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

Article pubs.acs.org/jpr

Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation Anna Meyfour,†,⊥ Hassan Ansari,‡ Sara Pahlavan,‡ Shahab Mirshahvaladi,† Mostafa Rezaei-Tavirani,⊥ Hamid Gourabi,§ Hossein Baharvand,*,‡,¶ and Ghasem Hosseini Salekdeh*,†,# †

Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, and §Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, 16656-59911 Tehran, Iran ⊥ Proteomics Research Center, Department of Basic Science, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, 19839-63113 Tehran, Iran ¶ Department of Developmental Biology, University of Science and Culture, 13145-871 Tehran, Iran # Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran ‡

S Supporting Information *

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 proposed reclassification of TBL1Y as “found missing protein” (PE1). Our results demonstrated 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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(PE2); five are at the homology base (PE3); and seven 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-determination.2 In addition to their roles in male infertility,3−5 we previously reported that the Y chromosome genes, including DDX3Y, a member of the

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 crossover.1 It is of particular interest due to the large number of “missing proteins”. The entire human proteome comprises 19 567 protein-coding genes at protein evidence levels 1−4 (PE1−4) from which 17 008 (87%) have been validated at the protein level (PE1) according to the NeXtProt release (2017−01). 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 © 2017 American Chemical Society

Special Issue: Chromosome-Centric Human Proteome Project 2017 Received: June 6, 2017 Published: August 30, 2017 4391

DOI: 10.1021/acs.jproteome.7b00391 J. Proteome Res. 2017, 16, 4391−4402

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Journal of Proteome Research

(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 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 the cardiac-induced aggregates were washed, 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.

DEAD-box RNA helicase family, are associated with neural induction in human embryonal carcinoma NTERA-2 cell line.6 Furthermore, KDM5D showed to exert number of functions related to different biological processes including RNA processing, protein synthesis, apoptosis, growth, and proliferation in DU-145 cell line indicating its potential role in prostate cancer.7 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 gender.7−14 Many cardiovascular diseases reside in the category of sexspecific differences according to in vivo evidence15 and clinical trial analysis.16−19 Gender-related transcriptome analyses in the healthy heart as well as nonischemic cardiomyopathy and heart failure reported the differences in expression levels of sex chromosome genes.15,20−22 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 Project.23,24 Human embryonic stem cells (hESCs) provide an enriched source of unprecedented low-abundance 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 Y-linked (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 sex-dependent cardiac developmental regulation that might underlie sexual dimorphism of cardiac diseases beyond a hormonal basis.

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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. Because of 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 described.3 The detailed information about the primers (target genes and transcript variants) and PE levels of MSY genes has been presented in Supplementary Tables 1 and 2. Immunostaining

For immunohistofluorescent analysis, samples were washed with PBS, fixed with 4% (w/v) PFA at 4 °C overnight, and prepared for paraffin-embedded tissue blocks. Paraffinembedded 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 heatmediated 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 X-100 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 described.26 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 al.3 Supplementary Table 3 lists the antibodies used in this study.

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 described.25 In brief, to initiate the dynamic culture, 2−3 × 105 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% nonessential amino acids (Gibco), 0.1% polyvinyl alcohol (PVA; SigmaAldrich), 10 mM ROCKi, and 12 mM of the small molecule 4392


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Figure 1. Characterization of differentiating human embryonic stem cells (hESCs) during cardiogenesis. (A) Schematic representation of the cardiac differentiation protocol in a dynamic suspension culture. hESCs were induced to mesendoderm and cardiac mesoderm using a cocktail of the small molecules (SMs) CHIR, SB431542, Pur, and IWP2. The cells subsequently differentiated to cardiac progenitor cells (CPCs) and cardiomyocytes. Samples were collected at mesendoderm (D1), cardiac mesoderm (D3), CPC (D6), and cardiomyocyte (D12) stages for further analysis. (B) Microscopic image of cardiospheres at day 7 when the first beating was observed. (C, D) Gene expression pattern of pluripotency, cardiac mesoderm, cardiac progenitor, and cardiac specific markers at various days of differentiation by quantitative real-time PCR (qRT-PCR) and immunostaining analyses; scale bar, 50 μm. Nuclear staining was performed using DAPI. Data are expressed as mean ± SEM and represent three biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Western Blot Analysis

ies 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.

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% SDSpolyacrylamide gel (SDS-PAGE) based on the molecular weight of target proteins and transferred to PVDF membranes (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 antibod-

siRNA (siTBL1Y) Design and Knockdown Procedure

We used ON-TARGET plus SMART pool siRNAs targeting human TBL1Y sequences that contained a mixture of four siRNAs: AUAUGAUGGUUUCGCAAGA; GGCACGACGUCCCAAGUAA; CCUGAUAGUUGCUGUGAUU; and AGGCAUCAGCAAUGGCAAA. Pooled siRNAs and scramble siRNA duplexes were synthesized, desalted, and purified by Thermo Fisher Scientific. At 36 h after differentiation initiation, 4393


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Journal of Proteome Research 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 cell cycle and molecular analyses. The efficiency of siRNA transfection was evaluated using FITC-conjugated siRNA (Invitrogen).

the gene expression results for developmental and cardiogenic markers (Figure 1D). MSY Genes Showed Remarkably Different Expression Pattern Compared to Their X Counterparts during Cardiogenesis

We analyzed the expression pattern of 43 MSY genes and their 15 X chromosome counterparts (Figure 2 and Supplementary

Fluorescence Activated Video Microscopy

siRNA treated cells were kept in culture until the first beating. Contraction analysis was 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

Figure 2. Expression of the male-specific region of the Y chromosome (MSY) genes altered at different stages of cardiac differentiation. D1, D3, D6, and D12 represent mesendoderm, cardiac mesoderm, cardiac progenitor cells, and cardiomyocytes, respectively. The relative expressions of all stages were compared to D0. The expression patterns of MSY transcripts were classified in five groups. Group 1 was composed of five genes, which were downregulated by differentiation initiation. Group 2 was composed of only XKRY, which showed significant increase in expression at D1. In Group 3, TBL1Y2 and PRY were overexpressed at D1 and D3. The expression level of transcripts in Group 4 significantly increased at D3. The expression level of transcripts in Group 5 was significantly upregulated at late cardiogenesis (D6 and D12). The data show that most Y chromosome genes are overexpressed at day 3, cardiac mesoderm stage. All experiments were performed in three biological replicates, and data were presented as mean ± SEM, ∗p < 0.05.

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 ± SEM from three biological replicates. Statistical significance was tested by using a twotailed unpaired student’s t-test in Graphpad Prism software (Graphpad Software, USA). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicated statistical significance.

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RESULTS Figure 1). The detailed information about the primers, their target transcripts and genes, and their PE levels have been presented in Supplementary Tables 1 and 2. Overall, 24 MSY genes encoding proteins at PE1, 14 genes encoding missing proteins (PE2+PE3), and five genes encoding uncertain proteins (PE5) were analyzed. The expression patterns of MSY transcripts could be classified in five groups. Group 1 was composed of five transcripts, TMSB4Y, NLGN4Y, TXLNGY, SRY, and VCY, which were downregulated by differentiation initiation (Figure 2). This might highlight their roles in embryonic characteristics with no prominent function in cardiac differentiation. Group 2 was composed on only XKRY, which showed significant increase in expression at mesendoderm stage. Group 3 included PRY and TBL1Y2, which were overexpressed at both the mesendoderm and cardiac mesoderm stages (days 1 and 3). In contrast, the expression level of Group 4, USP9Y, DDX3Y1, DDX3Y2, PCDH11Y, UTY, RPS4Y1, ZFY, TBL1Y1, TBL1Y3, KDM5D, KDM5D1, and KDM5D3, significantly increased at cardiac mesoderm stage (day 3). The expression level of transcripts in Group 5 including BCORP1, an uncertain protein (PE5), RBMY, HSFY, and HSFY1 was significantly upregulated at late cardiogenesis (D6 and D12) (Figure 2).

Generation and Characterization of hESC-Derived Cardiomyocytes

hESCs were cultured and passaged in dynamic suspension culture, and cardiogenic induction was carried out using a cocktail of small molecules (SMs). During differentiation, samples were collected at particular differentiation days (0, 1, 3, 6, and 12) for Y chromosome gene expression analysis (Figure 1A). We observed the onset of beating at day 7 in cardiospheres (Figure 1B) as presented in Supplementary Movie 1. The samples were assessed for pluripotency as well as the expression analysis of mesendoderm, cardiac mesoderm, progenitor, and cardiac-specific markers on the days of sample collection to confirm cardiogenic differentiation (Figure 1C). NANOG expression was reduced by differentiation initiation and remained low over the differentiation period, whereas cardiac specific markers TBX5, MYH6, and TNNT2 upregulated during cardiogenic induction (Figure 1C). Mesendoderm marker, T, showed a significant rise on day 1 followed by a substantial decline over the differentiation period. MESP1 had the highest expression on days 1 and 3 followed by a significant reduction on day 6 (Figure 1C). Immunofluorescent images of the cells during differentiation (D0, D1, D3, D6, and D12) confirmed 4394


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Figure 3. Results of immunostaining and Western blot analyses of MSY proteins, TBL1Y, HSFY, DDX3Y, RPS4Y1, KDM5D, and ZFY during cardiac differentiation. Immunostaining results have been presented in panels A and C (scale bar: 50 μm). Nuclear staining was performed using DAPI. The magnified and merged images of each protein have also been presented (scale bar: 10 μm). Western blot results have been presented in panels B and D. GAPDH was used as the loading control. D0, hESCs; D1, mesendoderm; D3, cardiac mesoderm; D6, cardiac progenitor cells; D12, cardiomyocytes.

Figure 1A). While the expression of RBMY transcript significantly increased during late cardiogenesis, RBMX transcripts showed opposite expression pattern and decreased (Supplementary Figure 1A). PRKY showed no significant changes in expression during differentiation, but the expression of its X counterpart, PRKX, significantly increased at mesendoderm stage (Supplementary Figure 1A). Furthermore, several MSY genes and their X chromosome counterparts showed similar expression patterns during differentiation including NLGN4Y/NLGN4X, DDX3Y/DDX3X, RPS4Y1/ RPS4X, ZFY/ZFX, KDM5D/KDM5C, and HSFY/HSFX at cardiogenesis (Supplementary Figure 1B). The expression level of seven X chromosome counterparts of MSY genes showed no significant changes during cardiac differentiation. These include PCDH11X, UTX, USP9X, DDX3X, TMSB4X, EIF1AX, and XKRX.

Overall, our results showed an association between the onset of cardiac mesoderm fate and upregulation of most Y chromosome genes. The expression level of 11 MSY genes including AMELY, PRORY, TSPY, TTTY10, TTTY13, CDY, BPY2, RPS4Y2, EIF1AY, TGIF2LY, and DAZ was below the detection level (data not shown). We also analyzed the expression pattern of X chromosome counterparts of MSY genes during cardiac differentiation. We observed that the expression patterns of some of the X chromosome genes were opposite to their Y counterparts (Supplementary Figure 1A). The three transcript variants of TBL1Y showed an overexpression pattern on days 1, 3, and 6 of differentiation, a totally opposite expression pattern to what was seen in its X counterpart, TBL1X, with the exception of variant 3, which showed a slight increase on day 3 (Supplementary 4396


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Figure 4. Downregulation of TBL1Y in differentiating human embryonic stem cells (hESCs). (A) Schematic representation of knockdown experiment during cardiac differentiation. At 36 h after differentiation initiation, 4 × 105 cells were transfected with TBL1Y siRNAs and scramble siRNA (siCtrl). After 48 h, the siTBL1Y and siCtrl treated cells from three biological replicates were collected for cell cycle and molecular analyses. Some siRNA treated cells were kept longer in culture for functional studies. (B) siRNA transfection efficiency evaluated by flow cytometry analysis determined to be >90%. (C−E) Downregulation of TBL1Y was further confirmed by quantitative real-time PCR (qRT-PCR), Western blotting, and immunostaining. The gene expression data were normalized to GAPDH and compared to siCtrl treated cells. GAPDH was used as the loading control in Western blotting. Nuclear staining was performed using DAPI. Gene and protein expression analysis confirmed downregulation of TBL1Y. (F) Microscopic images of siRNA transfected cells. (G, H) Contraction analysis in cardiomyocytes. (G) Representative traces of contraction recorded in siTBL1Y and siCtrl treated cardiomyocytes. (H) Statistical analysis of contraction showed a significant decrease in beating frequencies and a substantial increase in contraction durations of TBL1Y knockdown cardiomyocytes compared to control cells. All data were obtained from three biological replicates. Data are expressed as mean ± SEM, ∗∗∗p < 0.001. Scale bar, 50 μm. Abbreviations: siCtrl, cells treated with scrambled siRNA; siTBL1Y, cells treated with TBL1Y siRNAs.

Expression and Intracellular Localization of Several MSY Proteins Changed during Cardiac Differentiation

homemade polyclonal antibodies, which were generated and quality tested as described previously3 or commercial antibodies (Supplementary Table 3). TBL1Y showed a primary cytoplasmic localization with the onset of differentiation which turned to a complete nucleus

We selected some Y chromosome genes that remarkably upregulated during cardiogenesis to assess their protein levels by immunostaining and Western blot analyses. We used 4397


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Figure 5. Effects of TBL1Y knockdown in differentiating cells. (A, B) Cell cycle analysis in differentiating cells showed a significant rise in apoptosis of TBL1Y knockdown cells. (C) Immunoblotting of CtBP indicated its stabilization in TBL1Y knockdown cells compared to the control. (D) Gene expression analysis of some Notch signaling members as well as cardiac differentiation markers in siRNA treated cells. The gene expression data were normalized to GAPDH. Data are expressed as mean ± SEM and represents three biological replicates. ∗∗p < 0.01 and ∗∗∗p < 0.001. (E) Immunostaining of TBX5, GATA4, and NOTCH1 in siRNA transfected cells indicate fewer number of TBX5+/GATA4+/NOTCH1+ cells in TBL1Y knockdown cells (siTBL1Y). Nuclear staining was performed using DAPI. Scale bar: 50 μm. Abbreviations: siCtrl, cells treated with scrambled siRNA; siTBL1Y, cells treated with TBL1Y siRNA.

pattern at day 1 and partial nucleus localization at days 3 and 6. At late cardiogenesis, we observed TBL1Y only in the cytoplasm (Figure 3A). Immunoblotting data showed the highest expression of the TBL1Y protein at day 3 of cardiac differentiation (Figure 3B). The specificity of TBL1Y antibody was evaluated in both female and male hESC lines (RH5 and RH6) using immunostaining and Western blotting (Supplementary Figure 2). HSFY was immunostained in cytoplasm during cardiac differentiation with the exception of nuclear and cytoplasmic localization at day 12 (Figure 3A). Protein expression analysis showed an increase at days 6 and 12, which was compatible with HSFY transcript data during cardiac development. DDX3Y localized only in the cytoplasm over the entire course of cardiogenesis. We detected overexpression of

the DDX3Y protein on days 1, 3, and 6 of cardiac differentiation (Figure 3B). RPS4Y1 localized in the cytoplasm during cardiogenesis (Figure 3C). The protein expression pattern was similar to RPS4Y1 transcript data. KDM5D showed a cytoplasmic localization in a granular pattern at days 0, 3, 6, and 12 with the exception of prominent nuclear localization during the mesendoderm stage (Figure 3C). Western blotting data showed several bands that might reflect various isoforms of KDM5D (Figure 3D). Overexpression of this protein at day 3 was consistent with the transcript data. The ZFY protein raised at midcardiogenesis according to both immunostaining and immunoblot analyses (Figure 3C,D). These results were in agreement with gene expression data (Figure 2). According to 4398


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expressions remained fairly similar (Figure 5D). Immunostaining of siRNA transfected cells revealed active expression of TBX5, GATA4, and Notch in the control cells, while few of the siTBL1Y transfected cells stained for these proteins (Figure 5E). These results demonstrated that members of the Notch system actively expressed in the control but had relatively low expression in siTBL1Y transfected cells.

the results, protein expression and intracellular localization of some of the MSY genes changed during cardiac differentiation. Knockdown of TBL1Y in Cardiac Mesoderm Affected Cardiomyocyte Morphology and Function

A MSY missing protein, TBL1Y, showed a remarkably different expression pattern at both transcript and protein levels compared to its X counterpart. Furthermore, TBL1Y is a member of the WD40 repeat-containing protein family that regulate a wide range of cellular functions such as signal transduction, RNA processing, gene regulation, vesicular trafficking, and cytoskeletal assembly. Therefore, we further studied the function of this missing protein during cardiac differentiation of hESCs using siRNA approach. To achieve efficient siRNA transfection, the spheroids were enzymatically digested and plated as single cells 36 h after initiation of differentiation (Figure 4A). We evaluated transfection efficiency with FITC-conjugated siRNA, which was over 90% in the experimental samples (Figure 4B). At 48 h after siRNA transfection, we collected the treated cells for molecular and cell cycle analyses. A number of transfected cells were followed longer for functional analysis of their cardiac fate. The transfection of cells with TBL1Y siRNA resulted in significant TBL1Y reduction at both the transcript and protein levels (Figure 4C,D). Immunostaining of TBL1Y also confirmed efficient protein downregulation in the transfected cells (Figure 4E). TBL1Y knockdown resulted in morphological changes into more spindle-like cells (Figure 4F). We also studied the impact of TBL1Y knockdown on the function of differentiated cardiomyocytes. The first beating was observed in siCtrl transfected cells at day 7 after differentiation initiation (Supplementary Movie S2). However, siTBL1Y transfected cells did not start beating until day 14 (Supplementary Movie S3). There was significantly lower beating frequency in siTBL1Y knockdown cells (20.3 ± 0.8 beat/min) compared to the control cells (68 ± 4 beat/min). We also performed contraction analysis in siCtrl and siTBL1Y transfected beating cells. Relative to siCtrl, contraction of TBL1Y depleted cells had significantly longer durations (1.178 ± 0.05 s vs 0.789 ± 0.04 s, respectively; Figure 4G, H). siTBL1Y knockdown resulted in increased cardiomyocyte size compared to the control cells (Supplementary Figure 3). At 48 h after siRNA transfection, the collected cells were analyzed for cell cycle progression. Figure 5A shows the cell population in each cycle (G1, G2, S, and apoptosis). The siTBL1Y transfected cells had 10% more cells in the apoptosis stage compared to the siCtrl cells, whereas the percentages of cells at the other cell cycle stages remained similar (Figure 5B).

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DISCUSSION In this study, we initially investigated expression of MSY genes and proteins during cardiogenesis by taking the advantage of in vitro differentiation of hESCs into cardiomyocytes. The expression profiling during cardiac differentiation showed substantial alterations of MSY genes expression pattern along with differential cytoplasmic or nuclear localizations over the course of cardiogenesis. This finding suggests a possible role for Y chromosome genes in differentiation of hESCs into cardiomyocytes. Previously, the gene expression profiling of a healthy heart from mice as well as humans showed sexual dimorphisms at the transcriptional level, in particular the specific expression of MSY genes, KDM5D, DDX3Y, RPS4Y1, USP9Y, ZFY, UTY, EIF1AY, PRKY, and TXLNGY.15 We have also observed the highest expressional changes in the MSY genes that included significant upregulation of PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y1, DDX3Y, UTY, TBL1Y, PRY, and BCORP1 during cardiac mesoderm formation, which is a key transitional stage in cardiac development (Figure 2). There are 17 Y chromosome genes including our experimented ones that determined to be evolutionary conserved because they are required in specific doses for their important functions in the Central Dogma.2 Over-representation of MSY genes, RPS4Y, DDX3Y, ZFY, TBL1Y, KDM5D, UTY, and USP9Y that have been previously classified for Central Dogma functions, might reflect their role during the developmental stages, in particular the cardiac mesoderm fate. KDM5D, a male specific member of the ARID family that contains DNA binding motifs, is considered a H3K4 demethylase. Wamstad and colleagues reported that a multiple dynamic chromatin pattern such as H3K4 methylation existed during cardiac lineage specification and differentiation.28 Therefore, the alterations in chromatin regulatory genes such as KDM5D would be relevant during cardiac differentiation. Homozygous knockout of the Jumonji gene, as a member of the ARID family, resulted in impaired heart development in mice.29 UTY is an active lysine demethylase, which catalyzes demethylation of H3K27 peptides.30 Our results as well as other recent reports have suggested that UTY plays a role in mesoderm differentiation and cardiac development.31,32 Furthermore, UTX-deficient mice showed developmental defects in the heart, while UTY partially compensated for the loss of UTX in UTXΔ/y mice, which resulted in less mortality but severe cardiac malformation.31 Substantial over-representation of HSFY/X during cardiogenic differentiation of hESCs suggests them as putative active genes in cardiomyocyte differentiation. HSF2, a member of the HSF family, gets activated during murine heart development.33 To the best of our knowledge, this is the first study that showed BCORP1 expression at the transcript level and during cardiac differentiation. Its homologous gene on the Xchromosome, BCOR, was identified as an interacting transcriptional corepressor of BCL6. Mutations in BCOR resulted in a lethal syndrome, OFCD, and one form of Lenz microphthalmia syndrome.34,35 A total of 85% of OFCD patients show

TBL1Y Knockdown Negatively Impacted Cardiogenesis by Rescue of CtBP

TBL1X, the X chromosome homologue of TBL1Y, has been previously shown to activate Notch signaling via discharge of a member of the Notch corepressor, CtBP.27 We analyzed the protein expression of CtBP following TBL1Y knockdown and observed a significant increase in the quantity of CtBP protein in siTBL1Y compared to siCtrl transfected cells, which showed its stabilization in siTBL1Y affected cells (Figure 5C). The expression of some of the Notch signaling members (HES1, JAGGED1, and NOTCH1) along with cardiac specific markers (TBX5, GATA4, MEF2C, and MESP1) were assessed following TBL1Y knockdown. Interestingly, siTBL1Y transfection resulted in a substantial reduction of HES1, JAGGED1, NOTCH1, GATA4, and TBX5, whereas MEF2C and MESP1 4399


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abundance as well as downregulation of HES1 mRNA in siTBL1Y transfected cells. Therefore, we suggest that TBL1Y might promote discharge of CtBP, which would result in activation of Notch target genes. Notch signaling is believed to play an important role in developmental processes such as neurogenesis44 and cardiogenesis.45 It has been reported that Notch signaling promotes the cardiac differentiation of cardiac mesodermal cells. The active mRNA expression of Notch ligand, JAGGED-1, and the Notch target gene, HES1, was identified in KDR+/PDGFRα+ cells compared to hESCs.45 Notch timed activation via JAGGED-1 resulted in an eight-fold increment of cardiac differentiation.45,46 We observed significantly higher mRNA expressions of JAGGED1, NOTCH1, and HES1 as well as TBX5 and GATA4 in siCtrl compared to siTBL1Y, which suggested activation of Notch signaling in the course of cardiac differentiation as reported in other studies.45,46 Immunostaining analysis showed that NOTCH1, TBX5, and GATA4 actively expressed in control compared to TBL1Y knockdown cells. Altogether, these data showed that TBL1Y knockdown may negatively impact cardiogenesis by CtBP stabilization. The important role of Notch signaling in cardiac development has been further confirmed in in vivo and in vitro studies.47,48 Inhibition of Notch signaling and generation of transgenic mice such as Jag1flox, which resulted in dilated cardiomyopathy, are examples of these studies.47,48 Cardiac developmental impairments have been also identified in patients that harbor TBL1Y mutations, which resulted in nonsyndromic coarctation.49 We also observed delay in the initiation of spontaneous beating and an abnormal contraction pattern in siTBL1Y cardiomyocytes. According to previous findings, this might be related to cardiac developmental defects.47−50

developmental heart defects and the majority have septal malformations.34 Studies show the key role of BCOR as a transcriptional regulator during early embryogenesis, specifically cardiogenesis.35,36 TBL1Y is a member of the WD40 repeat-containing protein family. It has a high sequence similarity with its X counterpart. TBL1X reportedly has a number of important functions that include a component of the NCoR corepressor complex,37−39 a specific exchange factor of corepressors for coactivators,40 and even activation independent of the NCoR/SMRT/HDAC3 corepressor complex that results in ubiquitylation and discharge of recruited corepressors.40 In contrast to TBL1X, TBL1Y is considered a missing protein (PE2) since insufficient evidence has been produced as per the C-HPP metrics. TBL1Y has been designated as “marginally distinguished” in PeptideAtlas. This is due to the fact that TBL1Y is very similar to its X homologue counterpart, TBL1X. The alignment shows that in nearly all areas where you could distinguish TBL1Y from TBL1X, there are no peptides from the TBL1Y protein except for one peptide (NGNLASTLGQHK) that lets it to get its marginally distinguished status in PeptideAtlas. Furthermore, the only difference between TBL1Y and TBL1X in this peptide is a D→ N change, which can easily be explained by deamidation. Therefore, there is no credible evidence to suggest that TBL1Y can be distinguished from TBL1X with the mass spectrometry data. Families of proteins with high similarity account for many of the indistinguishable representative and marginally distinguished matches in PeptideAtlas and all other databases. These proteins may remain unidentifiable unless truly uniquely mapping or proteotypic peptides can be found to differentiate the family members.41 To address this challenge, we exploit genomics tool as a complement to protein studies. We presented several pieces of evidence to strongly support the reclassification of TBL1Y as a found missing protein (PE1). First of all, the TBL1Y antibody was developed using a unique nine amino acid peptide, TEASAMAKA. The expression of TBL1Y and TBL1X was analyzed in male and female hESCs at transcript level using qRT-PCR and at protein level using Western blot and immunohistostaining. We observed that while TBL1Y expressed sharply in XY hESCs (Figure 3A,B), no expression could be detected in XX hESCs (Supplementary Figure 2). TBL1Y also showed overexpression and a thoroughly different expression pattern compared to its X-homologue during cardiogenesis. Furthermore, we demonstrated that the protein abundance of TBL1Y declined in the siRNA transfected cells as measured by Western blotting and immunhistostaining (Figure 4). The concomitant downregulation of both TBL1Y transcript and protein after treatment of the cells by TBL1Y specific siRNA strongly support specificity and reliability of TBL1Y protein detection using homemade antibody. Altogether, we presented compelling experimental evidence to distinguish TBL1Y from TBL1X, and propose reclassification of TBL1Y as “found missing protein” (PE1). As described above, TBL1X acts independently in the discharge of different corepressors for signal transcriptional activation. TBL1X particularly interacts (derepresses) with CtBP, which results in discharge of the CtBP corepressor complex.40,42 CtBP has been identified as a key component of the RBP-J corepressor complex, which suppresses transcription of Notch target genes, Hey1 and HES1.40,43 In this study, we observed stabilization of the CtBP protein in terms of protein

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CONCLUSION The Chromosome-Centric Human Proteome Project (C-HPP) is designed to achieve a complete map of expression, quantitation, subcellular localization, and functional properties of the entire human proteome during developmental stages as well as adult life under various physiological and pathological conditions. The long-term goals not only would evolve human biological science, but also result in the discovery of target proteins for prognosis, diagnosis, prevention, and therapeutic purposes.51 The alterations of MSY genes expression during cardiogenesis represent a potential role for their genetic contribution in sex-dependent cardiac development outside of sex determination. We also presented compelling experimental evidence to distinguish TBL1Y from TBL1X and propose reclassification of TBL1Y as “found missing protein” (PE1). We suggest that our strategy can be applied to distinguish homologous proteins where mass spectrometry provides insufficient data.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.7b00391. List of specific primers designed for Y chromosome genes, their X homologue, cardiac differentiation markers, and Notch signaling members using Gene Runner and Vector NTI software; NeXtProt classifica4400


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tion of MSY genes analyzed in this study; list of primary and secondary antibodies used for immunostaining and Western blot experiments; Y chromosome genes with opposite or similar expression patterns compared to their X homologues during cardiogenesis; specificity of TBL1Y antibody evaluated in female RH5 hESC line using immunostaining and Western blotting; microscopic images of control and TBL1Y knockdown cardiomyocytes (PDF) Seven day beating spheroids generated from hESCs in dynamic suspension culture (MP4) Fourteen day beating cardiomyocytes differentiated from siCtrl transfected cells (MP4) Fourteen day beating cardiomyocytes differentiated from siTBL1Y transfected cells (MP4)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +98 21 22306485. Fax: +98 21 23562507. *E-mail: [email protected]. ORCID

Ghasem Hosseini Salekdeh: 0000-0002-5124-4721 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Human Y Chromosome Proteome Project is supported by a grant from Royan Institute and Iran Science Elites Federation to G.H.S. and H.B. and the Iranian National Science Foundation (INSF) to H.B. We are grateful to Mehdi Alikhani and Paria Pooyan for their technical assistance.

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

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