A Fresh Look at the Male-specific Region of the Human Y

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A Fresh Look at the Male-specific Region of the Human Y Chromosome Zohreh Jangravi,†,‡ Mehdi Alikhani,†,‡ Babak Arefnezhad,† Mehdi Sharifi Tabar,† Sara Taleahmad,† Razieh Karamzadeh,†,△ Mahdieh Jadaliha,† Seyed Ahmad Mousavi,† Diba Ahmadi Rastegar,† Pouria Parsamatin,† Haghighat Vakilian,† Shahab Mirshahvaladi,† Marjan Sabbaghian,∥ Anahita Mohseni Meybodi,⊥ Mehdi Mirzaei,# Maryam Shahhoseini,⊥ Marzieh Ebrahimi,¶ Abbas Piryaei,§ Ali Akbar Moosavi-Movahedi,△ Paul A. Haynes,■ Ann K. Goodchild,# Mohammad Hossein Nasr-Esfahani,○ Esmaiel Jabbari,▲ Hossein Baharvand,□,● Mohammad Ali Sedighi Gilani,∥ Hamid Gourabi,⊥ and Ghasem Hosseini Salekdeh*,†,$ †

Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran ∥ Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran ⊥ Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran # The Australian School of Advanced Medicine, Faculty of Human Sciences, Macquarie University, Sydney, NSW, 2109, Australia ¶ Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran § Department of Biology and Anatomical Sciences, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran △ Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran ■ Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW, Australia ○ Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran ▲ Biomimetic Materials and Tissue Engineering Laboratory, Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina, United States □ Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran ● Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran $ Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran S Supporting Information *

ABSTRACT: The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome’s length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the continued... Special Issue: Chromosome-centric Human Proteome Project Received: September 10, 2012 Published: December 20, 2012 © 2012 American Chemical Society

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dx.doi.org/10.1021/pr300864k | J. Proteome Res. 2013, 12, 6−22

Journal of Proteome Research

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brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein−protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping. KEYWORDS: human Y chromosome, male-specific region, Human Y chromosome Proteome Project

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INTRODUCTION Complete sequencing of the human genome has provided the scientific community with access to unprecedented numbers of human genes. However, about 30% of approximately 20300 genes currently lack any experimental proof at the protein level. Scant information is available related to protein function, abundance, subcellular localization, and interactions for numerous other proteins.1,2 The Chromosome-centric Human Proteome Project (C-HPP) has been designed to systematically map the entire human proteome in order to enhance our understanding of human biology and lay a foundation for diagnostic, prognostic, and therapeutic implications.1,3 C-HPP guidelines are set and international teams have selected chromosomes.4 Mapping the proteome of the Y chromosome will be conducted in Iran, and this will increase our understanding of the functions associated with proteins encoded by the Y chromosome. Furthermore, this project will attempt to establish a foundation for the development of diagnostic, prognostic, therapeutic, and preventive medical applications.

16 protein-coding genes: SRY, RPS4Y1, ZFY, AMELY, TBL1Y, PRKY, GYG2P1, USP9Y, DDX3Y, UTY, TMSB4Y, NLGN4Y, CYorf15A, KDM5D, EIF1AY, and RPS4Y2 (Figure 2). The X-transposed sequences have originated from an X-to-Y transposition 3−5 million years ago. The sequences encode only two protein-coding genes, TGIF2LY and PCDH11Y. The proteins located on these two regions have high sequence similarity with their homologues on the X chromosome (Supplementary Figure 1, Supporting Information). The ampliconic segments are composed of eight palindromes, three inverted repeats, and two arrays of no long open reading frames (NORF) and TSPY repeats. Genes located on ampliconic region were acquired from diverse source and then amplified. The ampliconic segments encode eight gene families (Figures 1 and 2). These include deleted in azoospermia (DAZ: DAZ1, DAZ2, DAZ3, DAZ4), chromodomain Y (CDY: CDY1, CDY1B, CDY2A, CDY2B), variable charge (VCY: VCY, VCY1B), heat-shock transcription factor Y (HSFY: HSFY1, HSFY2), RNA-binding motif Y (RBMY: RBMY1A1, RBMY1B, RBMY1D, RBMY1E, RBMY1F, RBMY1J), testis-specific protein Y (TSPY), basic protein Y2 (BPY2: BPY2, BPY2B, BPY2C), and PTP-BL related Y (PRY: PRY, PRY2, PRYP3, PRYP4). Among the three sequence classes in MSY euchromatin, the ampliconic sequences exhibit by far the highest density of both coding and noncoding genes. Overall, the ampliconic region contains 25 genes excluding TSPY. For TSPY, 27 to 40 gene copies have been reported in normal individuals.5 Currently, protein evidence exists for 22 genes: DAZ1, DAZ2, DAZ3, DAZ4, CDY1, CDY1B, CDY2A, CDY2B, HSFY1, HSFY2, RBMY1A1, RBMY1D, RBMY1F, RBMY1J, TSPY1, BPY2, BPY2B, BPY2C, PRY, PRY2, PRYP3, and PRYP4. In the ampliconic genes that contain two or more copies, the following family members encode similar proteins: CDY1, CDY2, HSFY2, PRY, and BPY2. The members of the RBMY family differ in only one or two amino acids. Members of the DAZ family represent highly similar sequences with different lengths due to different repeats of similar residues. Proteins located on the ampliconic region show lower similarity to their autosomal and X chromosome homologues compared with proteins in X-degenerate and X-transposed regions (Supplementary Figure 1, Supporting Information). While protein-coding families in the ampliconic regions are expressed predominantly or exclusively in the testes, most X-degenerate genes are ubiquitously expressed. The classification of the MSY protein-coding genes based on the number of genes in MSY sequence classes, the number of alternative splicing transcripts, protein isoforms (several different forms of the same protein) and gene copy number is presented in Supplementary Figure 2 (Supporting Information).

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STRUCTURE OF THE MALE-SPECIFIC REGION OF THE Y CHROMOSOME (MSY) Due to its distinctive role in sex determination, the Y chromosome has long attracted special attention from evolutionary biologists, geneticists, and even the public. The Y chromosome consists of regions of DNA that show quite distinctive genetic behavior and genomic characteristics. It is male-specific and constitutively haploid. Unlike other chromosomes, it largely escapes meiotic recombination. The Y chromosome is approximately 60 Mb in size and includes two segments, two pseudoautosomal regions, and the male-specific region of the Y chromosome (MSY). MSY, formerly known as the nonrecombining region (NRY), comprises 95% of the Y chromosome’s length, where there is no X−Y crossing over. MSY is flanked on both sides by pseudoautosomal regions of less than 3 Mb in length, where X−Y crossing over is a normal, frequent event in male meiosis.5 Overall, there are 60 genes (locus) on the MSY as reported in Ensembl v68. Currently, there is protein evidence for about 40 genes as described in NeXtProt, GPMdb with evidence code of green, PeptideAtlas with a false discovery rate (FDR) of 1%, or the Human Protein Atlas (HPA) with reliability score of high or medium. Of these, NeXtProt, GPMdb, PeptideAtlas, and HPA reported protein evidence for 31, 23, 17, and 2 genes, respectively (Figures 1 and 2). Figure 1 shows the MSY genes and indicates evidence available regarding their protein expression evidence, protein interaction, the availability of antibodies, and description of 3D structure, signal domain, motif, and Ncoil. No reliable protein evidence is available for 20 MSY genes: VCY1, VCY1B, XKRY1, XKRY2, AC006156.1, AC006156.2, SLC9B1P1, CYorf15B, CYorf17, BCORP1, TSPY2, TSPY3, TSPY4, TSPY8, TSPY10, RBMY1B, RBMY1E, TTTY10, TTTY12, and TTTY13. MSY consists of three different regions of euchromatic sequences: X-degenerate, X-transposed, and ampliconic5 (Figure 2). The X-degenerate sequences are relics of ancient autosomes that have evolved toward the sex chromosomes and encode

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GENE ONTOLOGY OF MSY We have annotated the molecular function, biological processes and subcellular distribution of the 60 MSY genes using Gene Ontology (GO), UniProt, NeXtProt, EMBL-EBI, and NCBI databases, in addition to other published data (Figure 3). On the basis of their molecular functions, MSY proteins can be divided 7



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Figure 1. Annotation of 60 MSY genes as reported in Ensembl v68. The expression evidence, protein interaction, availability of antibodies, and presence of 3D structure, signal domain, motif, and Ncoil are shown. The heatmap was constructed based on NeXtProt, GPMdb, ENSEMBL, HPA, MIM, Orphanet, Pfam, PRINTS, SMART, PROFILE, Interpro, Superfamily and PDB databases. The black square indicates no evidence and a green square indicates one or more than one pieces of evidence in the databases. In the protein evidence columns, a green square represents the protein level, a yellow square indicates transcript level, light green square indicates the protein level based on homology of TSPY gene family and red square indicates uncertainty at the protein level. 8



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Figure 2. Forty MSY protein-coding genes that have protein evidence in NeXtProt, GPMdb (with evidence code of green), PeptideAtlas(with a false discovery rate (FDR) of 1%), or Human Protein Atlas (HPA) (with reliability score of high or medium) (Figure 1). For each protein, database presenting evidence for protein, the number of possible transcripts and protein isoforms (several different forms of the same protein) and nonsynomous single-nucleotide polyphorphisms (nsSNP) assembled from data from the 1000 Genome Projects have been indicated. AZF region has been demarcated. The number of protein isoforms obtained from NeXtProt.

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MSY-ASSOCIATED TRAITS AND DISEASES The Y chromosome is not essential for life and cells with XO genotypes are often viable, unlike monosomics for other chromosomes. For quite some time, most regions of the Y chromosome were assumed to be functionally inert and sex determination was viewed as the sole function related to the Y chromosome.6 This theory changed the control of spermatogenesis was initially described and many genes associated were mapped to the Y chromosome.7 In recent years several MSY-associated diseases have been reported, such as prostate cancer, graft-versus-host disease (GVHD), autism, nonsyndromic speech delay, and gender differences in the disease pathophysiology of new-onset heart failure (HF) (Figure 4). Most of our information about the function of MSY genes are based on DNA analysis particularly DNA microdeletions (Supplementary Figure 3, Supporting Information). A major question for infertility clinics performing molecular screening for azoospermia factor region (AZF) deletions is, what is the functional contribution of the AZF encoded proteins to human spermatogenesis? Essentially as proteins provide the functional output of the cell information pertaining to them may provide the most relevant condition/disease related information. The analysis of proteins is particularly informative when interpretation of their expression, post-translational modifications, protein−protein interaction and proteins activity takes into account their dynamics in specific biological contexts.

into about 15 groups and these include protein binding (19.6%), DNA binding (10.7%), RNA binding (7.1%), metal ion binding (7.1%), miscellaneous functions (5.3%), transcription factor activity (5.3%), transcription regulator activity (5.3%), transferase activity (3.5%), ATP binding (3.5%), translation activator (3.5%), esterase activity (3.5%), oxidoreductase activity (3.5%), kinase activity (1.7%), protease activity (1.7%) and helicase activity (1.7%). Of the MSY proteins, 16.0% do not have a known molecular function (TSPY2, TSPY3, TSPY4, TSPY8, TSPY10, AC006156.1, AC006156.2, GYG2P1, VCY/VCY1B, XKRY1/2, BCORP1, PRY1, PRY2, PRY3, PRY4, CYorf15A/15B, CYorf17 and TTTY10/12/13) as no annotations could be located. Within the TSPY family, TSPY1 is the only protein for which a molecular function is described whereas the other TSPYs members are assumed to have similar functions based only on their similarities to TSPY1. MSY proteins have roles in a range of biological processes that include spermatogenesis (11.6%), transcription (10.0%), cell differentiation (8.3%), miscellaneous processes (8.3%), gonad development (6.6%), metabolic processes (6.6%), tissue development (5.0%), nucleosome assembly (5.0%), chromatin modification (5.0%), single fertilization (5.0%), translation (5.0%), sex differentiation (3.3%), cell adhesion (3.3%), RNA metabolism (1.6%), cell proliferation (1.6%), and sex determination (1.6%). No biological processes have been described for approximately 10% of the proteins (AC006156.1, AC006156.2, GYG2P1, VCY/VCY1B, BCORP1, PRY family, CYorf15A/15B, CYorf17 and TTTY10/12/13). MSY related proteins are predominantly localized in the nucleus (25.0%), other cellular components (21.8%) and the cytoplasm (6.2%) with about 21.8% of proteins located in both the nucleus and cytoplasm. The subcellular localization of 25.0% of proteins remains undescribed: PRKY, AC006156.1, AC006156.2, GYG2P1, VCY/VCY1B, BCORP1, the PRY family, CYorf15A/15B, CYorf17, and TTTY10/12/13.

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SEX DETERMINATION/REVERSAL

In 1959, the Y chromosome was identified as the main factor that determined maleness in humans and mice (for review, see refs 1, 13). This finding initiated a search for the testis-determining factor (TDF) on the Y chromosome. Several candidates such as ZFY, a zinc finger nucleic-acid-binding protein, were proposed and later refuted by genetic studies prior to the isolation of the sex-determining region of the Y chromosome (SRY). In 1990, SRY, located on the short arm of the Y chromosome, was identified and proposed to be a candidate for 9



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Figure 3. Heatmap presentation of gene ontology annotation of MSY proteins. Green squares indicate the presence of a particular molecular function, biological process, or cellular localization for the corresponding protein. Red square indicates that the gene ontology annotation may vary among members of protein families.

the elusive TDF.6 Point mutations were discovered within SRY in several XY females, which indicated that SRY was required for male development in humans. About 15% of all XY sex-reversed individuals have been known to carry SRY mutations.8,9 The SRY protein contains a sequence-specific high-mobility group (HMG) box, a conserved motif of DNA binding and DNA bending.10,11 In humans, SRY expresses in indifferent gonads at seven weeks postfertilization. By activating or coordinating the expression of genes such as SOX9, SRY causes differentiation of pre-Sertoli cells to produce a testis and suppress genes that favor the formation of the female gonad.12,13 Studies of human sexreversal patients have identified many point mutations that result in SRY amino acid substitutions. Most are within the HMG box but a few affect the N- and C-terminal nuclear localization signals (NLS) that flank the HMG box domain.14

Most sex-reversing mutations in the SRY result in impaired DNA binding and bending. However, several of the sex-reversing mutations which do not affect DNA binding have been mapped to one of SRY’s two independently functioning NLS that flank the HMG box domain. The translocation of SRY to the nucleus is critically dependent on these two NLS which are located in the C-terminal (β-NLS), that mediates nuclear transport through importin β1 (Impβ1; amino acids 128−134) and the N-terminal (CaM-NLS; amino acids 59−77) which is known to recognize the calcium-binding protein calmodulin (CaM).15,16 A sexreversing NLS mutation in the β-NLS specifically impairs Impβ1 binding and SRY nuclear localization indicating that nuclear translocation mediated by the β-NLS/Impβ1 is essential for sex determination.15 It has also been discovered that two sexreversing mutations in CaM-NLS did not affect Impβ1 binding, 10



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Figure 4. Graphical presentation of MSY proteins associated with diseases.

of both genes causes Sertoli cell−only syndrome, a condition characterized by the presence of complete Sertoli cells in the testes but a lack of spermatozoa in the ejaculate.20 However it has been demonstrated that the DBY gene has a more critical role in spermatogenesis than the USP9Y gene.25 This view is further supported by the observation that the DDX3Y protein is expressed in spermatogonia, whereas the USP9Y protein is found only in spermatids. Therefore, a deletion or dysfunction of DDX3Y may cause premeiotic disruption of spermatogenesis. Deletions or dysfunction of USP9Y would most likely cause only a postmeiotic germ line disruption.26,27 Further studies are required to determine the precise roles of each of these genes in fertility to develop more targeted Y chromosome screening process.28 AZFb microdeletions are found in approximately 1−2% of males with nonobstructive azoospermia.22 AZFb sequences contain RNA-binding motif protein Y chromosome (RBMY); heat shock transcription factor, Y-linked 1 (HSFY); PTPN13like protein Y-linked (PRY); lysine-specific demethylase 5D (KDM5D); testis-specific chromodomain protein Y 2 (CDY2); testis-specific XK-related protein, Y-linked (XKRY); eukaryotic translation initiation factor 1A (EIF1AY); and Y-chromosomal 40S ribosomal protein S4, Y isoform 2 (RPS4Y2) genes. The main male infertility-associated gene in this region is RBMY, an RNA-binding protein that is a splicing factor expressed in the nuclei of spermatogonia, spermatocytes, and round spermatids.20 HSFY proteins are expressed predominantly in germ cells and partial AZFb deletion has been attributed to its role in testicular pathology.29 PRY has a low degree of similarity to the protein tyrosine phosphatase, nonreceptor type 13. Four nearly identical copies of this gene exist within a palindromic region. This gene is involved in the regulation of apoptosis, an essential process that removes abnormal sperm from the population of spermatozoa. The PRY protein has been found in some spermatids and spermatozoa but not detected in premeiotic germ cells. Therefore, its contribution

implying that there are effects on nuclear translocation distinct from those that involve Impβ1.15

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SPERMATOGENESIS AND MALE INFERTILITY Infertility affects approximately 15% of couples and male causes of infertility are found in half of involuntarily childless couples.17 Chromosomal abnormalities, such as microdeletions, account for approximately 5% of infertility in males and the prevalence increases to 15% for azoospermia, which is the complete absence of sperm in the ejaculate of males. In oligozoospermic (less than 20 million sperm/mL) men, the prevalence of microdeletions is 5−10%.18,19 The role of the Y chromosome in male infertility has been extensively studied (for review, see refs 20, 21). However, our knowledge about the contribution of the Y chromosome genes in male infertility is based mainly on studies of microdeletions. Y chromosome microdeletions on long arm Yq are the most important causes of idiopathic infertility in males. This region on Yq is the AZF region that contains three different subregions (AZFa, AZFb, and AZFc). The genes in these regions are involved in the growth and development of sperm. Deletions in this region are specifically related to spermatogenesis failure.5,20 Figure 2 illustrates the different AZF regions of the MSY and the locations of genes to be discussed in the following paragraphs. An AZFa microdeletion is responsible for failure of spermatogenesis in approximately 1% of men diagnosed with nonobstructive azoospermia.22 The two genes located in the AZFa region are DEAD-box RNA helicase Y (DDX3Y) and the ubiquitin-specific protease 9Y gene (USP9Y).5 There is strong experimental evidence that DDX3Y, formerly known as the Dead Box Y gene (DBY), is the major male-associated gene in the AZFa region. This DDX3Y belongs to the large DEAD-box protein family and functions as an ATP-dependent RNA helicase.23 USP9Y, previously known as Drosophila fat-facets related-Y (DFFRY), encodes a protease with activity specific to ubiquitin and is involved in the regulation of protein metabolism (protein turnover).24 Deletion 11



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copy loss in the blood of men diagnosed with prostate cancer compared to those with no prostate cancer has been reported. With the average copy number of TSPY in the normal population being about 27 to 40,5 it has been shown that in both Caucasian and Hispanic men with 30 copies of TSPY was higher in normal controls compared to those with cancer.45 It is not clear whether the loss of TSPY copies seen in those with prostate tumors is the initiator of prostate tumorigenesis or the result of a prior genomic defect that occurs elsewhere in the genome.45 Analysis of expression of the Y chromosome genes in prostate cancer or prostatic hyperplasia samples using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) has shown aberrant expression of a small number of genes (TSPY, SRY, PRY, ZFY, KDM5D, EIF1AY and TMSB4Y), which suggests that they may play a potential role(s) in or be influenced by oncogenesis in the prostate.46,47 It has also been suggested that in high-grade melanomas TSPY undergoes epigenetic silencing.48 Protocadherin Y (PCDH11Y or PCDH-PC) has been proposed as a potential target for therapeutics designed to suppress prostate cancer aggressiveness. PCDH11Y is homologous (98.1%) to protocadherin X (PCDH11X), a gene product encoded by the human X chromosome (Supplementary Figure 1, Supporting Information), and differs from it as PCDH11Y lacks a small 13-bp continuous sequence.49 A comparative genetic analysis of apoptosis-resistant prostate cancer cell lines has led to the description of selective expression of PCDH11Y in apoptosisresistant and hormone-refractory cell variants.49 PCDH11Y expression is proposed to be a novel mechanism that promotes progression of human prostate cancer to its hormone resistant and aggressive states via the Wnt signaling pathway.50,51

to the testicular pathology of men who have an AZFb deletion (meiotic arrest) is questionable.30 It is likely that other AZFb genes may have important roles in the process of spermatogenesis. A small deletion in AZFb that included the KDM5D has been reported in a Romanian man diagnosed with azoospermia.31 However, KDM5D protein has yet to be reported in human male germ cells. AZFc deletions cause approximately 12% of nonobstructive azoospermia and 6% of severe oligozoospermia.32 The proximal part of the AZFc deletion interval overlaps with the distal part of the AZFb deletion interval.5 The AZFc region therefore also includes copies of some of the AZFb genes, including one of three copies of testis-specific basic protein Y2 (BPY2) and one of two copies of the testis-specific chromodomain protein Y1 (CDY1) and the DAZ1/DAZ2 gene doublet (Figure 2). DAZ genes are the major genes involved in fertility in the AZFc region and their deletions can cause a spectrum of phenotypes that range from oligozoospermia to azoospermia.33 Partial deletions of DAZ genes seem to be related to oligozoospermia and DAZ gene expression is reduced in azoospermic patients.32,34 DAZ genes are known to be involved in the spermatogenic process because they are expressed during all stages of germ cell development.35,36 DAZ proteins regulate translation, bind RNA in germ cells, and are involved in the control of meiosis and maintenance of the primordial germ cell population.35,36 CDY contains a chromodomain and a histone acetyltransferase catalytic domain and is expressed only in testis tissue after meiosis in spermatids.37 BPY proteins may contribute to the specific histone hyperacetylation process in late spermatids leading to a more open chromatin structure which is required to facilitate the spermatogenic histone replacement by transition proteins and protamines. The BPY2-encoded protein is present in the nuclei of spermatogonia, spermatocytes, and round spermatids.38 It interacts with ubiquitin protein ligase E3A and may be involved in maintaining the fertilization capacity of human sperm.39 For these reasons, BPY2 and CDY are interesting proteins to be further studied. It should be noted that geographical and ethnic differences (haplogroups) might affect the pattern and phenotype of these deletions in the AZFc region.40 In addition to AZF genes, TSPY genes have also been associated with male infertility. TSPY proteins have been identified in spermatogonia and may regulate the timing of spermatogenesis by signaling spermatogonia to enter meiosis.41 More copies of TSPY have been found in infertile patients.42 However further investigation is required to characterize the role of TSPY in infertility.

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GERM CELL TUMORS

In 1987, the description of a gonadoblastoma locus on the Y chromosome was proposed to explain the high frequency of gonadoblastoma in dysgenetic gonads of XY females.52 Gonadoblastoma, a special type of germ cell tumor, is histologically similar to testicular carcinoma in situ, which is the precursor for testicular germ cell tumors. The presence of the Y-located gonadoblastoma locus on Y chromosome gene(s) in intersex/sex-reversed patients could be considered as a gain of function(s) by the germ cells in a predominantly female environment. The availability of complete sequencing of the MSY revealed a TSPY gene cluster that encompasses approximately 0.7 MB of DNA, as the currently known functional genes within the gonadoblastoma locus on the critical region of the Y chromosome.53 According to research, TSPY is present and is expressed in XY females and intersex individuals diagnosed with gonadoblastoma.54,55 Localization of TSPY protein with established germ cell tumor markers in the same tumor cells of both gonadoblastoma and the precursor for germ cell tumors supports the candidacy of TSPY as the primary gene for the gonadoblastoma locus on the Y chromosome.56 TSPY gene expression is also present in seminomas, a subtype of testicular germ cell tumor.57 The coexpression of TSPY and the androgen receptor (AR) in testicular germ cell tumors in patients as well as in model cell lines has been observed, however such coexpression is not found in the normal testes of humans or mice. It is suggested that TSPY serves as a repressor in androgen-induced tumor development in testicular germ cell tumors.58 These findings raise the possibility

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PROSTATE CANCER Prostate cancer, as with numerous other cancers, is the result of genetic alterations that tend to accumulate during disease progression. Although genetic alterations on the Y chromosome are unknown, loss of Y chromosome material is reported to be one of the most common aberrations in prostate cancer.43 In an analysis of 50 human prostate cancer specimens, it was shown that at least one out of six analyzed Y chromosomespecific genes were deleted in most specimens. Loss of the SRY gene was shown in 38% of cases. In addition, there was an 18% loss in ZFY, 14% in BPY1, 52% in KDM5D, 32% in RBMY1A1 and 42% in BPY2. Multiple gene loss was most commonly associated with advanced stages and grades of prostate cancer.44 The TSPY gene arguably constitutes the most interesting gene on this chromosome whose deletion, aberrant and androgenresponsive expression may be involved in cell proliferation and oncogenesis in the prostate gland. A high incidence of TSPY 12



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female hormones protect the cardiovascular system from atherosclerotic changes in premenopausal women. In a recent study of patients with idiopathic-dilated cardiomyopathy (IDCM) and new-onset HF, Y chromosome specific differences in gene expression were described.80−82 There was an overexpression of 18 Y chromosome-related transcripts that included a greater than 10-fold change in expression of USP9Y, DDX3Y, RPS4Y1 and EIF1AY in males together with a range of gene expression changes in women. Of the overexpressed transcripts in men, 73% were found on the Y chromosome.83

that TSPY could be used as a clinical marker to assess the malignancy of testicular germ cell tumors.

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GRAFT-VERSUS-HOST DISEASE (GVHD) The histocompatibility locus antigen (HLA)-identical bone marrow transplantation (BMT) 1 is frequently implicated in GVHD or graft rejection. The complication is thought to be initiated by the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes originating from the recipient.59,60 The male-specific H−Y antigens are the most extensively studied minor histocompatibility antigens. Female patients who undergo BMT have a higher risk for graft rejection with male bone marrow compared to bone marrow from females.59,61The first identified gene that encoded for an H−Y antigen was 11-residue peptide derived from KDM5D, an evolutionarily conserved protein encoded on the Y chromosome. The protein from the homologous gene on the X chromosome, KDM5C, differs by two amino acid residues in the same region.62 Thus far, several other H−Y antigens have been identified, including USP9Y,63,64 UTY,65,66 RPS4Y,67 DDX3Y,68,69 and TMSB4Y.70

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PROTEIN−PROTEIN INTERACTIONS (PPIS) Proteins are the main structural elements, signaling messengers, catalysts, and molecular machines of biological systems.84 Over 80% of proteins are estimated to operate in complexes rather than alone.85 Protein−protein interactions (PPIs) regulate numerous cellular processes in a highly specific manner and the distortion of PPIs may lead to the development of numerous diseases. Therefore, detection and characterization of PPIs and their networks is essential for understanding the mechanisms of biological and disease processes at the molecular level.86,87 Several groups have studied the interaction of MSY proteins with other proteins and reported a biological function for some of the detected interactions (Table 1). However, no function has been assigned to many. The full list of interactions has been presented in the PPI section of Human Y chromosome Proteome Database (www.HUPO.ir). Among MSY proteins, the SRY protein interaction has been the best studied. As described above, SRY interacts with Impβ1, and CaM.88 The exact mechanism however by which they mediate or contribute to SRY nuclear translocation remains to be determined. CaM may facilitate nuclear import through direct interaction of SRY with the nuclear pore complex and subsequently, CaM release from SRY is likely to be affected by DNA binding in the nucleus, which has been demonstrated at least in vitro.16 DAZ and RBMY are RNA-binding proteins expressed exclusively in germ cells. It has been demonstrated that human DAZ/DAZL proteins can form a stable complex with human PUM2, a human homologue of Pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans.89 It has been suggested that PUM2 functions as a translational regulator of germ cell lineage in conjunction with DAZ and DAZL proteins.89 RBMY has been proposed to regulate splicing specifically in the germ line. This protein contains an RNA recognition motif (RRM) in the N-terminal part and a central region serine-arginine-glycine-tyrosine (SRGY) domain formed by four repeats of a 37 amino acid peptide that includes a total of five SRGY tetrapeptides. Several reports have demonstrated that RBMY directly interacts with a variety of factors involved in splicing regulation, including some serine/arginine-rich (SR) proteins, Sam68-like phosphotyrosine protein (T-STAR), APassociated tyrosine phosphoprotein p62 (Sam68), transformer-2 protein homologue (Tra2-β), and SR proteins 9G8 (also known as splicing factor, arginine/serine-rich 7). Most of these protein interactions are mediated by the SRGY boxes.90−92 TSPY interacts with eEF1A and enhances protein synthesis that is characteristic of actively growing and/or replicating cells.93TSPY also interacts with type B cyclins and enhances cyclin B-CDK1 kinase activity and a rapid G2/M transition in the cell cycle.94 TSPY also plays a role in chromatin modification and/or organization during spermatogenesis by interacting with core histones. TSPY traps androgen-bound AR and suppresses androgen signaling which results in a reduction in malignancy from seminoma to nonseminoma (NSE) testicular cancer.58

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GENDER DIFFERENCES IN DISEASE PATHOPHYSIOLOGY PCDH11Y and PCDH11X genes are members of the cadherin superfamily. Both are highly expressed in the fetal brain and spinal cord.71 These human genes are absent in the nonhuman primates Pan troglodytes and Gorilla gorilla, which supports the hypothesis that they possibly played a significant role in the evolutionary transition of language and brain development in modern humans.72 A small deletion within the PCDH11X/Y gene pair has been observed in a child with a significant nonsyndromic speech delay.73 The PCDH11X deletion was inherited from the (phenotypically normal) mother, but the PCDH11Y deletion was not present in the father and therefore appeared to be a de novo occurrence. The authors have postulated that the deletions interfered with the normal splicing, thus altering gene expression which resulted in a disruption in language development. This finding might support the hypothesis that PCDH11X/Y gene pairing has an influential effect on the capacity for language in humans. The incident of autism in males is four times higher compared to females.74 An explanation proposed for this discrepancy includes the role of genes on the X or Y chromosome, those associated with sex-related hormones, and intrauterine or postnatal hormonal influences. The potential Y chromosome effect has been investigated by haplotype analysis using a set of mostly intragenic markers. Single nucleotide polymorphism (SNP) analysis of the Y-linked transducin b-like 1 (TBL1Y) and neuroligin 4 (NLGN4Y) genes in 146 autistic cases and 102 controls of EuropeanAmerican origin have suggested a role for the Y chromosome haplotypes in autistic disorders.75 The role of the Y chromosome in autism has also been proposed due to the structural abnormalities and aneuploidy of the Y chromosome in boys with pervasive developmental disorders or autism.76 Males with a 47, XYY karyotype often exhibit an autistic phenotype77 and a cross-sectional, multicenter study of 95 males with XXYY syndrome showed autism spectrum disorders in 28.3% of cases.78 Gender differences in those with heart disease/conditions are a well-known phenomenon as described in animal models as well as clinical trials.79 However, the biological basis for those differences is yet to be elucidated. It is clear for example that 13



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Table 1. Interaction of MSY Proteins with Other Proteins and Reported Biological Function for the Detected Interactiona protein SRY

TSPY

protein partner

function

methods

HDAC3128 Importin-β,15,128 XPO4,129 Ep300,128 CALM1130 WDR5,131 AR,132 WT1,133 PKA,134 ZNF208,135 SLC9A3R2,136PARP-1137 RPL13a and RPS7138 SP1139 eEF1A,93 CyclinB194 AR58

Deacetylation and cytoplasmic delocalization Nuclear transport and localization Regulation of genes expression for sex determination and testis development Works as a splicing factor during testis determination Synergistically activate MAO A transcription May be in germ cell tumorgenicity Repressor of cell proliferation in germ-cell tumors by suppression of androgen signaling Phosphorylation and nuclear transport May function as a histone chaperone during maturation of the elongating spermatids in the testis May be involved in condensation of chromosome before entering meiosis in normal spermatogenesis Repress the target gene by chromatin modification Regulation of enzymatic activity of KDM5D UBE3A ubiquitination may be required for VCY2 function Control of the male germ cell cytoskeletal network Translational regulator in the germ cell lineage Histone-to-protamine transition in germ cell A eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome Might influence the apoptotic sensitivity of prostate cancer cells mRNA splicing

PD128 E-BBA,15 PD,128,129 Co-IP130 Y2H,132,133,136 Co-IP,131,134,136,137 PD135,137 Y2H138 Co-IP139 PD,93,94 Co-IP93,94 Co-IP58

CSNK2A1122 H2A, H2B, H3, H4140 KDM5D

BPY2 DAZ CDY EIF1AY

MSH5141 H3K4142 PCGF6142 UBE3A39 VCY2IP-1 (MAP1S)95 PUM289 H4 and H2A37 EIF5B143

PCDH11Y CTNNB149 RBMY SRP20,90 SAM68,144 T-STAR,144 TRA2B,91,92 Splicing factor 9G8120 UTY TLE1/2145

May mediate transcription repression

BA122 PD140 AP and Co-IP141 BA142 Co-IP142 Y2H and Co-IP39 Y2H, Co-IP, and PD95 Y2H, Co-IP BA37 NMR143 Co-IP49 Y2H,90,144,119 Co-IP120 Y2H145

a

HDAC3, histone deacetylase 3; KPNB1, importin subunit beta-1; XPO4, exportin-4; Ep300, histone acetyltransferase p300; WDR5, WD repeatcontaining protein 5; AR, androgen receptor; CALM1, calmodulin; WT1, Wilms tumor protein; PKA1, cAMP-dependent protein kinase type 1; ZNF208, zinc finger protein 208; SLC9A3R2, solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 2; PARP1, poly [ADPribose] polymerase 1; RPL13a, 60S ribosomal protein L13a; RPS7, 40S ribosomal protein S7; SP1, transcription factor Sp1; eEF1A, elongation factor 1-alpha 1; CSNK2A1, casein kinase II subunit alpha; H2A, histone H2A; H2B, histone H2B; H3, histone H3; H4, histone H4; MSH5: DNA mismatch repair protein MSH5; H3K4, histone H3 Lys 4; PCGF6, polycomb group RING finger protein 6; UBE3A, ubiquitin-protein ligase E3A; MAP1S, microtubule-associated protein 1S (VCY2IP-1); PUM2, pumilio homolog 2; EIF5B, eukaryotic translation initiation factor 5B; CTNNB1, catenin beta-1; SRSF20(SRP3), pre-mRNA-splicing factor SRP20; SAM68(KHDRBS1), src-associated in mitosis 68 kDa protein; T-STAR, RNAbinding protein T-Star; TRA2B, transformer-2 protein homolog beta; Protein PO, 60S acidic ribosomal protein PO; TLE1/2, transducin-like enhancer of split 1/2′; Y2H, Yeast Two-Hybrid; Co-IP, co-Immunoprecipitation; PD, pull-Down assay; E-BBA, ELISA-based binding assay; BA, biochemical activity; NMR, nuclear magnetic resonance; AP, affinity purification.

disorders.105 On the other hand, the protein structure−function relationships may have a key regulatory influence on biological activities. In addition, investigating the effect of protein−ligand interactions on the regulation of protein activity is another dynamic area of structural biology.97,99,106−109 To date, many techniques have been developed to expand our information about the process of protein structure−function relationships as well as protein folding and its interactions. Among the Y chromosome proteins, the structures of KDM5D, ZFY, RBMY1A1 and SRY have been the most studied (PDB IDs: KDM5D: 2E6R; ZFY: 7ZNF, 1KLS, 1XRZ, 5ZNF, 1KLR; RBMY1A1: 2FY1; SRY: 1J47, 1HRZ, 1J46, 1HRY) (Figure 1). Recently, the crystal structures of CDYs as a putative histone acetyltransferase have also been determined (PDB IDs: CDY1B: 2FBM; CDY2B: 2FW2).110 The ZFY protein is a member of the polydactyl zinc finger proteins, which may act as a transcription factor by interacting with the specific DNA sequence.111 This protein contains odd−even finger pairs within the 13-tandem zinc finger domain.112 Grants et al. have studied the mechanism by which odd−even repeats of the zinc finger domains interact with DNA. This study has demonstrated that DNA binding by ZFY is mediated exclusively by the interaction of zinc fingers 12 and 13 with an AGGCCY box from DNA.113−116 However, there are no significant contributions of even−odd repeats in the ZFY zinc finger domain to DNA binding.117 In the

BPY2 is a testis-specific protein that has no active X homologue. The functional role of the gene in spermatogenesis is unknown.37 Wong et al. have investigated the proteins that interact with BPY2 in order to understand its function. They observed the interaction of BPY2 with ubiquitin-protein ligase E3A (UBE3A) and have proposed that by interacting with BPY2, UBE3A may additionally interact with other proteins and target them for remodelling by ubiquitination in the testis during spermatogenesis and fertilization.39 These researchers have also noted that VCY2IP-1 is a protein partner of BPY2 and proposed that BPY2 is involved in the cytoskeletal network by its interaction with VCY2IP-1.95

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OVERVIEW OF THE STRUCTURAL STUDIES IN THE Y CHROMOSOME PROTEOME The biological functions of most proteins are carried out by the process of folding into precise three-dimensional conformations, known as the native structure.96,97 Structural biology with the aid of spectroscopic as well as thermodynamic procedures improves our knowledge about protein stability, folding and its interactions.98−104 Understanding the process of protein folding increases our knowledge about the stability of the protein structure in its native or misfolded forms, as the misfolded protein results in a range of diseases, including numerous neurodegenerative 14



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Figure 5. Some common and important post-translational modifications (PTMs) of MSY proteins. The PTMs were extracted from UniProt, Phosphositeplus, dbPTM, Phospho.ELM and GPMdb. Black boxes represent protein domains including Dead, DEAD-BOX domain and H−C, Helicase_C domain in DDX3Y; RS4NT, RS4NT domain; S4, S4 domain, Rib-S4, Ribosomal_S4e domain; and KOW, KOW domain in RPS4Y1 and RPS4Y2; WD40, WD40 domain in TBL1Y; eIF-1a, eIF-1a domain in EIF1AY; ARID, ARID domain; zf-C5HC2, zf-C5HC2 domain; and PLU-1, PLU-1 domain in KDM5D; Coes, Coesterase domain and TM, TM domain in NLGN4Y; Zfx-Zfy, Zfx_Zfy_act domain and zf-C2H2, zf-C2H2 domain in ZFY; ECH, ECH domain in CDY2B and CDY1B; TYM, Thymosin domain in TMSB4Y; Pkinase, Pkinase_AGC domain in PRKY; HMG, HMG-Box domain in SRY; HSF-DNA, HSF-DNA Bind domain in HSFY; NAP, NAP domain in TSPY.

PTMs. Although more than 300 different types of PTMs have been reported and many more are still being reported, a few are common and functionally important.121 Figure 5 illustrates the important modification sites and topology of the modified amino acids in the male specific region of the Y chromosome-linked proteins in six common PTM types. The experimental validated PTM data sources were extracted from Swiss-Prot, Phosphositeplus, dbPTM, Phospho. ELM and GPMdb. Among MSY protein coding genes 25 proteins showed 6 common types of PTM including phosphorylation, ubiquitination, acetylation, methylation, deamination, and glycosylation or glycation.121 The most abundant PTM found in DDX3Y (n = 67) which is subjected to 5 PTM types including phosphorylation (n = 27), deamination (n = 19), acetylation (n = 3), ubiquitination (n = 9) and methylation (n = 9). Many of reported PTMs are within functional domains, for example, 25 PTMs of DDX3Y are found

case of the SRY protein, thermodynamic and spectroscopic analysis of the SRY protein as another transcriptional regulator suggests that SRY’s regulatory function on gene expression requires both specific DNA binding and DNA bending.118−120 These and other studies have opened the way for future structural, kinetic, and thermodynamic studies of the Y chromosome proteome.

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POST-TRANSLATIONAL MODIFICATION OF MSY PROTEINS Among various regulatory mechanisms available to the cell, posttranslational modifications (PTMs) serve a particular niche in being highly dynamic and largely reversible. PTMs give rise to great proteome complexity and diversity of structure and function of proteins.121 Currently, large-scale protein identification projects allow identification and quantization of various PTMs. Such platforms allows identification and quantification of various 15



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within the DEAD - box domain meanwhile 10 of them lie within Helicase_C domain. It seems that DDX3Y is regulated by a vast and dynamic array of PTMs that would strictly control its structure and function in nucleic acid binding and helicase activity. MSY proteins including EIF1AY, RPS4Y1 and RPS4Y2 have all 6 type of PTMs. Krick et al. reported that after phosphorylation by CSNK2A1, TSPY transport into the nucleus and acts as motivator of cell growth and proliferation.122 Despite the great importance of PTMs for biological function, their study in MSY proteins has been very limited and biological implications of such key modifications have yet to be discovered. All in all, we do not realize the full extent and functional importance of MSY protein modifications in cell mechanism and function.

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MINING MSY TRANSCRIPTS IN RNA SEQUENCING DATA As the cost of the next-generation sequencing methods has significantly decreased in the past few years, RNA sequencing (RNA-seq) has become a popular method for transcriptome analysis.123 This approach has generated a wealth of data. We reanalyzed the RNA-seq data of 13 human tissues, as well as 10 differentiated and undifferentiated cell lines using all of the sequence reads generated by the BodyMap2 (ERP000546), Cancer Transcriptome (SRP005242), and Human Embryonic Stem Cell Transcriptome (SRP002079) projects. Expression levels were measured in sensitive and specific reads per kilobase of exon model per million mapped reads (RPKM), which is a measure of transcription activity.124 Results have indicated that over 50% of the MSY confirmed or putative protein coding genes have a testis-specific expression pattern at the transcript level (Figure 6). Likewise, CYorf17 was not detected in the cell lines or tissues at the cutoff value of 0.1 RPKM. Hierarchical clustering analysis was performed on the MSY-selected gene and a heat map was generated. Our analysis showed that among all analyzed cell lines, prostate cancer cell lines were enriched for RPS4Y1, USP9Y, KDM5D, PCDH11Y, and TBL1Y transcripts, whereas and hESC lines were enriched for PRKY, TMSB4Y, NLGN4Y, AMELY, and RPS4Y2 transcripts. As expected, comparison of various tissues showed that most of MSY transcripts were enriched in testis tissue. Moreover, we could identify several novel exons for the MSY genes including PCDH11Y, UTY, RPS4Y1 genes in hESC-H1 (data not shown). Analysis of RNA-seq data has also revealed transcript evidence for XKRY1, XKRY2, CSPG4P2Y, and GOLGA2LY1, TTTY10, TTTY12, and TTTY13 which warrants further investigation to determine the protein evidence for these genes (Figure 6).

Figure 6. Hierarchical clustering and the transcript profiles of MSY genes in the 14 human tissues and 11 cell lines based on RNA-seq data. We mapped the sequence reads to the reference human genome sequence (ENSEMBL GRCh37.64 assembly) using TopHat (version 1.3.2)126 and Bowtie (version 0.12.7).127 Then, we assembled the alignments into gene transcripts and calculated their relative abundances from known coding and noncoding regions of the Y chromosomes using Biocunductor.124 The value was calculated by dividing the number of reads mapping to the protein coding part of each gene by the length of the protein coding part of the gene and the total number of reads from the library to compensate for different read depths in different samples. The human embryonic stem cell (hESC) lines include undifferentiated hESCs (hESCs_ hESC_A_p1_1 and hESC_B_p1_1), early initiation of neural differentiation (N1_A_p1_1), differentiation into neuronal progenitor cells (N2_A_p1_1, N2_A_p1_1 and N2_B_p1_1), and differentiation into early glial-like cells (N3_A_p1_1). DU-145, VCap, LnCap, RWPE are prostate cancer cell lines.

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HUMAN Y CHROMOSOME PROTEOME PROJECT (Y-HPP) The Y-HPP working group has been established to organize a collaborative network among research teams comprised of geneticists, molecular biologists, anthologists, biochemists, cell biologists, pathologists and clinicians. This network aims to effectively integrate proteomics data into genomics and other laboratory and clinically relevant data with the intent to generate a comprehensive knowledge of MSY-associated biological systems. To facilitate the overall progress and management of the Y-HPP and make the data and metadata openly available, the Y-HPP web portal (www.hupo.ir) will be used with the intent to become a central focal point for the Y-HPP. This web portal is linked to the main C-HPP (www.c-hpp.org).

The C-HPP consortium is of the belief that high quality, extensive proteome maps are achievable within a two-phase plan to be conducted over a 10-year period. In phase 1, which is approximately 6 years, we plan to map all proteins that currently lack high quality protein and mass spectrometry evidence 16



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Figure 7. Overall plan of the YHPP project. In phase 1, which is estimated to take six years, YHPP starts with defining the list of proteins coded by the human Y chromosome and a list of missing or poorly characterized proteins. The project includes a systematic exploration of the human Y chromosome proteome using mass spectrometry and antibody-based proteomics. Specific antibodies are produced and validated. Efforts will be made to confirm all protein-coding gene predictions, and PTMs, alternative splicing transcription (AST) products, and many nsSNP sequence variants will be investigated. The characterizations will be followed by analyzing the expression, modifications and interaction of proteins in healthy and diseased tissues as well as cell lines. The study begins by analyzing normal prostate and prostate cancer tissues, and normal and azoospermia testes. Proteomic studies will be backed up with relevant genomics, transcriptomics, epigenomics data. In phase 2, which is approximately four years, identified proteins will be further studied in other tissues and diseases using additional proteomic measurements.

(Figures 1, 2, and 7). In addition, efforts will be made to confirm all protein-coding gene predictions, such as XKRY, CSPG4P2Y, and GOLGA2LY1 in MSY. We will also investigate PTMs, numerous representative alternative splicing transcription (AST) products, and many nsSNP sequence variants (Figure 2). Our strategy began with the definition of the proteins coded by the human Y chromosome and a list of missing or poorly characterized proteins as presented above (Figure 7). The project has been established to allow for a systematic exploration of the human Y chromosome proteome using mass spectrometry and antibody-based proteomics. Specific antibodies to human Y chromosome target proteins are being produced using a method that involves the cloning and protein expression of protein epitope signature tags. The homemade antibodies are listed in Figure 1. The antibodies are being subsequently validated using several approaches, including siRNA. The characterizations will be followed by antibody-based detection in selected tissues (such as testis

and prostate) and cell lines that include prostate cancer, human embryonic stem cell, and human embryonal carcinoma stem cell. The expressions of proteins will be analyzed in healthy and diseased tissues, such as normal prostate and prostate cancer tissues, and normal and azoospermia testes. We will also align the proteomics data set to the output of microarray, RNA-seq and real-time polymerase chain reaction analysis data with defined expression thresholds. The already available transcriptome data could help us to get information about the expression of Y chromosome genes and their contribution to disease and therefore better define targeted future proteomic experiments, For example, while most of published papers highlighted the association of Y chromosome genes in prostate cancer (Figure 4), the analysis of cancer microarray database (ONCOMINE)125 revealed that the expression of Y chromosome genes were mainly altered in kidney and testicular cancers. Furthermore, a coexpression of the ampliconic genes was observed in various cancers. This warrants 17



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further research on the genetic base(s) and functional consequences of coexpression of MSY genes in different diseases (data not shown). In phase 2, which is estimated to take four years, identified proteins will be further studied in other tissues and diseases using additional proteomic and antibody measurements3 (Figure 7).

Notes

CONCLUSION AND PERSPECTIVE The objective of Y-HPP is to map and annotate all proteins encoded by genes on the MSY sequences. Throughout this 10year project, the Y-HPP aims to generate information useful in the search for new biomarkers and drug targets, and in the study of disease gene families clustered in MSY. As outlined by Paik et al.,3 there are a number of short- and long-term challenges that need to be overcome. The short-term challenges include improved crossanalysis and integration of different data sets, handling of data variability, cost-effective technology development, and harmonization with Biology/Disease-HPP. Possible solutions have been proposed that include sharing resources, data, reagents (e.g., antibodies), and reference specimens; a standard data submission system or criteria; employing NeXtProt, dbSNP, GPMdb, and PeptideAtlas; and sharing data through the C-HPP portal and other public biology/ disease databases. Although it is more difficult to predict long-term challenges, they will include enhanced detection limits for low abundance (rare) proteins, sample biobanking, and maintenance and inclusion of complex PPI and PTM information in different data sets and biological functions. Potential solutions for the longerterm challenges may include development of new algorithms for inclusion of PPI and PTMs in the biological databases, miniaturization of sample preparation, multiplexed fractionation steps, and collaboration with government agencies for stable biobanking resources.3

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Y-HPP is supported by a grant from Royan Institute. P.A.H. acknowledges funding support from the Australian Research Council.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure 1. Similarity of MSY proteins with proteins on X and autosomal chromosomes. Similarity search was performed with BLAST (Basic Local Alignment Search Tool) against nonredundant protein database. The presented proteins of X and autosomal chromosomes showed the highest similarity with their corresponding Y chromosome proteins. Similarity has been presented as percent identity and percent positive. Percent identity means identical amino acids within the noted alignment length. Percent positive scores represent the given amino acid substitution that occurs more frequently in the alignment than expected by chance. Only one protein of multiple copy genes has been presented. Supplementary Figure 2. The classification of MSY protein-coding genes based on gene distribution in MSY sequence classes, alternative splicing transcript, protein isoforms (several different forms of the same protein), and gene copy number. Supplementary Figure 3. Number of papers published on MSY protein-coding genes. Only a small portion of papers include protein studies mainly related to SRY. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Legrain, P.; Aebersold, R.; Archakov, A.; Bairoch, A.; Bala, K.; et al. The Human Proteome Project: current state and future direction. Mol. Cell. Proteomics 2011, 10, M111 009993. (2) Paik, Y. K.; Jeong, S. K.; Omenn, G. S.; Uhlen, M.; Hanash, S.; et al. The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome. Nat. Biotechnol. 2012, 30, 221−223. (3) Paik, Y. K.; Omenn, G. S.; Uhlen, M.; Hanash, S.; Marko-Varga, G.; et al. Standard guidelines for the chromosome-centric human proteome project. J. Proteome Res. 2012, 11, 2005−2013. (4) C-HPP-Co-Chairs. C-HPP Overview paper http://www.c-hpp.org/. (5) Skaletsky, H.; Kuroda-Kawaguchi, T.; Minx, P. J.; Cordum, H. S.; Hillier, L. D.; et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 2003, 423, 825−837. (6) Sinclair, A. H.; Berta, P.; Palmer, M. S.; Hawkins, J. R.; Griffiths, B. L.; et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990, 346, 240−244. (7) Tiepolo, L.; Zuffardi, O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum. Genet. 1976, 34, 119−124. (8) Veitia, R. A.; Salas-Cortés, L.; Ottolenghi, C.; Pailhoux, E.; Cotinot, C.; et al. Testis determination in mammals: more questions than answers. Mol. Cell. Endocrinol. 2001, 179, 3−16. (9) Knower, K.; Kelly, S.; Harley, V. Turning on the male−SRY, SOX9 and sex determination in mammals. Cytogenet. Genome Res. 2003, 101, 185−198. (10) Gubbay, J.; Collignon, J.; Koopman, P.; Capel, B.; Economou, A.; et al. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990, 346, 245−250. (11) Ner, S. S. HMGs everywhere. Curr. Biol. 1992, 2, 208. (12) Harley, V. R.; Clarkson, M. J.; Argentaro, A. The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocrine Rev. 2003, 24, 466−487. (13) Gasca, S.; Cañizares, J.; de Santa Barbara, P.; Méjean, C.; Poulat, F.; et al. A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11199. (14) Wilhelm, D.; Palmer, S.; Koopman, P. Sex determination and gonadal development in mammals. Physiol. Rev. 2007, 87, 1−28. (15) Harley, V. R.; Layfield, S.; Mitchell, C. L.; Forwood, J. K.; John, A. P.; et al. Defective importin β recognition and nuclear import of the sexdetermining factor SRY are associated with XY sex-reversing mutations. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7045. (16) Harley, V. R.; Lovell-Badge, R.; Goodfellow, P. N.; Hextall, P. J. The HMG box of SRY is a calmodulin binding domain. FEBS Lett. 1996, 391, 24−28. (17) De Kretser, D. Male infertility. Lancet 1997, 349, 787−790. (18) Carrell, D.; De Jonge, C.; Lamb, D. The genetics of male infertility: a field of study whose time is now. Syst. Biol. Reprod. Med. 2006, 52, 269−274. (19) Foresta, C.; Moro, E.; Ferlin, A. Y chromosome microdeletions and alterations of spermatogenesis. Endocrine Rev. 2001, 22, 226−239. (20) Vogt, P. H. Azoospermia factor (AZF) in Yq11: towards a molecular understanding of its function for human male fertility and spermatogenesis. Reprod. Biomed. Online 2005, 10, 81−93.

AUTHOR INFORMATION

Corresponding Author

*Tel: +98 21 22306485. Fax: +98 21 23562507. E-mail: salekdeh@ royainstitute.org; [email protected]. Author Contributions ‡

These authors contributed equally to this work. 18



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A Fresh Look at the Male-specific Region of the Human Y

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