Chromosome-Centric Human Proteome Project Allies with

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Chromosome-Centric Human Proteome Project Allies with Developmental Biology: a Case Study of the Role of Y chromosome Genes in Organ Development Anna Meyfour, Paria Pooyan, Sara Pahlavan, Mostafa Rezaei-Tavirani, Hamid Gourabi, Hossein Baharvand, and Ghasem Hosseini Salekdeh J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00446 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Journal of Proteome Research

Chromosome-Centric Human Proteome Project Allies with Developmental Biology: a Case Study of the Role of Y chromosome Genes in Organ Development

Anna Meyfour1,2, Paria Pooyan1, Sara Pahlavan3, Mostafa Rezaei-Tavirani2, Hamid Gourabi4, Hossein Baharvand3,5*, Ghasem Hosseini Salekdeh1,6*

1

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

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

Proteomics Research Center, Department of Basic Science, Faculty of Paramedical

Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3

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

Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 4

Department of Genetics at Reproductive Biomedicine Research Center, Royan

Institute, Banihashem Sq., Banihashem St., Ressalat highway, Tehran, Iran 5

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

6

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

Karaj, Iran

*Corresponding authors: Ghasem Hosseini Salekdeh, Ph.D., Hossein Baharvand, Ph.D. Royan Institute, Banihashem Sq., Banihashem St., Ressalat highway, Tehran, Iran. Postal Code: 1665659911, P.O. Box: 16635-148, Tel: +98 21 22306485, Fax: +98 21 23562507. Emails: [email protected], [email protected] 1

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Abstract One of the main goals of Chromosome-Centric Human Proteome Project is to identify protein evidence for missing proteins (MPs). Here, we present a case study of the role of Y chromosome genes in organ development and how to overcome the challenges facing MPs identification by employing human pluripotent stem cell differentiation into cells of different organs yielding unprecedented biological insight into adult silenced proteins. Y chromosome is a male-specific sex chromosome which escapes meiotic recombination. From an evolutionary perspective, Y chromosome has preserved 3% of ancestral genes compared to 98% preservation of the X chromosome based on Ohno’s law. Male specific region of Y chromosome (MSY) contains genes that contribute to central dogma and govern the expression of various targets throughout the genome. One of the most well-known functions of MSY genes are to decide the malespecific characteristics including sex, testis formation and spermatogenesis which are majorly formed by ampliconic gene families. Beyond its role in sexspecific gonad development, MSY genes in co-expression with their X counterparts, as single copy and broadly expressed genes, inhibit haplolethality and play a key role in embryogenesis. The role of X-Y related gene mutations in the development of hereditary syndromes suggests an essential contribution of sex chromosome genes to development. MSY genes, solely and independent of their X counterparts and/or in association with sex hormones, have a considerable impact on fetus development. In this review, we present major recent findings on the contribution of MSY genes to gonad formation, 2


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spermatogenesis, and the brain, heart and kidney development and discuss how Y chromosome proteome project may exploit developmental biology to find missing proteins. Keywords: Chromosome-Centric Human Proteome Project (C-HPP), Y chromosome, gonad formation, organ development

3



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Introduction The chromosome-centric human proteome project (C-HPP) is designed to address complete map of expression, quantitation, sub-cellular localization, and functional properties of the entire human proteome. One of the main goals of this project is to identify protein evidence for missing proteins (MPs), an inevitably challenging job. The possible limiting factors might reside in I) the expression of MPs in unusual and rare organs or cell types which could be resolved by employing larger entity of samples, II) MPs of the low abundance or unsuitable for mass spectrometry which necessitates the development of more advanced techniques for their identification , III) MPs that are only expressed in specific or early developmental stages and are silent in adult cells 1. The adult silenced genes highlighted the importance of epigenetic regulation as many developmental specific genes become silent due to DNA-histone association and DNA methylation. Therefore, epigenetic manipulation of fully differentiated adult human cells might be a promising approach to discover more MPs involved in development as it has already resulted in identification of 29 MPs specific to spermatogenesis and development 2. Furthermore, another promising approach is to employ novel techniques such as human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). hPSCs can selfrenew indefinitely in culture while maintaining the ability to become almost any cell type in the human body.

4


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Here, we present a case study of the role of Y chromosome genes in organ development and introduce hPSC-based developmental studies as a novel potential approach for human proteome project to identify missing proteins which might function during organogenesis and are silent in adult fully differentiated cells. Y chromosome is a haploid, male-specific sex chromosome which escapes meiotic recombination. It has a nonrecombining region (NRY) flanked by two pseudoautosomal regions in either sides which altogether have a 60 Mb size. NRY which is also known as male-specific region of Y chromosome (MSY) comprises 95% of Y chromosome length 3, 4

. According to the last release of neXtProt (2017-0-4, v2.9.0), there are 48 coding

genes on Y chromosome from which, 26 have protein evidence (PE1), 22 do not have experimental protein evidence among which 10 have been validated at transcript level (PE2), 5 are at homology base (PE3) and 7 are at uncertain level (PE5) (www.neXtProt.org)(Table 1). From an evolutionary perspective, X and Y chromosomes are originated from autosomes since 300 million years ago and since then, Y retained only 3% of ancestral genes compared to 98% preservation of the X chromosome

5,

6

. Although Y

chromosome has rapidly lost most of ancestral genes, but remained stable over the last 25 years preserving a particular set of ancestral genes

7, 8

. The reason behind this non-

random preservation was explained in the report by Belllott et al 9. They reconstructed Y chromosome evolution across eight mammals and showed convergent conservancy of X-Y gene pairs in the placental and marsupial lineages, exhibiting long survival despite rapid decay and implicated coherent function in regulation of each central dogma stages. As stated for central dogma, these MSY genes contribute to transcription, 5



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translation, and protein stability and in a bigger picture they control the expression of 10, 11

several targets throughout the genome

. The most important mechanisms

underlying their survival are likely to be (1) amplification of multi-copy gene families that govern male gonad development and spermatogenesis

4, 9

, and (2) conservation of

single copies to maintain the correct concentration of extensively-expressed genes vital for both sexes

3, 10, 12

. The X homologue of most dose-sensitive single-copy genes with

breadth of expression escapes X chromosome inactivation

9, 13

and those which are

inactivated have Y homologues with different function. The Turner’s syndrome (monosomy X) is an example of the presence of dose-insufficient sex-related genes. One percent survived fetuses of monosomy X are often mosaic for whole or part of the second sex chromosome (X or Y) 9. Furthermore, some inherent diseases such as Kabuki syndrome, X-linked intellectual disability and autism result from mutations in sex chromosome genes UTX

14

, KDM5C

15

, NLGN4X/NLGN4Y

16

, respectively. The role of

X-Y related gene mutations in the development of these inherent syndromes suggest an essential contribution of sex chromosome genes to embryogenesis. Similarly, PCDH11Y/PCDH11X are sex-linked genes with high expression in the brain and spinal cord during embryonic development

17

and no expression in non-human primates

18

.

Mutations of PCDH11Y/PCDH11X result in disruption of language development and occurrence of non-syndromic speech delay

19

. Y chromosome genes in co-expression

with X homologues can greatly impact the development. X-Y pair genes are expressed during early embryogenesis even before gonad formation

20, 21

. The overexpression of

X-Y pair genes has been identified as early as the onset of zygote gene activation in mammalian development 9. Moreover, sexual dimorphism reportedly emerged in both 6


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genders physiology and pathophysiology particularly the incidence of some hereditary disorders such as autism with 4 times higher rate in males

22

. All these differences from

anatomy to sex-related diseases, originate from sex chromosomes. Additionally, MSY genes, solely and independent of their X counterparts and/or in association with sex hormones, regulate development. These findings necessitates a more focused and detailed study of Y chromosome genes function during development. Our recent findings also showed that Y chromosome genes play an important role during cardiac and nervous system formation

23, 24

. In this review, we discuss the major findings on the

role of Y chromosome genes in various organs development using conventional and novel technologies such as differentiation of hPSCs into multiple cell types of almost all human organs.

Y chromosome in testis formation The phenotypic sex of an individual is mainly determined by the type of gonad that develops in the embryo

25

. Gonad development is a sequential process triggered and

controlled by individual’s sex chromosome complement (specifically the presence or absence of a Y chromosome). This process is followed by the formation of the genital ridge, migration of the primordial germ cells, and the sexual dimorphic differentiation of the gonads

26, 27

. This developmental process directs the bipotential genital ridge to

develop one of the two functionally distinct organs, a testis or an ovary

27

. In human

embryos, formation of the bipotential gonad occurs 33 days post-ovulation (d.p.o.)25. Development of the genital gonads is governed by several central transcription factors that their mutation may inhibit further development of the bipotential gonad 25, 27. Studies 7



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on mammalian testis development, identified SRY (a gene located on the short arm of the Y chromosome) as a master gene initiating a cascade of events preceding testis differentiation and ovarian developmental inhibition

25, 28

. An initial SRY expression in

male gonadal ridge is observed during 41-44 d.p.o. that reaches a peak at 44 d.p.o., when sex cords are first visible. SRY expression remains at low levels within the newly formed sex cords throughout the embryonic period and after that

26

. It has been

suggested that SRY may act as a transcription repressor for pluripotency-associated genes and as a transcription activator for differentiation-related genes29. In male genital ridge, SRY expression at its critical level is accompanied by sexually dimorphic expression of its downstream target, SOX9

25

. Compared to SOX9

expression in males, its expression decreases in female developing gonads

30

. SRY

together with steroidogenic factor 1 (SF1, also known as nuclear receptor subfamily 5, group A, member 1 [NR5A1]) directly binds to a specific site (‘TESCO’, testis-specific enhancer of Sox9 core) located upstream of the transcriptional start site of SOX9 between _13 and _10 kb resulting in its upregulation. Upregulated SOX9 autoregulates its expression through binding to TESCO 25, 31. Consequently, the sequential expression of these sex-determining genes causes the activation of downstream targets such as Amh 32, Vnn 33, Pgds and Cbln4 34 (Fig. 1). In addition to activation of Sox9, Sry influences the expression level of various target genes leading to early events of testis differentiation

31

. This male-specific gene takes

part in Sertoli cell fate determination (by binding to and regulating Ptgds) 31, inhibition of ovarian development (by repressing Wnt signaling-dependent gene expression) Sertoli cell proliferation (by targeting Fgf9)

36

35

,

, M germ stem cell niche development (by

8


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targeting Gdnf)

37

, meiotic arrest in germ cells and their M lineage determination (by

activating Cyp26b1)38, and eventual formation of the testis cord and encapsulating the germ cells (by stimulating Wnt5a, Dhh and Ntf3)

31, 39

(Fig. 1). Despite wide range of

target genes and diverse functions of SRY, it is not thoroughly clear how it is being regulated. However, several regulators have been introduced including Gata4, Zfpm240 , Wt134 and Nr5a141, Cbx242 , Jmjd1a, Dax126.

Y chromosome in spermatogenesis Non-obstructive azoospermia in men (complete absence of sperm in the ejaculate) frequently harbours microdeletions within the Azoospermia Factor (AZF) regions (consisting of three sub-regions of AZFa, AZFb and AZFc) of the Y chromosome

43, 44

.

AZFa region of human Y chromosome plays an important role in spermatogenesis as deletions within it, result in irreversible spermatogenic defects causing lack of germ cells in the testis seminiferous tubules diagnosed as Sertoli-cell-only syndrome (SCOS) 45, 46. Further studies demonstrated that deletions within two AZFa genes namely, DDX3Y and USP9Y are responsible for SCOS phenotype role

47

with DDX3Y having a more critical

48

. DDX3Y is a RNA helicase belonging to DEAD-box family which has important

role in cell cycle control, programmed cell death and most of the processes of RNA metabolism including mRNA nuclear export, transcriptional regulation and translational initiation

49

. This gene is expressed in human pre-meiotic spermatogonia during early

stages of fetal testis development

50

(Fig. 1). Additionally, Gueler and colleagues

identified DDX3Y expression after birth in prepubertal Ap spermatogonia suggesting a key role for this protein in male germ cell proliferation and survival 9


50

. Furthermore,


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generation of a stable AZFa knockout iPSC line transfected with DDX3Y resulted in formation of prospermatogonial germ cell-like cells as well as germ cell differentiation indicating a regulatory role for DDX3Y in early male germline development 51. AZFb region of Y chromosome contains two main genes (RBMY and PRY) which function during spermatogenesis. While hypospermatogenesis is induced by deletions of all AZFb genes except RBMY and PRY, deletions of these two genes resulted in a complete meiotic arrest

52

. RBMY as a testis-specific splicing factor is expressed at all

stages of germ cell development including A and B spermatogonia, spermatocytes and round spermatids 53(Fig. 1). Its expression and colocalization by splicing factors mediate regulation of RNA splicing at early stages of spermatogenesis

53, 54

. Mice deficient in

Rbmy1 showed spermatogenic arrest which further introduces Rbmy1 as a crucial gene in normal spermatogenesis

55

. Furthermore, recent studies have assigned a role for

sufficient copy numbers of functional RBMY1 in sperm motility which might result in asthenozoospermia in conditions of insufficient copies expression56. PRY gene of AZFb encodes a protein phosphatase that plays a role in regulation of apoptosis required for eliminating non-functional spermatozoa during apoptotic degradation. Although PRY expression was identified in spermatids and spermatozoa, it was absent in premeiotic germ cells (Fig. 1). Therefore, its contribution to meiotic arrest in AZFb deletions is not clear 57. Deleted in Azoospermia (DAZ) genes are known to have crucial role in sperm development

43, 58

. DAZ proteins encoding genes, located within AZFc region, contain a

highly conserved RNA recognition motif (RRM) and a distinctive 24 amino acid sequence known as DAZ repeats

43, 58

. During germ cells differentiation, RNA binding 10


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proteins play crucial roles to store network of mRNAs and control translation timing. It seems that DAZ proteins as RNA binding proteins, take part in translation initiation of mRNA targets

59

. DAZ expression is almost seen at all stages of normal

spermatogenesis from primordial germ cells to spermatogonia, early and late spermatocytes, postmeiotic germ cells and eventually sperms

43

(Fig. 1). DAZ

overexpression resulted in differentiation of both hESCs and iPSCs into primordial germ cells like cells (PGCLCs) and facilitated their further maturation and development

60

.

Furthermore, overexpression of human DAZ in Dazl null mice could partially restore germ cell number

61

. Hence, DAZ proteins may lead to germ cells specification and

meiosis and maintain primordial germ cell population

59

. Chromodomain Y-linked (CDY)

gene of AZFc encodes a protein containing a chromodomain and a histone acetyltransferase catalytic domain essential for generation of protamine-based form of histone-based chromatin structures in late spermatid nuclei spermatogonia, spermatocytes and round spermatids

63

47, 62

. BPY2 is expressed in

(Fig. 1) and believed to interact

with ubiquitin protein ligase E3A which makes it a possible candidate for preservation of human sperm fertility

64

. Y chromosome has copies of TSPY genes which are

expressed in spermatogonia and they regulate the timing of meiosis initiation 65.

Y chromosome in nervous system development Human brain is the most complicated organ, which shows marked sex differences between males and females from anatomical, functional and biochemical perspectives 66

. Sex differences in the brain have been found at all stages of development and can

affect various parameters such as brain area volume, cell number and cytoarchitecture, 11



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neural functions, synaptic connectivity, perception, cognition and memory. Moreover, pathophysiology of neurological and neuropsychiatric disorders including the incidence, age of onset, prevalence and severity as well as their symptomatology are sexdependent

67, 68

. Indeed, males and females brains go through different developmental

paths 69. Brain development is a lifelong process that starts prior to the formation of gonads (testis or ovary) and production of sex hormones

69, 70

. Hence, brain’s sexual destiny

starts with the differences in male and female sex chromosomes. Although sex chromosomes account for the differences between the two genders’ brains, the direct roles of their genes are not well studied

66

. As an inevitable consequence, XY and XX

brain cells have distinct transcriptional patterns that dictate their development and function 71. Transcriptional studies revealed the expression of several MSY genes (SRY, RPS4Y1, ZFY, PCDH11Y, TBL1Y, PRKY, USP9Y, DDX3Y, UTY, TMSB4Y, NLGN4Y, HSFY, TXLNGY, KDM5D & EIF1AY) in numerous regions of the male brain at various developmental stages from week 7 post-gestation to adulthood 67, 72, 73. According to the developmental transcriptome data from the BrainSpan atlas (www.brainspan.org), most of the Y chromosome genes are expressed ubiquitously during brain development (Fig. 2). Among these ubiquitously-expressed genes (e.g. RPS4Y1, ZFY, PCDH11Y, TBL1Y, PRKY, USP9Y, DDX3Y, UTY, TMSB4Y, NLGN4Y, CD24P4, TXLNGY, KDM5D & EIF1AY), some keep their fine expression during the entire life (e.g. ZFY, PCDH11Y, TBL1Y, PRKY, USP9Y, DDX3Y, UTY, TMSB4Y, NLGN4Y, TXLNGY, KDM5D & EIF1AY). According to neXtProt, TBL1Y (PE2), PRKY (PE5), TXLNGY & TTTY10 (PE5) & RPS4Y2 (PE2) are all considered as missing proteins. While their gene expression 12


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data during brain development were presented in BrainSpan. Therefore, they can be considered as potential candidates to search for their protein evidence using hPSC derived neural cells which is in accordance with the main goal of Human Proteome Project. RPS4Y1 virtually maintains its sharp expression, prenatal and during infancy; consequently, it may play a role at early stages of brain development. Interestingly, CD24P4 (Known as CD24 in neXtProt), located within MSY region of the Y chromosome, encodes a signal transducer protein and retains the highest gene expression level during the embryonic and prenatal periods followed by a decrease over the rest of life (Fig. 2). However, our understanding of MSY genes function is restricted due to lack of comprehensive studies that should have been done on different periods of brain development. SRY is expressed within various regions of the mammalian male brain especially in dopamine and catecholamine-abundant regions such as the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA). SRY protein co-localization with TH (tyrosine hydroxylase)-positive neurons in SNpc and VTA may partly explain the male-specific regulation of dopamine-dependent functions such as the control of movement, attention, and reward-based learning in males

68, 70, 74

. SRY knockdown in

human neuroblastoma cell line (M17) resulted in reduction of enzymes regulating dopamine synthesis and metabolism and consequently dopamine production. However, SRY overexpression caused an increase in enzyme level followed by dopamine synthesis. It has been shown that SRY activates regulatory region of the human TH promoter

74

. These data were in agreement with other studies done in rodents which

indicated SRY conserved function among mammalians 70. 13



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Protocadherin 11Y (PCDH11Y) is a human-specific gene emerged on Y chromosome through a duplication and transposition of its paralog, PCDH11X, 6 million years ago

11,

75, 76

. PCDH11Y encodes a protein which consists of an extracellular domain with seven

cadherin repeats, a short transmembrane region, and a cytoplasmic tail

75, 76

. Based on

longitudinal gene expression profiling of males brain, PCDH11Y mRNA is highly expressed in infants while this expression level decreases in childhood and reaches the 72, 73

lowest expression level in adults

. PCDH11Y which is a cell adhesion molecule

takes part in cell survival, fate determination and organization of synapses in different neuronal populations

67

. Johansson and colleagues showed PCDH11Y expression in

NEUN+ neurons, SOX10+ oligodendrocyte precursor cells and ISL1+ motorneurons in different parts of the brain and the central nervous system (CNS)

67

. Blanco and

colleagues showed the upregulation of PCDH11Y in in vitro differentiation of prostate cancer cell lines towards neural lineage, induced by retinoic acid 77. It has been reported that cytoplasmic domain of PCDH11Y interacts with β-catenin resulting in its nuclear accumulation

78

. β-catenin as a component of canonical Wnt signaling pathway is

involved in the formation of the dorso–ventral (D–V) axis in several species such as zebrafish, Xenopus and mice

79, 80

. Wnt signaling is also required for the establishment

of the antero-posterior (A–P), and left–right (L–R) axes of the brain and body

81, 82

.

Interestingly, retinoic acid is also a crucial morphogen involved in the formation of the A–P axis and establishment of the D–V axis 76. NLGN4Y is a Y chromosome specific cell adhesion molecule, a member of neuroliglin (NLGN) family, and a postsynaptic transmembrane protein that interacts with neurexin (NRXN)83. Like other members of this family, NLGN4Y acts as an inducer and 14


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modulator of synaptic activity. NLGN4Y regulates synaptic adhesion and development through its extracellular domain which is an acetylcholinesterase (AChE) homologue without enzymatic activity

83, 84

. Like other cell adhesion molecules, therefore, NLGN4Y

is an essential factor for formation of functional synapses and its mutations as well as translation or function failure, may cause neurological disorders, such as autism. It has been shown that both NLGN4Y mutation in XY and overexpression in XYY, are associated with prominent risk of autism

85-87

. Another interesting finding on NLGN4Y

was that its expression reduced during brain development from early postconception weeks to adulthood 67, 72. In a study designed to address Y chromosome human proteome project, Vakilian and colleagues profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of a human embryonic carcinoma cell line, NTRA-2. The expression of RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY and ZFY were significantly increased during differentiation.

23

. Furthermore, DDX3Y knockdown in neural differentiation of NTRA-2

resulted in cell growth arrest at G1/S phase and overexpression of pro-apoptotic proteins 23.

Y chromosome in cardiac development Gender-related differences in basal cardiovascular function have been reported for two decades. There are physiological differences in left ventricular ejection fraction (LVEF) and stroke volume (SV), resulting in specific heart rates in men compared to women 89

88,

. These sex-related differences have been also observed at transcriptome level in 15



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human and rodents, many of which such as USP9Y, RPS4Y1, DDX3Y, XIST, TIMP1, UTY, ZFY and PRKY, are expressed on X-Y chromosomes

90

. Interestingly, sex

dimorphism has been observed in mitochondria transcriptome of rats heart as well

91

.

Moreover, there are gender-related differences in the prevalence of cardiovascular diseases

such

as

atherosclerosis,

myocardial

infarction,

myocarditis,

dilated

cardiomyopathy and heart failure with higher rates in men compared to hypertention in women

92-94

. Molecular biology studies showed that these sexual disparities present at

transcriptional level particularly in the expression of Y chromosome genes such as DDX3Y, RPS4Y1, USP9Y and JARID1D

95

. Therefore, sex can have an impact on

regulatory mechanisms of cardiovascular physiology and pathophysiology. While female protection may be in part due to estrogen influence

96

and male susceptibility due to

testicular hormone 97, but Y chromosome seems to play an important role in these malespecific cardiac functions

98

. A growing body of evidence has been emerged on Y

chromosome function by crossing Y chromosomes from different mouse strains onto a single background strain showing its impact on cardiac phenotype

99

. Llamas et al

exchanged the Y chromosome of C57BL (ChrYC57) with that of A/J mice (ChrYA), resulting in consomic strains (C57.YA and A/J.YC57)

100

. While both mice strains are

normotensive, C57BL mice hearts developed an eccentric hypertrophy phenotype compared to A/J mice

101

. Surprisingly, the cardiomyocytes size appeared to be smaller

in C57.YA compared to C57BL. On the other hand, A/J.YC57 obtained larger cardiomyocytes in comparison to A/J mice, indicating a contributory role for Y chromosome in the loci governing cardiomyocytes size in male mice. These findings provided valuable hints on Y chromosome genes contribution to cardiovascular 16


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phenotype. To further confirm this hypothesis, they studied the cardiomyocyte’s phenotype in consomic strains undergone pre-pubertal castration

102

. The strains

carrying YC57 showed a decrease in cardiomyocytes size in the absence of post-pubertal testosterone. However, YA failed to induce changes in cardiomyocytes size suggesting their insensitivity to the hypertrophic effects of post-pubertal testosterone. However, the transcriptome analyses showed different expression level for many cardiac genes in unaffected C57.YA cardiomyocytes even greater than that observed in C57BL mice. Furthermore, the cardiac transcriptome of castrated C57BL mice was compared to their sham-operated counterpart which showed a different expression of cardiac genes independent of testosterone

102

. These findings show the role of Y chromosome in

cardiac cell properties in the presence or absence of androgen regulations. To better identify how Y chromosome polymorphisms may impact cardiac characteristics, they further focused on these two male mice strains, C57BL and C57.YA103 and found different distribution of androgen receptors (ARs) in chromatins from neonatal male hearts of these two strains. Furthermore, they identified genes with AR occupancy which were enriched in biological processes related to heart development as well as hypertrophy. They also showed different chromatin accessibility and strain-specific H3K4me3-marked genes. Moreover, when they profiled Y chromosome genes in the heart of these two strains, they observed different expression of Jarid1d, Uty, Ddx3y and Eif2s3y among which Jarid1d and Uty displayed similar pattern in both heart and testis in a narrow window of 24 h after birth. These data, for the first time, provided a conceptual framework to unravel how Y chromosome regulates cardiac phenotype by affecting chromatin accessibility and neonatal programming 17


103

.

Androgens affect


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Page 18 of 44

cardiac circadian rhythm, expression of contractile genes and myocardial functional characteristics by influencing MSY genetic materials in the first 24 h of neonatal life. These findings introduce androgens as a necessary but not sufficient factor to some particular cardiac functions. Altogether, they suggested MSY as the origin of malespecific genetic factors which can modulate the androgen functions among individuals 98

.

As described previously, UTY was identified as one of MSY genes which may function in cardiac development. UTY and its X counterpart have been shown to be overexpressed during cardiac differentiation of ESCs

104

. These data identified a key

role for UTX in cardiac lineage differentiation of ESCs. UTX∆/∆ female mice died at embryonic stage; however, UTX∆/y male mice displayed various phenotypes including sever cardiac developmental defects suggesting a partial compensation of UTY for its X homologue. Furthermore, UTX-null mice and UTX-null ESCs provided information on UTX function as a coactivator of core cardiac transcription factors

104

. Another study

showed that male UTX knockout (KO) with UTY expression survived until birth, while female UTX KO embryos died from developmental defects in mesodermal lineages such as mesoderm-derived posterior notochord, cardiac and hematopoietic tissues. These results suggested a role for UTX/UTY in early embryonic development

105, 106

.A

recent study showed that UTX knockdown inhibited the direct cardiac reprogramming ability of a miR combo (miR-1, miR-133, miR-208 and miR-499) by restoring the level of H3K27me3 in fibroblasts 107. To better understand the Y chromosome function in cardiac development, the hormonefree models should be used. To address Y chromosome human proteome project, our 18


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group performed the first whole expression profiling of Y chromosome genes in a cellbased model of in vitro cardiac differentiation by using male hESCs. We identified alterations in gene expression of TBL1Y, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y, DDX3Y, PRY, XKRY, BCORP1, RBMY, HSFY and UTY (Fig. 3) which influenced protein expression and localization during cardiac differentiation. Among these, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y, DDX3Y, UTY, TBL1Y, PRY and BCORP1 showed overexpression at cardiac mesoderm development. Some other MSY genes such as RBMY, HSFY and BCORP1 were overexpressed during late cardiogenesis. All MSY genes which were expressed and showed alterations during cardiac differentiation of hESCs, are summarized in figure 3. Because TBL1Y overexpression appeared in a thoroughly different pattern compared to its X homologue24, we further studied the function of this missing protein in cardiac differentiation of hESCs. TBL1Y knock down negatively affected cardiac differentiation by stabilization of CtBP as a member of Notch co-repressor complex resulting in inhibition of Notch signaling activation24. Perissi and colleagues showed that Notch signaling activation requires the release of a dual repression caused by distinct corepressor complexes, CtBP and NCoR/SMRT

108

.

TBL1X/TBL1Y and TBLR1 are F box/WD40 containing factors that function as exchangers in the release of dual repression by dismissal of CtBP and NCoR/SMART from corepressor complex resulting in transcriptional activation of Notch target genes such as Hes124

108

(Fig. 4). Moreover, a delay in the onset of beating and an abnormal

contraction pattern was observed in cardiomyocytes lacking TBL1Y24 . Other reports also illustrated cardiac developmental defects in patients carrying TBL1Y mutations resulting in non-syndromic coarctation

109

. Furthermore, UTY overexpression during in 19



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vitro cardiac differentiation24 was in agreement with previous studies reporting UTY function in cardiac development

104-107

. Moreover, OFCD patients carrying BCOR

mutations show developmental heart defects with majority having septal malformations 110

. A growing body of evidence show a key role for BCOR as a transcriptional regulator

during early embryogenesis specifically cardiogenesis BCORP1

identification

at

transcript

level

and

111

its

. Based on our findings of overrepresentation

during

cardiogenesis, it may also impact cardiac development which has to be further investigated. These findings suggest that MSY genes either solely or in cooperation with their X homologue, regulate cardiogenesis. Therefore, there is an inevitable need for new experimental paradigm to increase the foundation of basic knowledge on MSY genes expression and their impact on gender-related developmental differences during cardiogenesis. Cardiogenic differentiation of hPSCs might be a possible approach for such cardiac developmental studies as well as congenital cardiovascular disorders.

Y chromosome in kidney development The kidney is an organ which filters wastes from body fluids, regulates blood electrolytes and maintains whole-body homeostasis. A key function of kidney is to regulate blood pressure by activation of renin-angiotensin system. Gender differences were described to affect the morphology and function of kidneys

112

. Despite similar

number of nephrons in both females and males, the mass of kidney is higher in the latter 113. Furthermore, it has been reported that the composition of renal transporters as well as infiltration of specific organic compounds were gender-related

112

. These sex-

related differences were reflected in the higher prevalence and progression of renal 20


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diseases in males as shown by 50% more men diagnosed with end stage renal disease (ESRD)

114

. Sex hormones might impact the progression of renal diseases in females

and males resulting in estradiol-related protection and testosterone-associated vulnerability, respectively

115

. However, these sex dimorphism might be also mediated

by the expression of sex chromosomal genes

116

. The gene expression of healthy

glomeruli and tubuli showed sexual dimorphism with enriched sex chromosome genes such as EHD2, DDX3Y, EIF1AY, CYorf14, ESPL1, KDM5D, PNPLa4, RPS4Y1 and XIST

117

. Sexual dimorphism of renal gene expression was also reported in ESRD

particularly the following nine genes: XIST, DDX3Y, KDM5D, CYorf14, EIF1AY, HDHD1A, RPS4Y1, STS and ZRSR2

117

. Moreover, renin-angiotensin system (RAS)

showed gender-related differences resulting in sex-specific blood-pressure regulation 118, 119

. Consomic rat models provided the first evidence on the contribution of Y

chromosome to regulation of blood pressure

120

. SRY was determined as a Y

chromosome gene responsible for elevation of the blood pressure by affecting RAS

121,

122

. SRY and its X homologue, SOX3, reportedly regulate renin and angiotensinogen 123

promoters

. Moreover, SRY expression/SOX3 absence in the kidney resulted in a

male-specific regulation of renin production. Therefore, SRY/SOX3-mediated regulation of RAS is a rate–limiting step in the kidney (due to SRY expression in kidney) resulting in a male-specific control of blood pressure 124. The overexpression of RAS components has been shown during kidney development. Furthermore, transgenic mice with targeted inactivation of renin kidney

125

or angiotensin

126

showed developmental defects in the

127

. Based on these findings, SRY might function in kidney development by

activation of SRY-mediated renin/angiotensin promoters. 21



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PRKX gene expression was identified in the fetal kidney as well as polycystic kidney, though it is absent in adult healthy kidney

128

. PRKX overexpression was shown to be

able to activate cellular migration and epithelial tubular formation in vitro

128

as well as

ureteric bud branching and glomeruli morphogenesis in embryonic mouse kidney explants suggesting its possible role in kidney development

129, 130

. Constitutive PRKX

activation in human autosomal dominant polycystic kidney disease (ADPKD) epithelial cell lines has shown to rescue adhesion and migration defects that could compensate PKD1 mutation in ADPKD phenotype

131

. Because PRKX escapes X inactivation,

therefore, it requires the expression of both alleles to exert its normal function. Furthermore, it has a highly similar sequence with its Y counterpart. Therefore, we suggest that PRKY might play an important role in male kidney development as well.

Conclusion and perspective Y chromosome governs male-specific characteristics including sex determination, testis formation and spermatogenesis. Most of Y chromosome genes contribute to these distinct male-specific functions particularly through ampliconic gene families (Table 1). Beyond its role in sex-specific gonad development, MSY genes in co-operation with their X counterparts as single copy and broadly-expressed genes prevent haplolethality and play a key role in fetus development. Moreover, Y chromosome genes can exhibit unique functions causing sexual dimorphism, independent or in association with sex hormones, dissimilar to their X-linked collective tasks during organ development. However, little knowledge exists on MSY proteins and their role during different organs development (Table 1). This might have been originated from the biological limitations 22


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and/or technical issues such as expression of MSY proteins in rare organs or cell types, low abundance of proteins, expression in specific or early developmental stages and being silent in adult tissues and cells, high similarity with X counterparts, and lack of specific antibodies again MSY proteins. In Y chromosome Human Proteome Project, we aimed to overcome these obstacles to unravel the gene expression and protein function of Y chromosome genes during development, taking the advantage of hPSCs. We aim to identify the existence, abundance and localization of MSY proteins during organ development specially MPs that are only expressed in early developmental stages and are silent in adult cells. Two unique characteristics of hPSCs make them well suited for these studies. First, hPSCs have unlimited self-renewal capacity, providing abundant material for mass spectrometry analysis. The discovery of patient-specific induced pluripotent stem cells (iPSCs) paved the way for developmental studies as well as end-stage pathophysiology of hereditary disorders and consequently, discovery of novel therapeutic. The access to the patient-specific iPSCs may also facilitate the discovery of missing proteins. Second, hPSCs have the potential to generate every adult cell type, offering an attractive window into proteins expressed during human development. Their in vitro culture system also provides a rapid, cost-effective way to interrogate the function of a gene during a specific developmental process and to identify highly similar missing proteins. Proteins such as RPS4Y2 and TBL1Y have been designated as “marginally distinguished” in PeptideAtlas due to their very high similar to their X homologue 23



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Page 24 of 44

counterpart. Therefore, there is no credible evidence to suggest that they can be distinguished from their X counterparts with the mass spectrometry data. Families of proteins with high similarity represent many of the indistinguishable and marginally distinguished matches in the databases.

They may remain unidentifiable, unless

uniquely-mapping or proteotypic peptides can be found to differentiate them 132. We recently exploited a genomics tool as a complement to protein studies to find TBL1Y, missing protein of Y chromosome24. We generated TBL1Y antibody using a peptide and showed that while TBL1Y expressed sharply in XY hESCs, no expression could be detected in XX hESCs. Furthermore, we observed that TBL1Y also showed overexpression and a thoroughly different expression pattern compared to its Xhomologue during differentiation of hESCs to cardiomyocyte. Using siRNA approach, we demonstrated that the RNA and protein abundance of TBL1Y declined in the siRNA transfected cells. All together, we could present compelling experimental evidence to distinguish TBL1Y from TBL1X, and propose reclassification of TBL1Y as ‘found missing protein’ (PE1). We expect that several Y chromosome missing proteins will be identified by mass spectrometry analysis of hPSC derived cells. Alternatively, in vitro culture system of hPSC can also provide a unique platform to identify the highly similar proteins and to interrogate their functions using various genomics and proteomics tools. The hPSCs, therefore, can act as a strong complement to C-HPP.

24


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Acknowledgment The Human Y Chromosome Proteome Project is supported by a grant from Royan Institute. Conflict of interest: The authors declare no competing financial interest.

25



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Figure legends

Figure 1. Y chromosome Genes and pathways required for the testis development and spermatogenesis. Bipotential gonadal ridge develops into one of two functionally distinct organs, a testis or an ovary. Genes such as Zfpm2, Wt1, Nr5a1, Cbx2, Jmjd1a and Dax1 are essential for the correct initiation and upregulation of SRY. SRY as a master gene initiates a cascade of events preceding testis differentiation and ovarian developmental inhibition by repressing Wnt signaling-dependent gene expression. In male genital ridge, SRY expression at its critical level is accompanied by sexually dimorphic expression of its downstream target, SOX9. In addition to SRY, Nr5a1 and Dax1 regulate SOX9 expression. Sex-determining genes activate the downstream targets such as Amh, Vnn, Ptgts, Cbln4, Fgf9, Gdnf, Cyp26b1, Wnt5a, Dhh and Ntf3 that are necessary for the regulation and maintenance of this crucial testis formation pathway. Human Y genes play an important role in spermatogenesis and their deletions result in spermatogenic defects. DDX3Y and TSPY is expressed in spermatogonia, RBMY, DAZ and BPY2 in spermatogonia, spermatocyte and round spermatid, USP9Y and CDY in spermatids, and PRY in spermatids and spermatozoa.

Figure 2. Heatmap presentation of the transcript profile of MSY genes during human brain development. All MSY gene expression data in male brain regions was downloaded from BrainSpan (atlas of developing human brain) covering prenatal (8-38 pcw), infancy (birth-18 mos), childhood (19 mos-11 yrs), adolescence (12-19 yrs) and adulthood (20-60+ yrs) developmental stages. The expression level of genes is shown 36


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in log2 RPKM (reads per kilobase per million) value. Abbreviations; pcw (post conception weeks), mos (months), yrs (years), DFC (dorsolateral prefrontal cortex), VFC (ventrolateral prefrontal cortex), MFC (medial prefrontal cortex), OFC (orbital frontal cortex), M1C (primary motor cortex), S1C (primary somatosensory cortex), IPC (posterior inferior parietal cortex), A1C (primary auditory cortex), STC (posterior(caudal) superior temporal cortex), ITC (inferolateral temporal cortex), V1C (primary visual cortex), HIP (hippocampus), AMY (amygdaloid complex), STR (striatum), MD (mediodorsal nucleus of thalamus) and CBC (cerebellar cortex), P (prenatal), I (infancy), C (childhood), T (adolescence or teenage years) and A (adulthood).

Figure 3. Visual representation of dysregulated MSY transcripts during cardiac differentiation of human embryonic stem cell (hESC). The Circos map compares the MSY gene expression pattern in hESC, mesendoderm, cardiac mesoderm, cardiac progenitor cell and cardiomyocyte. The inner colored segments below the outer segments are representative for each speciﬁc gene or differentiation stages; for instance, the red segment is related to the BCORP1 transcript, and the green segment corresponds to the mesendoderm stage. The outer segment of each gene demonstrates in which differentiation stages it’s mainly expressed. For example, BCORP1 mainly expressed in cardiac mesoderm stage (30%), cardiac progenitor stage (26%), cardiomyocytes (25%), respectively. The outer segment of each differentiation stage presents the contribution level of MSY genes in the expression of genes in each group (i.e. in cardiac mesoderm stage, among MSY genes, BCORP1 has the highest fold change ~ 11 %). The inner arcs link the expression of the genes to differentiation 37



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stages. The inner segment of genes sorted in a descending order for instance BCORP1 showed the maximal overexpression (0-50) specifically at cardiac mesoderm (~ 15%) while SRY showed the maximal downregulation (0-10) during cardiac differentiation. The inner segment of developmental stages show that most of Y chromosome genes overexpress at cardiac mesoderm stage (0-150) followed by other stages. Gene expression data obtained from REF.24.

Figure 4.

TBL1Y may play role in cardiac development by Notch signaling

activation. Cell to cell contacts and subsequent interaction of NOTCH receptor and its ligand, JAGGED 1, result in Notch signaling activation. Activation of this signaling requires the release of a dual repression caused by distinct corepressor complexes, CtBP and NCoR/SMRT/HDAC. TBL1X/TBL1Y and TBLR1 are F box/WD40 containing factors that function as exchangers in the release of dual repression by dismissal of CtBP and NCoR/SMART from corepressor complex resulting in transcriptional activation of Notch target genes such as Hes1. MAML stabilizes the RBP-J/NICD binding to DNA during activation of target genes.

38


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Table 1. Y chromosome genes, their status in neXtProt and findings on their roles in development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Y-Chr genes USP9Y VCY BPY2 EIF1AY PRY TMSB4Y UTY DDX3Y ZFY RBMY1A1 RBMY1C RPS4Y1 CD24 TSPY1 SRY DAZ2 RBMY1F DAZ4 TGIF2LY HSFY KDM5D PCDH11Y DAZ1 DAZ3 CDY2 CDY1 XKRY2 RBMY1B RBMY1E XKRY RBMY1D NLGN4Y RPS4Y2 AMELY TBL1Y PRORY TSPY2 TSPY3 TSPY4 TSPY8 TSPY10 SLC9B1P1 PRKY BCORP1 TTTY13 TTTY12 TTTY10 TXNLGY

PE level PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE1 PE2 PE2 PE2 PE2 PE2 PE2 PE2 PE2 PE2 PE2 PE3 PE3 PE3 PE3 PE3 PE5 PE5 PE5 PE5 PE5 PE5 PE5

Function ubiquitin-specific protease, Hydrolase unknown function Protein binding Translational initiation, Protein binding unknown function Actin monomer binding histone demethylase RNA helicase, DNA binding, RNA binding transcriptional activator RNA splicing, Protein binding RNA binding, RNA splicing RNA binding, rRNA binding Signal transducer activity, Protein binding, Protein binding Transcription factor, DNA binding RNA binding RNA binding, RNA splicing RNA binding DNA binding, protein binding Transcription factor, , protein binding Histone demethylase, DNA binding Calcium ion binding RNA binding, translation activator RNA binding Histone acetyltransferase Histone acetyltransferase unknown function RNA binding, mRNA splicing RNA binding, mRNA splicing unknown function RNA binding, mRNA splicing Cell adhesion molecule binding Translation. rRNA binding Structural constituent of tooth enamel Transcription activation/corepression unknown function unknown function unknown function unknown function unknown function unknown function proton antiporter activity Protein serine/threonine kinase unknown function unknown function unknown function unknown function Syntaxin binding

39


Human organogenesis Brain, spermatogenesis, heart spermatogenesis Brain spermatogenesis, heart Brain Brain, heart Brain, spermatogenesis, heart Brain, heart spermatogenesis, heart spermatogenesis, heart Brain, heart Brain spermatogenesis Brain, testis, kidney spermatogenesis spermatogenesis, heart spermatogenesis Brain, heart Brain, heart Brain, heart spermatogenesis spermatogenesis spermatogenesis spermatogenesis spermatogenesis, heart spermatogenesis, heart Single fertilization spermatogenesis, heart Brain Brain Tooth enamel development Brain, heart spermatogenesis spermatogenesis spermatogenesis spermatogenesis spermatogenesis Brain, kidney heart Brain Brain


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Y chromosome Genes and pathways required for the testis development and spermatogenesis. Bipotential gonadal ridge develops into one of two functionally distinct organs, a testis or an ovary. Genes such as Zfpm2, Wt1, Nr5a1, Cbx2, Jmjd1a and Dax1 are essential for the correct initiation and upregulation of SRY. SRY as a master gene initiates a cascade of events preceding testis differentiation and ovarian developmental inhibition by repressing Wnt signaling-dependent gene expression. In male genital ridge, SRY expression at its critical level is accompanied by sexually dimorphic expression of its downstream target, SOX9. In addition to SRY, Nr5a1 and Dax1 regulate SOX9 expression. Sex-determining genes activate the downstream targets such as Amh, Vnn, Ptgts, Cbln4, Fgf9, Gdnf, Cyp26b1, Wnt5a, Dhh and Ntf3 that are necessary for the regulation and maintenance of this crucial testis formation pathway. Human Y genes play an important role in spermatogenesis and their deletions result in spermatogenic defects. DDX3Y and TSPY is expressed in spermatogonia, RBMY, DAZ and BPY2 in spermatogonia, spermatocyte and round spermatid, USP9Y and CDY in spermatids, and PRY in spermatids and spermatozoa. 361x270mm (300 x 300 DPI)


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Figure 2. Heatmap presentation of the transcript profile of MSY genes during human brain development. All MSY gene expression data in male brain regions was downloaded from BrainSpan (atlas of developing human brain) covering prenatal (8-38 pcw), infancy (birth-18 mos), childhood (19 mos-11 yrs), adolescence (12-19 yrs) and adulthood (20-60+ yrs) developmental stages. The expression level of genes is shown in log2 RPKM (reads per kilobase per million) value. Abbreviations; pcw (post conception weeks), mos (months), yrs (years), DFC (dorsolateral prefrontal cortex), VFC (ventrolateral prefrontal cortex), MFC (medial prefrontal cortex), OFC (orbital frontal cortex), M1C (primary motor cortex), S1C (primary somatosensory cortex), IPC (posterior inferior parietal cortex), A1C (primary auditory cortex), STC (posterior(caudal) superior temporal cortex), ITC (inferolateral temporal cortex), V1C (primary visual cortex), HIP (hippocampus), AMY (amygdaloid complex), STR (striatum), MD (mediodorsal nucleus of thalamus) and CBC (cerebellar cortex), P (prenatal), I (infancy), C (childhood), T (adolescence or teenage years) and A (adulthood). 361x270mm (300 x 300 DPI)



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Figure 3. Visual representation of dysregulated MSY transcripts during cardiac differentiation of human embryonic stem cell (hESC). The Circos map compares the MSY gene expression pattern in hESC, mesendoderm, cardiac mesoderm, cardiac progenitor cell and cardiomyocyte. The inner colored segments below the outer segments are representative for each speciﬁc gene or differentiation stages; for instance, the red segment is related to the BCORP1 transcript, and the green segment corresponds to the mesendoderm stage. The outer segment of each gene demonstrates in which differentiation stages it’s mainly expressed. For example, BCORP1 mainly expressed in cardiac mesoderm stage (30%), cardiac progenitor stage (26%), cardiomyocytes (25%), respectively. The outer segment of each differentiation stage presents the contribution level of MSY genes in the expression of genes in each group (i.e. in cardiac mesoderm stage, among MSY genes, BCORP1 has the highest fold change ~ 11 %). The inner arcs link the expression of the genes to differentiation stages. The inner segment of genes sorted in a descending order for instance BCORP1 showed the maximal overexpression (0-50) specifically at cardiac mesoderm (~ 15%) while SRY showed the maximal downregulation (0-10) during cardiac differentiation. The inner segment of developmental stages show that most of Y chromosome genes overexpress at cardiac mesoderm stage (0150) followed by other stages. Gene expression data obtained from REF.24. 199x199mm (300 x 300 DPI)


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Figure 4. TBL1Y may play role in cardiac development by Notch signaling activation. Cell to cell contacts and subsequent interaction of NOTCH receptor and its ligand, JAGGED 1, result in Notch signaling activation. Activation of this signaling requires the release of a dual repression caused by distinct corepressor complexes, CtBP and NCoR/SMRT/HDAC. TBL1X/TBL1Y and TBLR1 are F box/WD40 containing factors that function as exchangers in the release of dual repression by dismissal of CtBP and NCoR/SMART from corepressor complex resulting in transcriptional activation of Notch target genes such as Hes1. MAML stabilizes the RBP-J/NICD binding to DNA during activation of target genes.

468x417mm (144 x 144 DPI)



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for TOC only 190x275mm (300 x 300 DPI)


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Chromosome-Centric Human Proteome Project Allies with

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