XY

Abstract

The X chromosome is one of the two sex chromosomes in humans. It is highly conserved among other mammalian species. The X chromosome accounts for about 5% of the total human genome and contains upward of 1200 genes. Many X chromosome genes, about one-fifth, appear to play a role in human cognition and brain development. The X chromosome is also unusual in that in females, who have two of these chromosomes, one undergoes inactivation as a means of dosage compensation since males have only one X chromosome (their other sex chromosome being a Y chromosome).

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X Chromosome

Y. Boyd, in Encyclopedia of Genetics, 2001

Human X-Linked Disease

The most common human syndromes associated with the X chromosome are anomalies in sex chromosome number that arise through nondisjunction at meiosis. Turner syndrome occurs in approximately 1 in 2000 female births and is caused by the loss of an entire chromosome leading to an XO karyotype. To explain why the presence of a single X chromosome is deleterious in XO females but not in XY males, it has been proposed that Turner syndrome is caused by a single, not double, dose of one or more of the few genes that normally escapes from X-inactivation. This is in tune with the observation that mice with an XO karyotype do not have an overt phenotype and that there are fewer mouse genes reported that escape X-inactivation. An additional X chromosome is present in the 1 in 600 males that are Klinefelter syndrome patients, who have an XXY karyotype. More rarely, females have also been identified with XXX and XXXX complements. Mutations, or rearrangements, in genes that are important in primary or secondary sex determination can give rise to females with an XY chromosome complement and males with an XX complement.

Mutations in single X-linked genes are fully expressed in males and give rise to 'sex-linked' disorders, for example, Duchenne muscular dystrophy which has an incidence of around 1 in 3000 males and the fragile X-linked mental retardation syndrome which has an incidence of around 1 in 10000 males. As a result of the random inactivation of one of their two X chromosomes in early development, all females are mosaics of two populations of cells and the relative numbers of cells in these two populations will differ between individuals. Often females heterozygous for a mutated gene are completely unaffected as the population of cells expressing the nonmutated allele either provides a sufficient quantity of normal gene product, or, during development or lineage differentiation, predominates over the population of cells carrying the mutant allele. However, some female carriers for X-linked 'recessive' diseases manifest some disease symptoms because of a natural skew in favor of cells with the mutated X as the active chromosome. Very occasionally, carrier females may manifest the same severity of disorder as that seen in males. Mutations in X-linked genes may also give rise to X-linked 'dominant' disorders found only in females and in these instances it is assumed that affected males die before birth. The most common example of an X-linked dominant is Rett syndrome, a severe progressive neurological disorder affecting approximately 1 in 20000 females, which has recently been associated with mutations in the gene encoding methyl-CpG-binding protein.

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Genetics as a Tool in Neurology

Dennis R. Johnson, Fuki M. Hisama, in Molecular Neurology, 2007

3. Sex-linked Inheritance

The X and Y chromosomes determine sex. In addition, the human X chromosome contains hundreds of other genes. Because females have two copies of the X chromosome, whereas males have only one (they are hemizygous), diseases caused by genes on the X chromosome, most of which are X-linked recessive, predominantly affect males.

Examples of X-linked recessive diseases include adrenoleukodystrophy, Fragile X syndrome, Duchenne muscular dystrophy, and red-green color blindness.

The pedigree in these disorders is characterized by affected males related through carrier females, and absence of father-to-son transmission. If females are affected, their symptoms are usually milder, and they are termed manifesting heterozygotes. This situation typically arises from skewed X-inactivation with preferential inactivation of the normal X chromosome in their cells. In the normal situation, X chromosome inactivation is random, with inactivation of a woman's paternal X chromosome in some cells, and inactivation of her maternal X chromosome in others. In other, rare cases, women with only a single copy of the X chromosome (45, X) or with structural abnormalities of the X chromosome may manifest an X-linked recessive condition.

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Mammalian Preimplantation Development

R.A. Reijo Pera, L. Prezzoto, in Current Topics in Developmental Biology, 2016

3.2 X-chromosome Inactivation

X-chromosome dosage is compensated between both sexes in mammals via inactivation of one of the two parental X chromosomes while maintaining active expression of a subset of genes on both chromosomes. Genes that escape X inactivation are generally those associated with Y-chromosome homologs. The process of XCI has been most thoroughly studied in mice (Disteche & Berletch, 2015; Kamikawa & Donohoe, 2014). In mice, the paternal X chromosome is imprinted to be silenced during early embryo development via a mechanism whereby imprinted expression is established by expression of the noncoding RNA, Xist, that represses transcription from the paternal X chromosome (Okamoto et al., 2011). Subsequently the paternal X chromosome reactivated in the inner cell mass of the blastocyst and random XCI selection ensues as differentiated cell lineages form. In contrast, in other species including the rabbit and human, Xist is not imprinted and XCI begins later in development than occurs in mice and Xist is expressed from either the maternal or the paternal X chromosome in some cases resulting in transient inactivation of both X chromosomes (Okamoto et al., 2011). Subsequently in the rabbit as development ensues, the choice of which X chromosome will become inactive occurs downstream of Xist expression; furthering defining species-specific differences is the skewing of X inactivation in some tissues and the identity of the individual genes on both X chromosomes that escape XCI (Deng, Berletch, Nguyen, & Disteche, 2014).

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X-Chromosome Analysis

John M. Butler, in Advanced Topics in Forensic DNA Typing: Methodology, 2012

Applications of ChrX Testing

X-chromosome STR typing can be helpful in some kinship analysis situations particularly with deficient paternity cases where a DNA sample from one of the parents is not available for testing. For example, if a father/daughter parentage relationship is in question, X-STRs may be helpful due to the 100% transmission of the father's X-chromosome to his daughter (Table 15.1). On the other hand, in a father/son parentage question, Y-chromosome results would be helpful (see Chapter 13). Table 15.2 lists several applications for X-chromosome DNA testing. ChrX testing can be especially helpful in some missing persons or disaster victim identification situations (see Chapter 9) where direct reference samples are not available and biological relatives must be sought to aid human identification.

Table 15.2. Applications of X-Chromosome Analysis.

•

Complex kinship cases involving at least one female

•

Disputed paternity to a daughter (especially in motherless cases)

•

Half-sister testing where the father is the common relative

•

Grandparent—grandchild comparisons

•

Paternity testing in incest cases (see Figure 15.2)

(See Figure 15.1 for Illustration of Example Pedigrees)

X-chromosome markers can help infer parent-offspring relationships that involve at least one female, such as mother-daughter, mother-son, and father-daughter duos (illustrated in Figure 15.1). In complicated kinship scenarios, such as incest (Figure 15.2), ChrX markers may aid sorting out difficult relationship questions.

Figure 15.1. Some example pedigrees where ChrX testing can be helpful.

Figure 15.2. Use of ChrX testing in an incest case to help distinguish whether the victim's father (H1) or brother (H2) fathered the victim's daughter. The mother passes a combination of her X-chromosomes (XB, XC) on to her son (XB,C). If either XB or XC is more abundant in the victim's daughter, then H2 is more likely (her brother is the father). If XA is more abundant in the victim's daughter, then H1 is more likely (her father is the father). Autosomal genetic markers would probably not be very helpful in this situation due to the high degree of allele sharing expected among close relatives.

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Sexual Differentiation

Richard E. Jones PhD, Kristin H. Lopez PhD, in Human Reproductive Biology (Fourth Edition), 2014

X Chromosome

The X chromosome is a fairly large chromosome (Figures 5.1 and 5.3) and contains over 1000 genes (out of the 20,000–23,000 human genes). Most of these are essential genes unrelated to sex determination. Some are metabolic enzymes essential for life (so-called "housekeeping genes"). Because females have two X chromosomes and males have only one, it might be assumed that female cells make twice the quantity of proteins encoded in the X chromosome compared with males. This is not the case; in fact, males and females have similar expression of genes on the X chromosome. This is because, early in embryonic development, a female shuts down one X chromosome in each of her cells. In 1949, Murray Barr discovered that female cells, but not male cells, contain a small dot of condensed material that represents one of the X chromosomes that has been inactivated (X-chromosome inactivation). This inclusion is called the Barr body or sex chromatin (Figure 5.2). In 1961, Mary Lyon proposed that one X chromosome is inactivated randomly in female embryonic cells so that a double gene dosage is avoided. All cells that follow a particular cell line have the same X chromosome inactivated. This means that, in some regions of a woman's body, the X chromosome she inherited from her mother is active whereas only the one inherited from her father is active in other regions (i.e. women are mosaics with respect to X chromosome expression). If more than two X chromosomes are present, as in some conditions produced by errors of fertilization (discussed later in this chapter), all of the X chromosomes are inactivated except one (Figure 5.2).

FIGURE 5.2. Possible variations in number of sex chromosomes in males and females and the resultant number of sex chromatins, or Barr bodies. Note that the number of Barr bodies is one less than the number of X chromosomes.

FIGURE 5.3. Location of genes involved in gonadal sex differentiation. The sex-determining region of the Y (SRY) gene codes for the production of the SRY protein, which causes testis differentiation. Absence of this gene in an individual lacking the Y chromosome results in the formation of ovaries. The DAX-1 gene on the X chromosome suppresses SRY gene expression, but the normal interaction of DAX-1 and SRY has not been fully discovered.

The presence of the Barr body is used to determine the sex of fetuses during procedures such as amniocentesis and chorionic villus sampling (see Chapter 10). Also, cells from the lining of the mouth membrane can be checked for the presence of a Barr body (the buccal smear test). Fluorescent dyes can be used to identify X and Y chromosomes.

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Biology/DNA

V. Pereira, L. Gusmão, in Encyclopedia of Forensic Sciences (Second Edition), 2013

The X-Chromosome

Origin and Evolution

The X-chromosome is one of the sex chromosomes in humans. With around 155 million base pairs and 1100 genes, it represents about 5% of the total human genome that comprises 20 000–25 000 genes.

The two sex chromosomes have originated from an ancestral autosomal pair (Figure 1). Around 300 Mya, this pair of autosomes started to accumulate differences and suffered structural and functional changes throughout the evolution, originating two different chromosomes – X and Y.

Figure 1. Evolution of the sex chromosomes.

Even though they have a common origin, the X- and the Y-chromosomes followed different evolutionary paths: the Y-chromosome has lost most of its genomic material and does not suffer recombination in 95% of its extension, while the X-chromosome still retains traces of its autosomal past, suffering recombination along its entire length during female gametogenesis. Homology between the two chromosomes is still present in the telomeric pseudoautosomal regions (PAR 1 and PAR 2; Figure 1).

Characteristics

The particular characteristics of the X-chromosome make it an interesting subject for genetic studies. Many genetic conditions have already been described as being related to mutations in specific genes on the X-chromosome. Hemophilia is a classic example of an X-chromosome-associated disease, among others.

In the last decade, the interest in the study of the X-chromosome markers as tools for forensic and population genetic studies has been growing, as they can help to detect underlying patterns of genetic differentiation that are not usually captured by the traditionally analyzed autosomal markers.

Humans have one pair of the sex chromosomes, but the number of X-chromosomes present in each cell varies between males and females. Females have two copies, while males have one X-chromosome and one Y-chromosome. Therefore men inherit their X-chromosome from their mothers, whereas women inherit one X-chromosome from each parent (Figure 2). The paternal X-chromosome they receive does not suffer recombination (except in the PARs) and is transmitted directly to the daughters. The maternal X-chromosome contains combined information from the two X-chromosomes present in the mother.

Figure 2. Genetic transmission of X- and Y-chromosomes.

Given the difference in copy number in males and females, the study of the X-chromosome in men allows direct access to their haplotypes. As recombination only occurs in females, in each generation, only two-thirds of X-chromosomes recombine. The X-chromosome is thus disproportionately influenced by female demography, making the study of the X-chromosome particularly useful for detecting subtle differences between the two genders.

In a population with equal number of female and male individuals, there will only be three X-chromosomes for every four autosomes present. Furthermore, for each three X-chromosomes in a population, one will be paternally and two maternally inherited. This will lead to clear differences not only in the recombination but also in the mutation rates between the two, as in the X-chromosome the rates will be lower due to the higher mutation rate in male gametogenesis.

Owing to these characteristics, and also due to its younger age, the diversity on the X-chromosome is expected to be lower than on the autosomes. As a consequence, and looking at it from a population genetic point of view, the effects of selection, genetic drift or substructure in a given population are more pronounced. The same is observed in the linkage disequilibrium (LD) patterns. LD can be defined as the nonrandom association of alleles at two or more loci. Several factors are responsible for breaking the extent of LD in a chromosome, such as the physical distance between the markers and recombination and mutation rates. Given that recombination only occurs in females, only one half of the X-chromosomes in a population will recombine in each generation, and therefore it will necessarily take more time to break down LD by recombination. As a result, LD is greater when compared to the autosomes and the size of regions with a single genetic history is larger, making it a better and more accurate tool to detect patterns of LD in populations.

The comparative analysis between the autosomes and the X-chromosome can also be used to reveal differences in demographic histories, migration, and breeding patterns of females and males. The usual studies of gender-biased demographic events compare information obtained from the Y-chromosome and mitochondrial deoxyribonucleic acid (mtDNA) markers. Unlike mtDNA and Y-chromosome analyses that inform about the history of female or male lineages respectively, the X-chromosome allows the simultaneous study on both genders, making it an ideal system for studying population genetic differences between males and females regarding mutation rates and patterns of recombination. Owing to recombination, the X-chromosome is composed by a block-like pattern with different chromosomal regions being informative of distinct genetic histories, unlike uniparental markers that are transmitted as a single locus and where all the markers share the same genealogic history.

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Mental Disorders and Transgenerational Epigenetics

Takeo Kubota, ... Kunio Miyake, in Transgenerational Epigenetics, 2014

X Chromosome Inactivation

The X chromosome has a large number of genes, whereas the Y chromosome has relatively few. Thus, females (XX) have more genes than males (XY). To minimize this sex imbalance, one of the two X chromosomes in females is inactivated by an epigenetic mechanism.41 Improper X chromosome inactivation (XCI) is thought to be an embryonic lethal condition, as suggested by the findings that a majority of aborted embryonic clones produced by somatic nuclear transfer showed failure of XCI,42,43 although it is difficult to directly demonstrate failure of XCI in human aborted embryos.

Even when failure of XCI occurs in women with one normal X chromosome and a small X chromosome due to a large terminal deletion, and, thus, the overdosage effect of X-linked genes is small, such affected women show severe congenital developmental delays,44 indicating that proper epigenetic regulation of the X chromosome is essential for normal development (Figure 24.1B).

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Epigenetics in pervasive developmental disorders: translational aspects

T. Kubota, in Neuropsychiatric Disorders and Epigenetics, 2017

5.4.2 Abnormalities in X-Chromosome Inactivation

The X chromosome is much larger than the Y chromosome; therefore it carries substantially more active genes than the Y chromosome. Consequently, females with an XX karyotype could have higher gene expression from their two X chromosomes than males with an XY karyotype that have a single X. However, this potential imbalance between females and males is prevented by the epigenetic inactivation of one of the X chromosomes in females. If X-chromosome inactivation (XCI) does not occur properly, it can cause lethality in the affected female embryo; this effect is evident in embryonic clones produced by somatic nuclear transfer in which the majority of clones abort due to the failure of XCI in mice [31] (Fig. 5.1B).

In humans, if one of the X chromosomes in a female is very small (e.g., a ring X chromosome generated by a chromosomal rearrangement that deletes the XCI locus (XIST gene) at Xq13, the female has a normal and a small X chromosome that are both active. Although this does not result in embryonic lethality, affected females generally show extremely severe neurodevelopmental delay [32]. These results indicate that the proper inactivation of genes is essential for normal birth and development.

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CG-CNVs

Thomas Liehr, in Benign & Pathological Chromosomal Imbalances, 2014

2.7.2.2 X-Chromosome

For the X-chromosome, the Lyon-hypothesis has to be observed. Every human, even every mammalian female cell, inactivates transcription in one of both X-chromosomes to balance X-linked gene dosage between male and female. In male and female all (additional) X-chromosomes except one undergo X-chromosome inactivation. This epigenetic event leads to gene silencing along almost the entire X-chromosome. But not all genes on the inactive X-chromosome are inactivated: genes in the pseudo-autosomal regions, the regions of the X-chromosome homologous to the Y-chromosome and responsible for XY-pairing during meiosis, as well as 15 to 20% of individual genes on the X-chromosome are not inactivated; another 10% of the genes escape inactivation partially. X-chromosome inactivation needs for its initiation the so-called X-inactivation-specific transcript (XIST) gene in Xq13.2 [Pontier and Gribnau, 2011].

Balanced and unbalanced translocations involving the X-chromosome including the XIST-gene region need to be checked carefully for their clinical relevance, since X-inactivation may also spread to genetic regions not derived originally from the X-chromosome itself. Thus, in large part and otherwise not viable, UBCA without severe clinical consequences may be observed [Stankiewicz et al., 2006]. Accordingly, even though additional copies of an X-chromosome may not be considered as CG-CNV, even large unbalanced translocations involving the XIST-gene may behave exactly like this.

For loss of parts or of an entire X-chromosome, what was stated in section 2.7.2.1 for loss of euchromatic Y-chromosome material is valid. (Partial) monosomy of the X-chromosome is normally associated with features of Turner syndrome.

Abstract

The X chromosome is known to contain the largest number of immune-related genes of the whole human genome. For this reason, X chromosome has recently become subject of great interest and attention and numerous studies have been aimed at understanding the role of genes on the X chromosome in triggering and maintaining the autoimmune aggression. Autoimmune diseases are indeed a growing heath burden affecting cumulatively up to 10% of the general population. It is intriguing that most X-linked primary immune deficiencies carry significant autoimmune manifestations, thus illustrating the critical role played by products of single gene located on the X chromosome in the onset, function and homeostasis of the immune system. Again, the plethora of autoimmune stigmata observed in patients with Turner syndrome, a disease due to the lack of one X chromosome or the presence of major X chromosome deletions, indicate that X-linked genes play a unique and major role in autoimmunity. There have been several reports on a role of X chromosome gene dosage through inactivation or duplication in women with autoimmune diseases, for example through a higher rate of circulating cells with a single X chromosome (i.e. with X monosomy). Finally, a challenge for researchers in the coming years will be to dissect the role for the large number of X-linked microRNAs from the perspective of autoimmune disease development. Taken together, X chromosome might well constitute the common trait of the susceptibility to autoimmune diseases, other than to explain the female preponderance of these conditions. This review will focus on the available evidence on X chromosome changes and discuss their potential implications and limitations.

Highlights

► The X chromosome contains the largest number of immune-related genes of human genome. ► X chromosome defects may explain the female preponderance in autoimmune diseases. ► High number of X monosomy cells was found in women with autoimmune diseases. ► X-linked genes dosage may play a role in loss of tolerance. ► X-linked microRNAs may be involved in development of autoimmune diseases.

Keywords

Sex chromosomes

Genetic factors

Autoimmunity

Female preponderance

Etiopathogenesis

Abbreviations

AID

autoimmune diseases

GWAS

genome-wide association study

PBC

primary biliary cirrhosis

MHC

major histocompatibility complex

PID

primary immunodeficiency syndromes

XCI

X chromosome inactivation

SLE

systemic lupus erythematosus

SSc

systemic sclerosis

AITD

autoimmune thyroid disease

HIGM

X-linked hyper-IgM syndrome

IPEX

Polyendocrinopathy, and enteropathy, X-linked syndrome

WAS

Wiskott-Aldrich syndrome

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Biochemistry and Biophysics Reports

Volume 15, September 2018, Pages 86-92

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Author links open overlay panelThi Hai YenTrana1KojiHisatakea

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Abstract

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.

Abbreviations

iPSCs

induced pluripotent stem cells

ESCs

embryonic stem cells

XCR

X chromosome reactivation

XCI

X chromosome inactivation

EGFP

enhanced green fluorescent protein

hKO

humanized Kusabira Orange

Xist

X-inactive specific transcript

inactive X chromosome

active X chromosome

Keywords

X chromosome reactivation

Reprogramming

Live cell imaging

CRISPR/Cas9

1. Introduction

iPSCs, generated by introduction of defined reprogramming factors into somatic cells [1], hold great promise for regenerative medicine and drug development [2]. However, iPSC generation is beset by inefficiency of reprogramming and heterogeneity of obtained cell populations [3]. It is therefore important to improve the efficiency of somatic cell reprogramming and select for iPSCs with full pluripotency.

iPSCs and ESCs display two distinct phases of pluripotency, the primed and naive states [4]. The ESCs in the naive state show higher ability to differentiate than those in the primed state [4], and generation of iPSCs with the pluripotency equivalent to the naive state is critical for their application in regenerative medicine. The naive state of pluripotency can be distinguished from the primed state by cell morphology, gene expression pattern, dependence on growth factors as well as, in the case of female cells, the presence of two active X chromosomes [4].

In eutherian mammals, female cells possess two X chromosomes, one of which is epigenetically inactivated during the early phase of embryonic development by a dosage compensation mechanism termed XCI [5]. XCI strictly depends on the X-inactive specific transcript (Xist) gene encoding a non-coding RNA, which plays a central role in inactivating the X chromosome in cis. Female somatic cells possess one active X chromosome (Xa) and one Xi, but once reprogrammed into the fully pluripotent state, female somatic cells reactivate Xi by a reverse process termed XCR [6]. Recent studies showed that XCR is closely coupled to acquisition of pluripotency by iPSCs [6], [7]. Thus, monitoring XCR may enable evaluation of pluripotency acquisition by iPSCs during somatic cell reprogramming.

Here we used the CRISPR/Cas9 system [8], [9] to generate female ESCs that carry the EGFP gene on one X chromosome and the hKO gene on the other. The obtained ESC clones expressed both EGFP and hKO, one of which was repressed in a random mode upon differentiation, concurrent with up-regulation of the Xist expression. The derived somatic cells that expressed only hKO were reprogrammed by Sendai virus expressing OCT4, SOX2, KLF4, and c-MYC [10], and found to initiate expression of EGFP when pluripotency marker genes were induced.

2. Materials and methods

2.1. Plasmids and guide RNAs

pX330-U6-Chimeric_BB-CBh-hSpCas9 (#42230) was purchased from Addgene. pPyCAG-EGFP-IP and pPyCAG-EGFP-IZ were generous gifts from Dr. Hitoshi Niwa (RIKEN CDB). Guide RNAs (gRNAs) were designed using CRISPRdirect (https://crispr.dbcls.jp), and the gRNAs that had the minimum potential off-target effects were chosen for the S and T locus (Fig. 1A, B and Supplementary Table 1). The B6N mouse Bac clones B6Ng01-177J10 (for the S locus) and B6Ng01-316J16 (for the T locus) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.

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Fig. 1. Knock-in of the EGFP and hKO genes driven by the human EF-1α promoter into the X chromosomes of mouse female ESCs. (A) Locations of the S and T loci on the mouse X chromosome are indicated by red bars. The location of the Xist gene is also indicated. The black arrows indicate the position and orientation of the guide RNAs (gRNA1 and gRNA2) used for the CRISPR/Cas9 system. (B) The intergenic sites between the Gm16459 and Syap1 genes ("S locus" in this study) and between the Taf1 and Ogt genes ("T locus" in this study) on the mouse X chromosome were chosen for insertion of the EGFP-IRES-Puror or hKO-IRES-Zeor cassette, which is driven by the human EF-1α promoter to express EGFP or hKO, respectively. The positions of the primers for genomic PCR are indicated by red arrows (a-d, m-p). (C) Genomic PCR analyses of the inserted fluorescent protein genes at the S locus in isolated ESC clones. BRC6 indicates the original female mouse ESCs used to insert the fluorescent protein genes. The primer sets used for PCR analyses are shown on the left. (D) Detection of random integration of the targeting vectors in the genome of isolated clones. The positions of primers for PCR analyses are indicated by red arrows (e-h for phEF1-EGFP-IP-Syap1, i-l for phEF1-hKO-IZ-Syap1). (E) Genomic PCR analyses of the inserted fluorescent protein genes at the T locus in isolated ESC clones. (F) Detection of random integration of the targeting vectors in the genome of isolated ESC clones. The positions of primers for PCR are indicated by red arrows (q-t for phEF1-EGFP-IP-Taf1, u-x for phEF1-hKO-IZ-Taf1).

2.2. Construction of plasmids

Complementary pairs of oligonucleotides encoding the gRNAs were annealed and inserted into the BbsI site of pX330-U6-Chimeric_BB-CBh-hSpCas9 to prepare Cas9/gRNA-expression vectors. The targeting vectors to knock-in the fluorescent protein genes into the S or T locus were constructed using pPyCAG-EGFP-IP and pPyCAG-EGFP-IZ. The CAG promoters were replaced by the human elongation factor-1α (EF-1α) promoter, and the EGFP gene in pPyCAG-EGFP-IZ was replaced by the hKO gene. The DNA fragments spanning the target site of gRNA1 (S locus) or gRNA2 (T locus) were isolated from the B6N mouse Bac clones and inserted into the upstream of the fluorescent protein gene and downstream of the drug-resistant gene (Supplementary Table 2).

2.3. Transfection of female mouse ESCs

Female mouse ESCs, BRC6 (RIKEN BRC, AES0010), were seeded at 5 × 105 cells/well on SNL feeder cells harboring the puromycin-resistant gene in a 6-well plate and cultured at 37 °C under 5% CO2 for 5 h in DMEM supplemented with 1 mM sodium pyruvate (Nacalai tesque, Inc.), 15% KnockOut Serum Replacement (KSR) (Thermo Fisher Scientific Inc.), nonessential amino acids (NEAA) (Wako Pure Chemical Industries, Ltd.), 0.1 mM 2-mercaptoethanol (2-ME) (Thermo Fisher Scientific Inc.) and 1000 U/mL LIF (Wako Pure Chemical Industries, Ltd.). Two micrograms each of pX330-Cas9/gRNA expression vectors and two different targeting vectors (phEF1-EGFP-IP-Syap1 and phEF1-hKO-IZ-Syap1, or phEF1-EGFP-IP-Taf1 and phEF1-hKO-IZ-Taf1) were mixed with 10 μl of Lipofectamine 2000, and the mixture was added to the ESCs. The cells were treated with 1 µg/mL puromycin for 5 days followed by treatment with 50 µg/mL zeocin for 3 days to isolate EGFP+/hKO+ ESC clones.

2.4. Genotype analysis of isolated ESC clones

Genomic DNAs were extracted from the isolated EGFP+/hKO+ ESC clones, which were cultured without feeder cells for 5 days prior to DNA extraction to avoid contamination with feeder cells. The ESCs were lysed in the presence of 0.5 μg/μl proteinase K, and the genome DNA was extracted by phenol/chloroform/isoamyl alcohol and precipitated by isopropanol. The location and sequences of primer sets used for PCR-based genotype analyses are shown in Fig. 1 as well as in Supplementary Table 3.

2.5. Differentiation of the EGFP+/hKO+ ESC clones

The EGFP+/hKO+ ESC clones were grown on SNL feeder cells in a 100 mm dish until ~80% confluency, and then trypsinized and suspended in the DMEM supplemented with 20% Fetal Bovine Serum (FBS), NEAA and 0.1 µM 2-ME. The cell suspension was transferred to a 100 mm cell culture dish and incubated for 20 min to remove feeder cells. Then, the supernatant containing the EGFP+/hKO+ ESCs was collected and plated into a 100 mm non-coated bacterial dish (AGC TECHNO GLASS CO., LTD.) for formation of embryoid bodies (EBs). After 5 days, EBs were trypsinized and filtrated through a 100 µm-cell strainer (BD Falcon). The filtrated cells were cultured on a collagen Type I-coated dish (AGC TECHNO GLASS CO., LTD.) in the presence of 50 µg/mL zeocin to select hKO+ cells.

2.6. Reprogramming of the EGFP+/hKO+ ESC-derived somatic cells

The isolated hKO+ cells were seeded in a 24-well plate at 2.5 × 104 cells/well in DMEM plus 10% FBS and cultured at 37 °C under 5% CO2 for 12 h. The cells were then infected with the Sendai virus which expresses KLF4, OCT4, SOX2 and c-MYC (SeVdp(KOSM) [11]) for 16 h at 32 °C to induce reprograming. The virus-infected cells were trypsinized and cultured on SNL-feeder cells in Knockout DMEM (Thermo Fisher Scientific Inc.) supplemented with 15% KSR, 2 mM GlutaMAX (Thermo Fisher Scientific Inc.), NEAA, 55 μM 2-ME, 100 units/mL penicillin, 100 µg/mL streptomycin (Nacalai tesque, Inc.) and 1000 U/mL LIF for 7 days. The culture medium was replaced by 1:1 mixture of DMEM/F12 (Nacalai tesque, Inc.) and Neurobasal medium (Thermo Fisher Scientific Inc.) supplemented with N2 supplement (Thermo Fisher Scientific Inc.), B27 supplement (Thermo Fisher Scientific Inc.), 2 mM GlutaMax (Thermo Fisher Scientific Inc.), 0.1 mM NEAA, 0.1 mM 2-ME, 0.05% BSA (Thermo Fisher Scientific Inc.), 1000 U/mL LIF, 1 μM MEK inhibitor PD0325901, 3 μM GSK3β inhibitor CHIR99021, 100 units/mL penicillin and 100 μg/mL streptomycin for continuous culture of iPSCs.

2.7. Reverse transcription and quantitative real-time PCR

Total RNA was extracted from the iPSCs using Sepasol-RNA I Super G (Nacalai tesque, Inc.) according to the manufacture's instruction. To avoid contamination with feeder cells, the hKO+ cells-derived iPSCs were cultured without feeder cells for 5 days prior to RNA extraction. Reverse transcription was performed using Superscript III First-Strand Synthesis System (Thermo Fisher Scientific Inc.), and the mRNA levels of various marker genes were measured by quantitative real-time PCR using GoTaq qPCR Master Mix (Promega Corp.). The mRNA level of γ-tubulin was used to normalize the obtained data. The primers used in this study are listed in Supplementary Table 4.

3. Results

3.1. Knock-in of fluorescent reporter genes into both X chromosomes of female ESCs

To visualize XCR in live cells during somatic cell reprogramming, we first generated female ESCs that express EGFP from one X chromosome and hKO from the other. To insert the EGFP and hKO genes into the genome, we avoided protein-coding genes as an insertion site because of their potential effect as a facilitator or inhibitor on the reprogramming process when iPSCs are generated [12]. Instead, we chose two intergenic sites near the Syap1 or Taf1 gene on the X chromosome (Fig. 1A). These sites were chosen because the insertion sites, which we term S and T loci, are near the genes, Syap1 and Taf1, respectively, that are subject to XCI [13]. In addition, database search of National Center for Biotechnology Information (NCBI) showed that the genes surrounding the S locus (Syap1, Txlng, Rbbp7, and Ctps2) and the T locus (Taf1, Nono, Zmym3, and Med12) do not exhibit strong tissue- or developmental stage-specific expression pattern. Moreover, the GeneProf database (http://www.geneprof.org/) [14] showed that these loci are sandwiched between CTCF binding sites together with at least one of these surrounding genes. Thus, the EGFP and hKO genes that are inserted into the S and T loci were expected to obey XCI and XCR in a similar manner to the surrounding genes.

We inserted the EGFP and hKO gene into each S locus of both X chromosomes in mouse female ESCs, using the CRISPR/Cas9 system [8], [9]. The gRNA1/Cas9 expression vector was introduced into female ESCs together with two different targeting vectors, each of which contained either the EGFP or hKO gene driven by the human EF-1α promoter as well as a drug-resistant gene, puromycin- or zeocin-resistant gene, respectively (Fig. 1B). The single-colored ESCs were removed by sequential selections with puromycin and zeocin to obtain EGFP+/hKO+ ESC colonies. Among the isolated 50 clones, 33 clones that grew well were genotyped, and most of the isolated clones had the inserted gene(s) at the S locus. However, only five ESC clones (No. 20, 21, 29, 36, and 40) had the EGFP and hKO genes at each S locus on both X chromosomes (Fig. 1C) while other clones had only the EGFP or hKO gene on both X chromosomes. PCR analysis using the primer sets shown in Fig. 1D showed that phEF1-EGFP-IP-Syap1 was randomly inserted in the genome in three clones (No. 21, 36, and 40) (e+f and g+h) and phEF1-hKO-IZ-Syap1 in one clone (No. 40) (i + j and k + l) (Fig. 1D). These results show that two ESC clones (No. 20 and 29, hereafter called "S20″ and "S29″) have the EGFP and hKO genes at each S locus on both X chromosomes without any random insertion in the genome.

We also inserted the EGFP and hKO genes into another intergenic site near the Taf1 gene (T locus) (Fig. 1A and B). After co-transfection of mouse female ESCs with the gRNA2/Cas9 expression vector together with the two different targeting vectors harboring the EGFP-IRES-Puror or hKO-IRES-Zeor gene (Fig. 1B), EGFP+/hKO+ colonies were selected sequentially by puromycin and zeocin. Genotyping revealed that four ESC clones (No. 26, 31, 36, and 38) had the EGFP and hKO genes at each T locus on both X chromosomes (Fig. 1E). However, as shown in Fig. 1F, only one ESC clone (No. 36, hereafter called "T36″) was free of a randomly inserted vector. Thus, we obtained three ESC clones that expressed both EGFP and hKO from the S locus (S20 and S29) or the T locus (T36) using the CRISPR/Cas9 system.

3.2. The EGFP and hKO genes at the intergenic loci on the X chromosome are subject to XCI inactivation during ESC differentiation

To confirm that the EGFP and hKO genes inserted into the intergenic sites are subject to XCI, we differentiated the three ESC clones (S20, S29, and T36) through embryoid body (EB) formation into monolayer cells (Fig. 2A). Quantitative RT-PCR analyses of the embryoid bodies and monolayer cells showed that expression of pluripotency marker genes (Nanog, Oct4, Fbxo15, Esrrb, and Cdh1) decreased (Fig. 2B) while that of somatic cell marker genes (Cdh2, Tgfb1, and Thy1) increased (Fig. 2C), indicating differentiation of ESCs. Expression of the Xist gene also increased in EBs and monolayer cells derived from ESC clones (S20, S29, and T36), indicating that XCI occurred during differentiation (Fig. 2D). As shown in Fig. 2E, ESC clones (S20, S29, and T36), which initially expressed both EGFP and hKO, gradually lost expression of either EGFP or hKO during EB formation. When the EB-derived cells were allowed to further differentiate as a monolayer, the cells expressed only EGFP or hKO, showing that one of the fluorescent protein genes on the X chromosome was inactivated due to XCI. Some somatic cells differentiated from the T36 ESC clone were found to lose expression of both EGFP and hKO (Fig. 2E, white dotted line), suggesting that the gene inserted into the T locus may be repressed independent of XCI. These results indicate that the fluorescent protein genes driven by the human EF-1α promoter are subject to XCI even when inserted into intergenic sites of the X chromosome, and the fluorescent genes at the S locus may be more suitable than those at the T locus for observing XCI.

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Fig. 2. Observation of XCI in the ESCs carrying the EGFP and hKO genes on the X chromosome. (A) Differentiation of the EGFP+/hKO+ ESCs via embryoid body formation into monolayer cells and the fluorescent patters of the derived somatic cells. ESCs expressed both EGFP and hKO before differentiation, and either EGFP or hKO became inactivated randomly by XCI upon differentiation. (B-D) Expression of pluripotency marker genes (B), somatic cell marker genes (C) and the Xist gene (D) in S20, S29, and T36 clones during differentiation (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). BRC6 indicates the original female mouse ESCs. (E) Morphology and fluorescent images of the S20, S29, and T36 clones during differentiation. A white-dotted area indicates the cells that expressed neither EGFP nor hKO.

3.3. Live cell imaging of XCR during reprogramming

XCR is a reverse process of XCI and occurs in female cells during somatic cell reprogramming [6]. To examine whether the S29 ESCs could be used for detecting XCR in live cells in vitro, we isolated hKO-positive (hKO+) differentiated cells and performed reprogramming. As shown in Fig. 3A, the EB-derived somatic cells were selected by zeocin to isolate only hKO+ cells, which carried the hKO gene on the Xa and the EGFP gene on the Xi. The isolated cells expressed a somatic cell marker gene (Cdh2) but not a pluripotency marker gene (Oct4) (Fig. 3B). The hKO+ cells were then infected with Sendai virus that expresses KLF4, OCT4, SOX2, and c-MYC to induce reprogramming [10]. The virus-infected cells formed colonies, which showed repression of somatic cell marker genes (Thy1, Cdh2, and Tgfb1) (Fig. 3C) and induction of pluripotency marker genes (Nanog, Oct4, Fbxo15, Esrrb, Cdh1, Rex1, and Sox2) (Fig. 3D). Moreover, expression of the Xist gene was down-regulated to a similar level of female ESCs (Fig. 3E). These results indicate that the hKO+ somatic cells were reprogrammed into iPSCs and underwent XCR, which is indicative of the fully pluripotent state [15]. Observation of EGFP and hKO signals in these cells revealed that some colonies started to show the EGFP signal by day 15, which gradually became more homogeneous by day 17, indicating XCR occurred between day 15 and day 17 of reprogramming (Fig. 3F). Thus, the S29 ESCs can be used to monitor the dynamic process of XCR during reprogramming in live cells in a non-invasive manner.

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Fig. 3. Live cell imaging of XCR during reprogramming. (A) Schematic illustration of reprogramming somatic cells derived from S29 ESCs. The hKO+ monolayer cells derived from S29 ESCs were selected by zeocin and then reprogrammed into iPSCs by infection with the Sendai virus expressing KLF4, OCT4, SOX2, and c-MYC (KOSM). (B) Expression of somatic cell marker (Cdh2) and pluripotency marker (Oct4) genes in the differentiated hKO+ cells derived from S29 ESCs. (C-E) Expression of somatic cell marker genes (C), pluripotency marker genes (D) and the Xist gene (E) in iPSCs reprogrammed from hKO+ somatic cells (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (F) Morphology and fluorescent images of iPSCs reprogrammed from hKO+ somatic cells at day 0, day 15 and day 17 of reprogramming. The white arrow indicates an EGFP+ colony, which appeared around day 15.

3.4. Visualizing XCR to monitor acquisition of pluripotency during reprogramming

When we isolated iPSC colonies reprogrammed from hKO+ somatic cells that were derived from S29 ESCs, we obtained colonies that were morphologically indistinguishable but nonetheless showed different expression patterns of EGFP (Fig. 4A and B). Given that XCR is a late event in reprogramming when iPSCs acquire pluripotency [15], we further examined the relationship between expression of both fluorescent protein genes and the acquisition of pluripotency. We isolated three iPSC clones generated from hKO+ somatic cells (Fig. 4A and B); S29-20 colonies did not express EGFP at all whereas S29-3 and S29-6 colonies expressed EGFP in a mosaic or homogeneous pattern, respectively. All three clones showed low expression of somatic cell marker genes (Thy1, Cdh2, and Tgfb1) (Fig. 4C), indicating that they progressed past the early stage of reprogramming [16]. By contrast, the three clones showed increased expression of pluripotency marker genes (Fig. 4D), which are induced during reprogramming [16]. Except for Nanog, there was no significant difference in the expression levels of the pluripotency marker genes between S29-3 and S29-6 (Fig. 4D), which show mosaic and homogeneous EGFP fluorescence, respectively (Fig. 4B). However, S29-20 iPSCs, which failed to express EGFP, had significantly lower expression levels of Nanog, Oct4, Esrrb, Cdh1, and Rex1 (Fig. 4D). Expression of the Xist gene was significantly decreased in S29-3 and S29-6 but not in S29-20, indicating that XCR occurred in S29-3 and S29-6 but not in S29-20 during reprogramming (Fig. 4E). These results indicate that EGFP expression from the Xi, even if it is mosaic, reflects XCR and acquisition of pluripotency at a late stage of reprogramming.

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Fig. 4. XCR and acquisition of pluripotency in female iPSCs. (A) Schematic illustration of isolation of iPSC clones reprogrammed from S29-derived hKO+ somatic cells. (B) Morphology and fluorescent images of isolated iPSC clones that exhibit no expression (S29-20), heterogeneous expression (S29-3), or homogeneous expression (S29-6) of EGFP. The colonies were isolated at day 19 of reprogramming. (C-E) Expression of somatic cell marker genes (C), pluripotency marker genes (D) and the Xist gene (E) in the iPSC clones (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

4. Discussion

Here we describe female ESCs that carry the EGFP and hKO genes at intergenic sites on both X chromosomes. The EGFP and hKO genes inserted into an intergenic site near the Syap1 gene are subject to both XCI during ESC differentiation and XCR during somatic cell reprogramming and thus allow live cell monitoring of XCI and XCR.

Previous studies reported insertion of fluorescent genes into the coding region of the Pgk1 and Hprt genes, which obey XCI during mouse development [17], [18]. In these studies, inserting the fluorescent genes into the Pgk1 and Hprt genes does not appear to compromise normal mouse development. However, many genes have been shown to potentially influence the reprogramming efficiency [19]. Therefore, to avoid inadvertent effects on iPSC generation by fluorescent protein genes inserted within a gene, we tested the feasibility of inserting the fluorescent protein genes into intergenic sites where neighboring genes are subject to XCI. Although the EGFP and hKO genes inserted near the Taf1 gene showed unstable expression upon differentiation (Fig. 2E), the fluorescent protein genes inserted near the Syap1 gene underwent XCI and XCR in a predicted manner. Thus, if properly chosen and experimentally tested, an intergenic site on the X chromosome allows a foreign gene to obey XCI and XCR.

During reprogramming of S29 ESC-derived somatic cells, reactivation of the EGFP gene accompanied down-regulation of the Xist gene and induction of pluripotency marker genes, demonstrating that acquisition of pluripotency is visualized in live cells by fluorescence imaging of EGFP and hKO (Fig. 3, Fig. 4). Moreover, iPSCs that fail to activate the EGFP gene from Xi showed low expression of pluripotency marker genes, consistent with the close coupling between pluripotency and XCR [15]. Thus, the ESCs with the EGFP and hKO genes on both X chromosomes, especially the S29 ESCs described here, may become an important tool for high-throughput screenings for factors and culture conditions that promote the acquisition of pluripotency by iPSCs.

Conflicts of interest

The authors declare no conflict of interest.

Funding

This work was supported by JSPS KAKENHI Grant Numbers JP16K07244, JP17H05063, and JP16K08610 (to K.N., Y.H., an A.F., respectively), and JP17H04036 and JP17K19339 (to K.H.) as well as by Program to Disseminate Tenure Tracking System by MEXT (to K.N.).