Y Chromosome Missing Protein, TBL1Y, May Play an Important Role

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Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation Anna Meyfour, Hassan Ansari, Sara Pahlavan, Shahab Mirshahvaladi, Mostafa RezaeiTavirani, Hamid Gourabi, Hossein Baharvand, and Ghasem Hosseini Salekdeh J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00391 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Journal of Proteome Research

Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation Anna Meyfour1,2, Hassan Ansari3, Sara Pahlavan3, Shahab Mirshahvaladi1, Mostafa Rezaei-Tavirani2,

Hamid

Gourabi4,

Hossein

Baharvand3,5*,

Ghasem

Hosseini

Salekdeh1,6*

1

Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

2

Proteomics Research Center, Department of Basic Science, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 4

Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

5

Department of Developmental Biology, University of Science and Culture, Tehran, Iran

6

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

*Corresponding authors: Ghasem Hosseini Salekdeh, Ph.D., Hossein Baharvand, Ph.D. Royan Institute, Banihashem Sq., Banihashem St., Ressalat highway, Tehran, Iran. Postal Code: 1665659911, P.O. Box: 16635-148, Tel: +98 21 22306485, Fax: +98 21 23562507. Emails: [email protected], [email protected]

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Abstract Despite evidence for sex-specific cardiovascular physiology and pathophysiology, the biological basis for this dimorphism remains to be explored. Apart from hormonal factors, gender related characteristics may reside in the function of sex chromosomes during cardiac development. In this study, we investigated the differential expression of the male-specific region of the Y chromosome (MSY) genes and their X counterparts during cardiac differentiation of human embryonic stem cells (hESC). We observed alterations in mRNA and protein levels of TBL1Y, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y1, DDX3Y, PRY, XKRY, BCORP1, RBMY, HSFY, and UTY which accompanied changes in intracellular localization. Of them, the abundance of a Y chromosome missing protein, TBL1Y, showed a significant increase during differentiation while the expression level of its X counterpart decreased. Consistently, reducing TBL1Y cellular level using siRNA approach influenced cardiac differentiation by reducing its efficacy as well as increasing the probability of impaired contractions. TBL1Y knockdown may have negatively impacted cardiogenesis by CtBP stabilization. Furthermore, we presented compelling experimental evidence to distinguish TBL1Y from TBL1X, its highly similar X chromosome homologue, and propose reclassification of TBL1Y as ‘found missing protein’ (PE1). Our results demonstrate that MSY proteins may play an important role in cardiac development.

Keywords: Chromosome-Centric Human Proteome Project (C-HPP), Y chromosome, Human embryonic stem cell, Cardiac differentiation, TBL1Y


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Introduction The Y chromosome is a haploid, male-specific chromosome that escapes meiotic recombination. The male-specific region of the Y chromosome (MSY) holds 95% of its length and does not show any X-Y crossover1. It is of particular interest due to the large number of “missing proteins”. The entire human proteome comprises 19,567 proteincoding genes at protein evidence levels 1-4 (PE1-4) from which 17008 (87%) have been validated at the protein level (PE1) according to the NeXtProt release (201701).This statistics is rather decreased to 63% for Y chromosome protein entries although it is one of the smallest in the human genome. According to NeXtProt, there are 48 predicted protein-coding genes on the Y chromosome from which 26 have been validated at the protein level (PE1); 22 do not have experimental protein evidence among which 10 have been validated at the transcript level (PE2); 5 are at the homology base (PE3); and 7 are at an uncertain level (PE5) (www.nextprot.org). One of the major challenges of the Y chromosome project is to explore the Y chromosome “missing proteins”. Y chromosome genes and their X homologues are evolutionary conserved genes. Despite the small number of Y chromosome genes, their adequate expression is required for regulation of transcription, translation, and protein stability of male individuals beyond sex-determination2. In addition to their roles in male infertility3-5, we previously reported that the Y chromosome genes, including DDX3Y, a member of the DEAD-box RNA helicase family, are associated to neural induction in human embryonal carcinoma NTERA-2 cell line6. Furthermore, KDM5D showed to exert number of functions related to different biological processes including RNA processing, protein 3 ACS Paragon Plus Environment


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synthesis, apoptosis, growth and proliferation in DU-145 cell line indicating its potential role in prostate cancer7. Therefore, Y chromosome has an inevitable role in the sexual dimorphism of healthy and disease phenotypes, in addition to its more studied sex determination responsibility. Numerous studies show the different prevalence and severity of diseases based on gender7-14. Many cardiovascular diseases reside in the category of sex-specific differences according to in vivo evidence15 and clinical trial analysis16-19. Gender-related transcriptome analyses in the healthy heart as well as non-ischemic cardiomyopathy and heart failure reported the differences in expression levels of sex chromosome genes15, 20-22. Despite great experimental and clinical evidence, the biological basis for such dimorphism that leads to functional differences remains to be further investigated. The cell-based models are appropriate tools which enable targeted gene manipulations and subsequent functional studies in the Human Proteome Project23,

24

. Human

embryonic stem cells (hESCs) provide an enriched source of unprecedented lowabundance proteins that particularly express during development of target tissues. Here, we initially studied Y chromosome genes expression during cardiogenic differentiation of hESCs on specific days of cardiac development. We identified the protein expression and intracellular localization of a number of MSY genes. Of them, the expression of a Y chromosome missing protein at PE2 level, transducin beta like 1 Ylinked (TBL1Y), increased during differentiation while its X counterpart showed the opposite expression pattern. The function of TBL1Y in cardiac development was further studied by knocking down its expression using siRNA. Our results suggest a possible


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sex-dependent cardiac developmental regulation that might underlie sexual dimorphism of cardiac diseases beyond a hormonal basis. Materials and Methods Expansion and cardiogenic differentiation of hESCs in dynamic suspension culture Two hESC lines (female Royan H5 [RH5] and male Royan H6[RH6]) were expanded in static suspension culture. After one passage, we transferred the cells to 125-mL spinner flasks (Cellspin; Integra Biosciences AG, Switzerland) with a 100-mL working volume at a 40 rpm agitation rate for large-scale expansion as previously described25. In brief, to initiate the dynamic culture, 2-3x105 single cells/mL were transferred to 100 mL of hESC medium that was conditioned on human foreskin fibroblasts and contained freshly added 100 ng/mL bFGF and 10 mM rho-associated protein kinase inhibitor (ROCKi; Sigma-Aldrich, Germany). The seeded spinner flasks were incubated at 37°C and 5% CO2. We refreshed the medium after 48 h of culture. Induction of cardiomyocyte differentiation from hESCs in the dynamic suspension culture was performed by treating 5-day-old RH6 hESC size-controlled aggregates (average size: 175±25 µm) for 24 h in differentiation medium (Gibco, Germany) supplemented with 2% B27 minus vitamin A (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% non-essential amino acids (Gibco), 0.1% polyvinyl alcohol (PVA; Sigma-Aldrich), 10 mM ROCKi, and 12 mM of the small molecule (SM) CHIR99021 (CHIR; Stemgent, USA). After 24 h, the aggregates were washed with 10 mL of Dulbecco’s phosphate buffered saline (DPBS) and then maintained in fresh differentiation medium without SM for 1 day. At day 2, the medium was exchanged for new differentiation medium that contained 5 5 ACS Paragon Plus Environment


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mM IWP2 (Tocris Bioscience, UK), 5 mM SB431542 (Sigma-Aldrich), and 5 mM purmorphamine (Pur; Stemgent) supplemented with B27 minus insulin (Gibco). The aggregates were cultured for 2 days in this medium. After washing the cardiac-induced aggregates, we added fresh differentiation medium (100 mL) to the culture, which was totally refreshed every 2–3 days until the end of the differentiation process (day 12). Samples were collected at different time points (days 0, 1, 3, 6, and 12) of differentiation from three biological replicates for further analysis. RNA isolation and quantitative RT-PCR (qRT-PCR) RNA isolation was carried out using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. We removed any potential DNA contamination by treating the extracted RNA with RNase-free DNase (Takara, Japan). The resultant RNA was reverse-transcribed into cDNA and then diluted to 25 ng/µL for quantitative real-time PCR (qRT-PCR) in the Rotor Gene 6000 (Corbett, Australia). The calculation was performed using REST analysis software (QIAGEN, Germany). GAPDH was the housekeeping gene. Due to the high similarity in sequences of the Y chromosome genes and their X homologues, particularly their variants, the primer design appeared challenging. Therefore, we designed highly specific primers using Vector NTI software (Life Sciences, USA) as previously described3. The detailed information about the primers (target genes and transcript variants) and PE levels of MSY genes have been presented in Supplementary Table 1 and 2. Immunostaining For immunohistofluorescent analysis, samples were washed with PBS, fixed with 4% (w/v) PFA at 4ºC overnight, and prepared for paraffin-embedded tissue blocks. Paraffin-


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embedded spheroids were cut into 6-µm sections using a microtome (MicromHM325; Thermo Scientific, Germany) and kept at room temperature until use. For staining, we dewaxed and hydrated the spheroid section slides, followed by heat-mediated antigen retrieval using a Dako target retrieval solution (Glostrup, Denmark). Sections were washed with washing buffer (PBS/0.1% Tween 20), permeabilized with 0.5% Triton X100 in PBS for 15 min, and blocked with 5% (v/v) bovine serum albumin for 1 h. Primary antibodies diluted in blocking buffer (1:100) were added followed by an overnight incubation at 4°C. Secondary antibodies diluted in blocking buffer (1:500) were used for 1 h at room temperature. The sections were incubated with DAPI for nucleic acid staining and imaged with a fluorescent microscope (IX71, Olympus, Japan). For immunocytofluorescent analysis, the seeded cells were washed once with PBS and fixed with 4% (w/v) PFA at room temperature for 15 min. Permeabilization, blocking steps, and incubation with primary and secondary antibodies were performed as previously described26. In order to provide high quality and quantity of antibodies for the Y-HPP project, we generated homemade antibodies and evaluated their specificity in female hESC line (RH5). The production and validation steps were performed as described by Rastegar et al3. Supplementary Table 3 lists the antibodies used in this study. Western blot analysis Protein extractions were performed using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. Protein quantification was carried out by Bradford assay. Proteins (40 µg) were separated on 8%-12% SDS-polyacrylamide gel (SDS-PAGE) based on the molecular weight of target proteins and transferred to PVDF membranes



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(Bio-Rad, USA). The blots were blocked with TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) that contained 5% BSA. The membranes were incubated with individual antibodies overnight at 4°C. Subsequently, the membranes were washed three times with TBST for 15 min and then incubated with peroxidase-labeled secondary antibodies for 1 h at room temperature. After three washes in TBST, the blots were incubated with chemiluminescent peroxidase substrate (Sigma-Aldrich, Germany) in a dark room and exposed to X-ray films (GE healthcare, UK). Supplementary Table 3 lists the antibodies. siRNA (siTBL1Y) design and knockdown procedure We used ON-TARGET plus SMART pool siRNAs targeting human TBL1Y sequences that

contained

a

mixture

GGCACGACGUCCCAAGUAA;

of

four

siRNAs:

AUAUGAUGGUUUCGCAAGA;

CCUGAUAGUUGCUGUGAUU;

and

AGGCAUCAGCAAUGGCAAA. Pooled siRNAs and scramble siRNA duplexes were synthesized, desalted, and purified by Thermo Fisher Scientific. At 36 h after differentiation initiation, 4 × 105 cells were plated in each well of a six-well tissue culture plate at 80% confluency. Each well contained antibiotic-free RPMI supplemented with B27 minus vitamin A and 1% BSA. A total of 50 nM siRNA from pooled TBL1Y siRNAs and scramble siRNA (siCtrl), 5 µL of lipofectamin-3000 reagent (Invitrogen), and 100 µL of RPMI were preincubated for 20 min, then mixed with 900 µL of RPMI to prepare a transfection mixture. At 12 h after transfection, the medium was replaced with fresh 2 mL of RPMI that contained B27 minus insulin and 5 mM IWP2/SB431542/Pur. After 48 h, the siTBL1Y and siCtrl treated cells from three biological replicates were collected for


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cell cycle and molecular analyses. The efficiency of siRNA transfection was evaluated using FITC-conjugated siRNA (Invitrogen). Fluorescence activated video microscopy siRNA treated cells were kept in culture until the first beating. Contraction analysis were performed using fluorescence activated video microscopy. We loaded cells with Fura-2 AM (1 µM) and video imaged them using a fluorescent microscope (Olympus, IX71). The movies were analyzed in a custom-made Matlab macro. Beating frequencies and contraction durations were statistically analyzed. All measurements were performed at 37°C. Cell cycle analysis siRNA treated cells were detached and fixed with 500 µL of 70% ethanol overnight at −20°C. Cells were washed twice with PBS and stained with PI (50 µg/mL PI and 100 µg/mL RNase A in PBS) for 30 min at 37°C. Cell cycle analysis was performed on a BD FACSCalibur flow cytometer and the Cell Quest program (Becton-Dickinson, USA). Statistical analysis Data are presented as mean ± S.E.M from three biological replicates. Statistical significance was tested by using a two-tailed unpaired student’s t-test in Graphpad Prism software (Graphpad Software, USA). p

Y Chromosome Missing Protein, TBL1Y, May Play an Important Role

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