DDX3Y, a Male-Specific Region of Y Chromosome Gene, May

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DDX3Y, a Male Specific Region of Y Chromosome Gene, may modulate neuronal differentiation Haghighat Vakilian, Mehdi Mirzaei, Mehdi Sharifi Tabar, Paria Pooyan, Lida Habibi Rezaee, Lindsay Parker, Paul A. Haynes, Hamid Gourabi, Hossein Baharvand, and Ghasem Hosseini Salekdeh J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00512 • Publication Date (Web): 05 Jul 2015 Downloaded from http://pubs.acs.org on July 8, 2015

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Journal of Proteome Research

DDX3Y, a Male Specific Region of Y Chromosome Gene, may modulate neuronal differentiation Haghighat Vakilian1, Mehdi Mirzaei2, Mehdi Sharifi Tabar1, Paria Pooyan1, Lida Habibi Rezaee1, Lindsay Parker2, Paul A. Haynes2 , Hamid Gourabi3, Hossein Baharvand4,5 and Ghasem Hosseini Salekdeh1,6* 1.

Department of Molecular Systems Biology at Cell Science Research Center, Royan

Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 2.

Department of Chemistry and Biomolecular sciences, Macquarie University, Sydney,

NSW, 2109, Australia 3.

Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute

for Reproductive Biomedicine, ACECR, Tehran, Iran 4.

Department of Stem Cells and Developmental Biology at Cell Science Research Center,

Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 5.

Department of Developmental Biology, University of Science and Culture, ACECR,

Tehran, Iran 6.

Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran,

Karaj, Iran

*Corresponding authors: Ghasem Hosseini Salekdeh, Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Tel: +98 21 22306485, Fax: +98 21 23562507, Email: [email protected]

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Abstract Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells at three different developmental stages using qRT-PCR, western blotting and immunofluorescent. The expression level of 12 Y-linked genes significantly increased over neural differentiation. Including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Labelfree quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared to mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing and apoptosis, among other biological functions. Our results suggest that MSY genes may play important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development in probably a sexually dimorphic manner. Key Words: Chromosome Centric Human Proteome Project (C-HPP), Cell-based Human Proteome Project, Y-linked genes, DDX3Y, Shotgun Proteomics, Apoptosis, Cell Cycle, RNA metabolism. 2


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1. Introduction The male-specific region of the Y chromosome (MSY) includes 60 genes which are responsible for regulating critical biological roles such as transcription, RNA metabolism, nucleosome assembly, translation, cell differentiation, cell adhesion, cell proliferation, metabolic processes, tissue development, sex differentiation, spermatogenesis and sex determination1,2. In sexually dimorphic non-gonadal tissues, such as the brain, the function of the human Y chromosome has been debatable but must influence neuronal development through a combination of regulating gonadal secretions that produce sex-specific effects on the brain and acting to differentiate XX and XY neurons1,

3-7

. As the severity and onset of some psychiatric disorders such as

schizophrenia8, 9 and other neurally mediated diseases such as autism or Parkinson’s are more prevalent in men9, 10, 10-12 , the human Y chromosome could potentially contribute to a variety of biological mechanisms leading to such dimorphic pathophysiological outcomes. In fact, in the “four core genotypes” mouse model, which separates the genetic sex of the brain (XY versus XX) from the gonadal phenotype (testis versus ovaries), the XY mouse develops more tyrosine hydroxylase enzyme (TH) compared to XX line13 indicating evidence of a sexually dimorphic expression of dopaminergic brain neurons correlating with Y-linked gene expression in normal versus Parkinson’s brains9, 14. In 2009, Reinius and Jazin reported that 10 MSY genes were highly expressed in prenatal human brains15 suggesting that these may constitute ideal targets for studying the influence of the human Y chromosome on dimorphic neuronal development. Recently, several findings have linked DDX3X also known as DDX3 gene, a DEAD box RNA helicase enzyme, to DNA damage, cell cycle regulation, apoptosis, Wnt-β-catenin signaling, tumorigenesis, and viral infection16, 17, 18, 19

. DDX3X is ubiquitously expressed in various tissues while expression of DDX3Y is 3



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restricted to male germ cells20, spermatogenesis22,

23

21

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where it influences germ cell development and

. DDX3X is particularly interesting as it functions as a regulator of the

Wnt/B-catenin network, which is required to rescue midbrain dopaminergic progenitors and promote repair of neurons19. Thus, given its gender specific expression, it may contribute to dimorphic male susceptibility to Parkinson’s or other neutrally mediated diseases. Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of MSY gene expression during neural development. Unfortunately, there are also limited functional protein descriptions of MSY proteins based on proteomic profiling as the sensitivity of currently available techniques is a major challenge in detecting low abundance Y-linked protein expression in target tissues such as testis. Therefore, alternative human cell-based approaches can be used to provide valuable data that contribute to the HPP24. We thus hypothesized that in the NTERA-2 human embryonal carcinoma cell line (NT2), studying MSY genes and proteins during the differentiation towards mature neurons would be an interesting model to study possible functions of the human Y chromosome throughout neurogenesis. Additionally, knowing that the MSY paralogue gene DDX3Y seems to function in a gender specific manner, we knocked down DDX3Y expression in neural progenitor cells using the siRNA approach to provide the first functional description for the MSY gene DDX3Y in neurogenesis. We examined the expression of a selection of MSY and their X-linked homologue genes across multiple time points throughout neuronal differentiation by quantitative PCR. We then characterized

the

localization

of

some

of

these

MSY

protein

products

using

immunofluorescence. The MSY gene expression profiling data collected in this study indicates 4


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that male specific genes that are known to be expressed in testis appear to be expressed in cells with a neuronal phenotype as well. We also provide the first proteomic description of cells with a neuronal phenotype after DDX3Y downregulation, and have demonstrated that it disturbs neural progenitor cell growth, viability and proliferation, while also increasing cellular apoptosis.

2. Materials and methods 2.1. NT2 cell culture and neural differentiation The human NTERA2 clone D1 EC stem cells (NT2) were grown and maintained in DMEM medium (Gibco; 21331-020) supplemented with 10% fetal bovine serum (FBS; 16141_079; Gibco), 2 mmol.L-1 L-glutamine (Gibco; 25030-024), 0.1 mM β-mercaptoethanol (Sigma; M7522) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco; 15070-063) under an atmosphere of 5% CO2 at 37°C. Generation of neural tubes was performed using the differentiation protocol in free floating aggregates in stirred suspension bioreactor, according to Abbasalizadeh et al.25. Briefly, 5×106 NT2 cells were cultured in 50 ml medium in spinner flask (Cellspin; Integra Biosciences) and agitation rate was 40 rpm. Retinoic Acid (RA) (10 µM) treatment started from day 2 and medium was changed every other day. After 6-7 days, formed spheres (neural tubes) were transferred and seeded into T75 cell culture flasks (Falcon, Franklin Lakes, N.J., USA) and subsequently cultured for another 6 days. The neural-like tube structures were dissociated into single cells by 0.05% trypsin (27250_018;Gibco) and 2 mM disodium EDTA (108454; Merck, Darmstadt, Germany), then re-plated on laminin (5 mg/mL; L2020; Sigma) and poly-L ornithine (15 mg/mL; P4957; Sigma)-coated tissue culture dishes in medium without RA for up to 24 days. 2.2. Specific primer design for neural markers and X and Y homologue genes 5



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The chromosome Y and X homologues are extremely similar thus to distinguish between them we designed highly efficient and specific primers using Gene Runner software (Supplementary Table 1). Basic Local Alignment Search Tool (BLAST) was applied to check whether each primer pair was specific for a particular gene. Gel electrophoresis and melt curve analysis confirmed the accuracy of the amplicon size and primer efficiency, respectively. 2.3. Immunofluorescent staining of the cells Immunofluorescence was conducted according to manufacturer (abcam) protocol. First, NT2, neural progenitor and mature cells were fixed with 5% paraformaldehyde (Sigma-Aldrich, P6148) for 1 min, membranes were permeabilized by 0.3% Triton X-100 (Sigma-Aldrich, T8532) and then blocked with 10% host serum in 1% bovine serum albumin (Sigma-Aldrich, A3311). The cells were placed 12-16 h at 4oC with the primary antibodies (Supplementary Table S2), which were diluted in goat serum blocking solution (1:250; Sigma-Aldrich -Aldrich, G9023). Washing was performed three times with 0.1% Tween 20 (Sigma-Aldrich -Aldrich, P7949) in PBS, and cells were incubated at 37oC with the following secondary antibodies; Goat anti-Rabbit dylight594 (1:500; Abcam, ab98507), Goat anti-Rabbit FITC (1:200; Sigma-Aldrich -Aldrich, F1262) and Goat anti-Mouse FITC (1:200; Sigma-Aldrich -Aldrich, F0257) for 45 min. Nuclei were counterstained with DAPI (1:1000; Sigma-Aldrich -Aldrich, D8417) and analyzed with a fluorescence microscope (Olympus, IX71). 2.4. RNA Isolation and quantitative RT-PCR RNA isolation was carried out using Trizol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. To remove any potential DNA contamination, the extracted RNA was treated with RNase-free DNase (Takara, Japan). A quantity of 3 µg of total RNA was reverse 6


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transcribed into cDNA and then diluted up to 25 ng/µl to be used in qPCR. All experiments were repeated three times in ABI system. The calculation was performed using REST analysis, taking primer efficiencies into account. Calculations were normalized using GAPDH as a housekeeping gene. To find the transcripts that up- and down-regulated between conditions, t-tests were performed. The transcripts with t-test p-value less than 0.05 were considered to be differentially expressed. 2.5. siRNA (siDDX3Y) design and knockdown procedure We used ON-TARGETplus SMART pool siRNAs targeting human DDX3Y sequences that contained

a

mixture

of

four

siRNAs

AGACUUAGAUAAACGGUCA;

including:

GAGCAAGUACAGCGAGCAA;

CUGGAUAGGCACUUGGGAA;

and

CUACAGGCCUGGUUGGAUU. Pooled siRNAs as well as nonspecific control siRNA duplexes were synthesized, desalted, and purified by Thermo Fisher Scientific. 2 × 105 neural precursor cells were plated in each of six-well tissue culture plates at 80% confluency. Each well contained antibiotic free normal medium supplemented with 5% FBS. To prepare a transfection mixture, 50 nm siRNA from pooled siRNAs and nonspecific control siRNA (Mock), 4 µl of Lipofectamin-2000 reagent (Invitrogen), and 150 µl of Opti-MEM (Invitrogen) were preincubated for 20 min and then were mixed with 850 µl of DMEMF12 culture medium. Six hours after the transfection, the medium was replaced with fresh 1000 µl DMEM/F12 medium containing 10% FBS. After 24 h, pooled siRNA and mock treated cells from three biological replicates were collected for RT-PCR. The RT-PCR analysis of DDX3Y and some neural markers were performed as described above.

2.6. Western blot analysis 7



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Western blot analysis was conducted using protein samples prepared from three independent replications of neural precursor cells twenty-four hours after the transfection with pooled siRNAs and nonspecific control siRNA (Mock).

Briefly, the samples were run in a 12% SDS-

polyacrylamide gel, then proteins were blotted onto a PVDF membrane (Bio-Rad) by a wet transblotting apparatus following manufacturer instructions (Bio-Rad). Membranes were blocked for 1.5 h using western blocker solution (Sigma-Aldrich -Aldrich W0138) and incubated overnight at 4 °C with the respective primary antibodies, monoclonal anti-DDX3Y (1:1000; Sigma-Aldrich -Aldrich, WH0008653M1) and anti-β Tubulin produced in mouse (1:10000; Sigma-Aldrich -Aldrich, T8328). Membranes were incubated with the peroxidase-conjugated secondary antibodies, rabbit anti-mouse IgG (1:100,000, Sigma-Aldrich -Aldrich, A9044) as appropriate for 1 h at room temperature. Antibody binding was visualized by the ECL® system (Sigma, CPS-1-120) and the film was scanned with a densitometer (GS-800, Bio-Rad).

2.7. Apoptosis and cell cycle assay The cell cycle distribution was analyzed by flow cytometry. A density of 2×105 siRNA treated and untreated (mock) cells per well were seeded into a 6 well plate and incubated for 24 h. Then cells were harvested and washed twice with cold PBS. In the next step, cells were fixed by adding two volumes of cold, absolute ethanol at -4oC overnight. After fixation, cells were plated and re-suspended in staining solution (50 µg/ml propidium iodide, 100 µg/ml RNase A in PBS). Prior to analysis, samples were stored at 4°C in the darkness for at least 1 h. Cell cycle analysis was performed on BD FACSCalibure flow cytometer and Cell Quest program (BectonDickinson, San Jose, CA). All analyses were performed using three independent replications of

8


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neural precursor cells twenty-four hours after the transfection with pooled siRNAs and nonspecific control siRNA (Mock). Student t-tests were used to compare two groups.

2.8. Protein separation by SDS-PAGE and trypsin in-gel digestion Extracted proteins form each sample (150 µg) was fractionated on a 10% Bis-tris polyacrylamide gels. After staining and washing, each lane was cut into 16 equal slices from top to bottom and each piece was further sliced into smaller pieces and placed into the well of a 96-well plate. Gel pieces were washed briefly with 100 mM NH4HCO3 and with 200 µL of 50% ACN/100 mM of 50% NH4HCO3, twice for 10 min before dehydration with 100% ACN. In the next step, samples were air dried, reduced with 50 µL of 10 mM DTT/50 mM NH4HCO3 for 1 h at 37˚C and alkylated with 50 µL of 50 mM iodoacetamide/50 mM NH4HCO3 for 1 h at room temperature in the darkness. Samples were subsequently digested with 20 µL of trypsin (12.5 ng/mL of 50 mM NH4HCO3) for 30 min on ice and then were incubated overnight at 37˚C. Resultant peptides were extracted twice with 30 µL of 50% ACN/2% formic acid, dried, vacuum centrifuged and reconstituted to 10 µL with 2% formic acid. 2.9. Nanoflow liquid chromatography - tandem mass spectrometry Peptides were analyzed by nanoLC-MS/MS using an LTQ-XL ion-trap MS (Thermo, San Jose, CA). Reversed-phase chromatography columns were packed to approximately 7 cm in 100 µm I.D. tubing using 100 Å, 5 µM Zorbax C18 resin (Agilent Technologies, Santa Clara, CA) in a fused silica capillary with an integrated electrospray tip. An electrospray voltage of 1.8 kV was supplied via a liquid junction upstream of the C18 column. To inject samples onto the C18 column, a Surveyor autosampler (Thermo, San Jose, CA) was used. Each sample was loaded 9



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onto the C18 column followed by an initial wash with buffer A [5% (v/v) ACN, 0.1% (v/v) formic acid] for 10 min at 1 µL/min. Peptides were subsequently eluted with 0-50% buffer B [95% (v/v) ACN, 0.1% (v/v) formic acid] over 58 min at 500 nL/min followed by 50%-95% buffer B over 5 min at 500 nL/min and then directed into the nanospray ionization source of the mass spectrometer and scanned in the spectral range of 400 - 1500 amu. Tandem MS of the top six most intense precursor ions at 35% normalization collision energy was performed using Xcalibur software (version 2.06, Thermo, San Jose, CA.). Normalized Spectral Abundance Factor (NSAF) values were calculated for each reproducibly identified protein as previously described26. Proteins that were present in all three biological replicates with a summed spectral count of at least 6, were considered to be reproducibly identified and were included in the final dataset.To compensate for null values and allow log transformation of the NSAF data prior to statistical analysis, a spectral fraction of 0.5 was added to the total spectral counts for each protein in the data set. Statistical analysis was performed on log-transformed NSAF data using the Statistics Software Package (Student’s t-test) and P

DDX3Y, a Male-Specific Region of Y Chromosome Gene, May

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