsm2, Psmd8, Inpp5a, Amn1, Cntn1 and Parpbp were also specifically expressed in Sertoli cells, which are located at the base of the epithelium and visually form ring-like staining around edge of the seminiferous tubules. Itgb3bp, Etfa, Hcfc2, Mphosph8, and Ncbp1 were more abundant towards the lumen and the signal was seen in spermatogonia and spermatocytes. Svil and Alb showed no expression in testes (Fig. 5B). In analysed ovaries, Fam208a had a strong signal in granulosa cells surrounding the oocyte itself. The majority of our probes exhibited this pattern within the ovary. Parpbp, Gpsm2, Etfa, and Cntn1 had the strongest ovarian signal (Fig. 5C). The expression profiling in the brain revealed that Fam208a is strongly expressed in granular cells within the cerebellum. Similar signal was detected with Svil, Alb, Gpsm2, Etfa, Parpbp, Mphosph8, and Ncbp1. To conclude, the general expression pattern of Fam208a and its interacting partners suggests the contextual role of interaction in 9

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Fig. 5. Fam208a is differentially co-expressed with putative partner proteins in adult murine tissues. A) Complex heat map based on qRT-PCR data showing expression patterns of 18 genes within 20 different murine tissues with dark blue representing relatively low expression and bright red representing relatively high expression of pre-selected genes. X axis is divided into columns representing genes and Y axis forms rows dedicated for murine tissues. ‘F’ states for female and ‘M’ for male samples. B) In situ hybridisation staining of paraffin sections of testes and C) ISH of ovaries, NC = negative control.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

staining). Thus, our data suggests that FAM208a in somatic cell lines is responsible for epigenetic silencing via complex together with MPHOSPH8. Although no clear viability phenotype of FAM208a KO cell lines points to compensatory mechanisms that overcomes the ablation

of the protein. In conclusion, FAM208a is involved in regulation of cleavage during early zygote development, although its removal in stable somatic lines does not impair the cell cycle nor cell division. It is also possible that both suggested roles, cleavage regulation and epigenetic silencing, are 10

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Fig. 6. Ablation of FAM208a and MPHOSPH8 in somatic cells does not impair the cell division processes. A) Relative fold ratio of expression levels of HUSH complex proteins in mutated Hek293t cell lines. The levels of FAM208a in Fam208a mutants and MPHOSPH8 in Mpp8 mutants are below the detection limit. There is almost no effect on PERIPHILIN levels (PPHLN1), but the levels of SETDB1 could not be traced down in any of affected cell lines. B) To study the effect of downregulation of FAM208a and MPHOSPH8 in somatic cell lines, analyses of live cell concentration, total viability, and cell size measurements were performed.

related and interlinked either via Fam208a itself or some other regulatory proteins.

activated during further development as E12.5 heterozygous embryos are indistinguishable from wild types. Even homozygous embryos contain subset of wild type mRNA from heterozygote female (maternal products) and the usage of wild type RNA in first zygotic events results in a delayed effect of Fam208a ablation. RNA-seq data from zygotes indeed show the presence of maternal Fam208a RNA from maternal to zygotic transition of embryonic transcription at 4-cell stage. There is also high-levels of Fam208a in Metaphase I stage oocytes, showing clear involvement of maternal-originating molecules deposition [24]. To overcome this problem, we downregulated maternal RNA with the pool of siRNA directly in fertilized eggs. This manipulation resulted in increased incidence of multiple spindle formation and higher risk of cell division arrest. Very similar effect was observed in murine oocytes where Gpsm2 was downregulated via siRNA of [22]. Thus, we conclude that Fam208a together with Gpsm2 cooperate together to properly establish spindle apparatus poles. None of the control groups developed spindle apparatus with multiple poles, however, zygotes with downregulated Fam208a had impaired poles in 9 out of 11 detected spindles. Based on this and on RNA-seq data, we suggest that Fam208a is critical for early zygotic division processes, spindle dynamics and

4. Discussion Our findings suggest a new putative role of Fam208a in organisation of the spindle apparatus and an indirect role in the maintenance of genome stability via interaction with MPHOSPH8 in HUSH complex. In fact, the heterochromatin instability can be tightly linked with spindle apparatus establishment and cell division. So it is possible that these function are related and create a complex orchestration strategy involving Fam208a. We created a new mouse model with complete loss of the critical first exon of Fam208a, which caused functional ablation of the protein. The homozygous stat of this mutation is embryonically lethal. Moreover, we observed the delayed dosage effect in E9.5 stage heterozygous embryos with 22 somites compared to the wild type littermates with 28 somites. The fact, heterozygotes are born fully developed and viable suggests that Fam208a role is crucial mostly in the very early stages of embryogenesis. Interestingly, compensatory mechanisms are 11

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Fig. 7. Ablation of FAM208a and MPHOSPH8 in somatic cells did not impair the cell division processes A) Hek293t cells were mutated with the CRISPR/Cas9 system targeting either the Fam208a or Mphosph8 gene. LC-MS was used to identify changes implicated by these mutations. Normal wt Hek293t cells (HekWT) were used as a control and standard sample; the HekMT line does not carry any mutations and was used as a control of the mutagenesis process, which could also have introduced changes; lines Fam-a1, Fam-a2 and Fam-a3 carry different deletions in FAM208a and lines Mpp8-a and Mpp8-b had knocked out MPHOSPH8 protein. B) Expression profiles of cell lines differ in approximately one fourth of the proteins identified by LC-MS. C) Ontology graph representing 127 affected proteins grouped according to their ontologies. Proteins might be involved in more categories simultaneously, and numbers above lines represent common proteins for the linked groups. The first number in brackets represents upregulated while the second one shows downregulated protein.

establishment of bi-polar apparatus. Our data show that Fam208a null mutants die between E9.5 and E12.5. This is remarkably later than in ENU mutagenesis induced L130P mutants (point mutation of Fam208a leading to amino acid substitution – MommeD6), which are fully absorbed by E9.5 [25]. There might be several reasons for a milder effect in the complete knock out. L130P

mutants have so far not been fully characterized and it has not been reported whether the Fam208a mutation fully eliminates the endogenous protein. Thus, the presence of remaining and maybe not fully active Fam208a might cause a dominant negative effect. In addition, murine zygote deficient for Fam208a may develop compensatory mechanisms (HUSH2, Kap1) and use pathways, which provide partial 12

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rescue for embryos. Moreover, the presence of maternal RNA leads to the production of Fam208a protein in oocytes and thus postpones the onset of effects caused by Fam208a zygotic mRNA decay. By this act, maternal Fam208a helps to overcome the first rounds of zygotic division [26]. Using the Y2H system, we identified and verified 16 putative Fam208a interaction partners. Five of these proteins (Gpsm2, Eml1, Svil, Amn1 and Itgb3bp) are directly linked with spindle apparatus establishment and correct functioning and might be more important for interaction with Fam208a during the early rounds of zygotic divisions. One of the identified proteins is Gpsm2, a protein member of cortical complex (consisting of NUMA and Dynactin/dynein) [27 2004] that plays a key role in establishing proper spindle orientation [28]. Another interesting protein is Supervillin which co-localises with endogenous myosin II and EPLIN in the cleavage furrow during early cytokinesis [29]. Eml1 is critical for correct formation of cleavage plane [30]. The function of Amn1 is linked with both, spindle assembly and nuclear orientation checkpoints [31]. Itgb3bp (CENPR) is a core centromere protein, which prevents pre-mature separation [32]. Considering the fact, that almost one third of identified interaction partners is involved directly in cell division mechanism, we propose that Fam208a is most likely involved in this process as well. To further investigate the interaction partners of Fam208a in tissues, we performed expression profiling in murine organ samples, showing variable expression pattern in different tissues. Therefore, it is possible that the cell specific role of Fam208a is governed by different interactions. To map possible interactions among Fam208a and its partners in specific tissues, we performed in situ hybridisation with RNA probes (Suppl. Table 1). The hybridisation revealed that co-localisation and co-expression of Fam208a and its putative interaction partners is strongly tissue- and cell type-specific. Therefore, in hyper-proliferative cells, Fam208a might play a different role compared to slowly proliferative tissues. To further describe a role of Fam208a in the spindle apparatus assembly, we used CRISPR-Cas9 to prepare stable somatic cell lines with mutations in Fam208a and Mphosph8, which was identified as one of the interaction partners using Y2H system. This interaction has previously been described and verified [11]. Fam208a, Mphosph8, Periphillin, and Setdb1 were designated as HUSH complex, whose function was linked with gene silencing. To investigate whether these partners are also involved in mitotic cell division, we prepared knock down cell lines. No viability defects were observed in Fam208a mutant cells indicating that in comparison with the described effect during early zygotic division, the described machinery in somatic cells might not be effected at all. Crucial difference between cleavage of zygotic cells and normal cell division is that there is no increase of cytoplasmic mass in dividing cells in comparison with an increase of nuclear mass and overall cell number [33]. Cleavage cycle is completely omitting G1 and G2 phases and it only consists of quick sets of S and M phases [34]. Due to these differences, various proteins are orchestrating this zygote specific division. While Dnmt3a (methyltransferase cooperating with Fam208a) that recruits HUSH complex to its active sites is not necessary during zygotic divisions [35], LGN (GPSM2) is important for nuclear positioning and cellular polarity establishment particularly during embryonic cleavage [33]. Thus Fam208a seems to be acting, beside the HUSH complex, during early zygotic division and cleavage. Data from expression profiling were supported by LC-MS proteomics, showing similar interaction variety. However, it appears that both experimental setups, embryonic and cellular, point to the involvement of Fam208a in DNA/RNA binding and transcription regulations as majority of effected proteins are functionally linked to these ontologies. While the analysis of Fam208a interactions in cell lines revealed that protein partners are acting more as cell cycle regulators, the analyses performed in embryos identified a protein group participating in epigenetics processes. Therefore, it seems that epigenetic machinery in cell lines is more stable even if Fam208a is downregulated, while in

embryos, Fam208a removal causes accumulation of errors. In summary, the analysis of the proteomics data suggests that Fam208a has a crucial role during zygotic cleavage. Its epigenetic regulation role becomes a key function once the methylation processes are needed, i.e. the epigenetic machinery in cell lines is not influenced by the ablation of Fam208a while Fam208a removal in embryos causes gastrulation arrest and primitive streak formation failure. Thus, the depletion of Fam208a does not seem to affect standard mitotic division. In case of impairment of HUSH complex (Fam208a KO) function, cells can operate through other mechanism, e.g. through Kap1 complex [36 2013]. However, when MPHOSPH8 is downregulated and thus excluded from HUSH and other methylation complexes, e.g. with Dnmt3a [37], cells are barely able to cope with this lost. On the contrary, ablation of MPHOSPH8 predominantly causes an increase in proliferation followed by lower viability and higher sensitivity to cell death. Altogether, we identified 16 putative Fam208a-interacting partners, which besides the HUSH complex, create a novel protein network (Eml1, Svil, Gpsm2, Amn1, and Itgb3bp), linking Fam208a to the maintenance of the genome stability via controlling the function of spindle apparatus. This new role of Fam208a within unique complex appears to affect processes of an early embryonic division when the HUSH function is paused. The epigenetic role of Fam208a seems to be more crucial during and after the differentiation process and biological functions of this dual acting protein could be not only for developmental process but they seem to be cell-type and tissue specific. Molecular mechanism is yet to be discovered, nevertheless, based on other putative interaction proteins, we conclude that Fam208a is a crucial protein involved in several mechanisms maintaining genome stability. 5. Conclusions In summary, we demonstrated that Fam208a exerts an important role in early embryonic development and it is involved in the organisation of spindle poles and spindle apparatus assembly. To perform this role, distinct interaction partners must be present besides those responsible for epigenetic silencing (HUSH complex). We identified 16 proteins as putative interaction partners and 5 of them are linked with cell division processes. This library was profiled and showed high tissue specificity. Thus, the roles of Fam208a may differ in a cell-specific manner, as it is likely dependent on availability of its interaction partners. Altogether, our work provides important insight in understanding the molecular landscape of Fam208a as a multi-interacting protein affecting genome stability, playing an essential role in the initial steps of embryonic development via orchestrating spindle pole assembly and chromosome segregation. Author's contributions VG performed the experiments and prepared the manuscript, VN was responsible for statistical analyses of high through output methods (LC-MS, BIOMARK), IJ prepared murine Fam208a KO strain, MP dissected mice and isolated embryos, SB assisted with data interpretation, JP and RS designed the experiments, proofread, and corrected the manuscript. All authors read and approved the final manuscript. Conflicts of interest The authors declare that they have no competing interests. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. 13

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Consent for publication

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The authors agree with publishing this manuscript. Ethics approval and consent to participate Mice were bred and housed in accordance with animal welfare rules in a pathogen-free facility. All procedures used in this study were in accordance to applicable local laws and in conformity with animal welfare regulations of the Czech Republic. All animal models and experiments of this study were ethically reviewed and approved by the Institute of Molecular Genetics approved performed experiments (c.j.115/2016). Funding The study was supported by RVO 68378050 by Academy of Sciences of the Czech Republic and by LM2015040 (Czech Centre for Phenogenomics), CZ.1.05/2.1.00/19.0395 (’Higher quality and capacity for transgenic models’), CZ.1.05/1.1.00/02.0109 (BIOCEV Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University), LQ1604 (National Sustainability Program II project BIOCEV-FAR) funded by the Ministry of Education, Youth and Sports and the European Regional Development Fund. Acknowledgements Acknowledgment to Karel Harant and Pavel Talacko from Laboratory of Mass Spectrometry, Biocev, Charles University, Faculty of Science, where proteomic and mass spectrometric analysis had been done. We also thank to Dr. Epp Trevor for his help with Y2H experimental design. We would like to acknowledge Dr. Peter Solc for discussion about oocyte staining and fluorescent labelling. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yexcr.2019.05.018. References [1] T. Masuda, M. Tomita, Y. Ishihama, Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis, J. Proteome Res. 7 (2) (2008) 731–740. [2] I. Iraqui, et al., Recovery of arrested replication forks by homologous recombination is error-prone, PLoS Genet. 8 (10) (2012) e1002976. [3] A.C. Peters, et al., Mammalian DNA mismatch repair protects cells from UVB-induced DNA damage by facilitating apoptosis and p53 activation, DNA Repair 2 (4) (2003) 427–435. [4] D.O. Ferguson, et al., The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations, Proc. Natl. Acad. Sci. U. S. A. 97 (12) (2000) 6630–6633. [5] I.A. Tchasovnikarova, et al., G.E.N.E. SILENCING, Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells, Science 348 (6242) (2015) 1481–1485. [6] Q. Li, H. Wen, S. Ao, Identification and cloning of the cDNA of a Rb-associated protein RAP140a, Sci. China C Life Sci. 43 (6) (2000) 637–647.

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