Interdisciplinary Centre for Innovation in Biotechnology and Neuroscience, University of Sri Jayewardenepura, Nugegoda, Sri Lanka

Corresponding Author: Ranil de Silva, Interdisciplinary Centre for Innovation in Biotechnology and Neuroscience, University of Sri Jayewardenepura, Nugegoda- 10250, Sri Lanka. Tel: 94112758697; E-mail: ranil@sjp.ac.lk.

Funding: Ministry of Primary Industries through National Science Foundation, Sri Lanka, Grant no- SP/CIN/2016/02 and World Class University grant Project University of Sri Jayewardenepura, Grant no- WCUP/PhD/19 & WCUP/PhD/19B.

	ABSTRACT
	INTRODUCTION
	CONCLUSION
	ACKNOWLEDGMENT
	CONFLICT OF INTEREST
	REFERENCES

	ABSTRACT

Mammalian dosage compensation is a complex mechanism allowing inactivation of single X chromosome of the female to compensate to that of the X chromosome of the male. The mechanism includes many long non-coding RNA; mainly XIST, a noncoding RNA which coats the X chromosome to be inactivated and TSIX, another noncoding RNA act as a negative regulator of XIST preventing inactivation of the second X chromosome. Both XIST and TSIX and several other transcription factors along with polycomb proteins (PRC) work together in controlling the inactivation of one X chromosome while the other X chromosome remains active. This is facilitated by the sensing mechanism called the n-1 theory, induced by the X pairing region (XPR) allowing X chromosome pairing before inactivation. X inactivation occurs randomly and begins at the late blastocyst stage of an embryo when the cells start to differentiate by losing pluripotency. Therefore, pluripotent factors play an important role in inducing X chromosome inactivation. Once X chromosome is inactivated it is passed along cell division and maintained throughout life. This review discusses up-to-date discovered pathways involved in mammalian dosage compensation, from initiation to maintenance of the X chromosome inactivation and potential therapeutic effects for X chromosome-linked disorders.

	INTRODUCTION

   The X chromosome has many genes that are important for both females and males, therefore maintaining the correct chromosome dosage is important for many species. Male species with single X chromosome equalizes X chromosome-linked transcription levels to that of the females by a mechanism known as the dosage compensation (1). Discoveries of various theories for the past few years had allowed a better understanding of dosage compensation in mammals.

   However, dosage compensation differs from one species to another and is maintained throughout the lifetime of species. Drosophila, a genus of small flies achieves dosage compensation by doubling the transcription of genes in the X chromosome of the males (2) while Caenorhabditis elegans, a non-parasitic nematode halves the transcription of both X chromosomes genes in females (3). Mammals interestingly maintain correct chromosome dosage by inactivating one of the X chromosomes in females during the development of the embryo (1). There are three subclasses of mammals as follows, the eutherian who are also known as the placental mammals eg: humans, secondly, the marsupials whose young are born in the immature state and become matured in pouch such as Kangaroos and then the third subclass of mammals, the Prototheria who lay leathery-shelled eggs but feed their young with milk from belly pores eg: platypus (4) . The divergence between eutherians and marsupials suggests that mechanisms of dosage compensation have evolved independently in both lineages (5).

   Sex determination of eutherian mammals occurs at the fertilization stage with the inheritance of paternal X or Y chromosome in which the Y chromosome contains the gene Sry for development of male characteristics during organogenesis (6). As a result, the eutherian female mammal has two X chromosomes while a male has one X chromosome and a Y chromosome. The Y chromosome contains few genes; mostly male-specific, in contrast to X chromosome with 1000 genes (6) therefore it is important to maintain a balance in the expression of X chromosome genes attained by transcriptional silencing genes in one of the X chromosome in the female mammals. The mechanism which equalized the X linked genes in females to that of males is known as the dosage compensation. Dosage compensation is attained when one of the X chromosome is inactivated randomly at the late blastocyst stage of the developing embryo in the female eutherian mammal (7) by the X chromosome inactivation center (XIC) found in the X chromosome which contains the elements required for X chromosome inactivation (8–12).

   However, X inactivation exists in two lineage-specific forms known as “imprinted” and “random” XCI (24, 111).

   Random X inactivation is described to as one of the either maternal X chromosome or the paternal X chromosome being silenced throughout development of a female mammal. Interestingly humans show randomness in X inactivation giving rise to chimerism hence being better protected from X linked disorders. It is said that the X-chromosome-controlling element to play an important role in the choice of X to be inactivated or to remain active such that in females heterozygous for different Xce alleles, the X chromosome that carries a strong Xce allele is more likely to remain active than one that carries a weak Xce allele, thereby leading to skewed X inactivation (115) leading to mosaicism. Hence females tend to be more protective against X linked disorders due to the presence of extra X chromosome to cope up with the defective X compared to males who have a single X chromosome (91). Thereby which means either the paternal or maternal X chromosome with a better Xce allele is chosen to be active throughout a female human being. Interestingly both imprinted and random X inactivation process takes place via the XIC where the imprinted form silenced the paternal X chromosome while the random inactivation causes silencing of either the maternal or the paternal X chromosome (111).

   The X chromosome inactivation (XCI) produces a non-coding RNA, the X inactive specific RNA (XIST) which is only expressed when there are at least two X chromosomes in the somatic cell in mammals (6, 13–15). XIST with cis and trans acting activators and repressors and polycomb proteins (PRC) causes epigenetic changes in silencing the X chromosome, resulting in heterochromatin formation and condensed chromosome (14, 16–18). The resulting heterochromatic X chromosome is known as a Barr body which is moved to the nuclear envelope during interphase of cell division. Once Barr body forms it is maintained throughout cell division (19). Interestingly in humans, paternal and maternal X chromosomes are both active until random X inactivation occurs in the late blastocyst stage (20–22) whereas in mice, there is no randomness hence the paternal X chromosome is inactivated in the early embryo, and remains inactivated for the rest of embryogenesis in the placenta (23–25).

   X inactivation Imprinted or Random

   The imprinted form occurs in the extraembryonic tissues, the placental lineages and the random X inactivation occurs in the embryo (111, 112). Both imprinted and random X inactivation can be observed in mouse models. That is during early embryogenesis paternal X chromosome is silenced and in the inner cell mass of the blastocyst the paternal XCI is reversed and random XCI subsequently occurs in the epiblast (122).

   Humans do not undergo imprinted form of X inactivation instead recently it was suggested that the X chromosome dampening occurs during pre-implantation in a female embryo. Petropoulos and colleagues using transcriptomic studies showed that immediately after zygotic gene activation (ZGA) at E4, female embryos had almost double expression of X-linked genes compared with males, consistent with two X chromosomes in females (124, 125). But however surprisingly with increasing developmental time from E4 to E7, the expression of X linked genes decreased and by the E7 stage this doubling in females decreased to same level as that in males, slightly before implantation. Surprisingly, this decrease in X linked genes to compensate for that of males was purely not due to X inactivation because allelic expression analysis by single-cell RNA-sequencing revealed that both X chromosomes were active at all times (125). Which suggested a theory of X chromosome dampening. However, the role of XIST and the underlying mechanism of X-dampening remains unclear (123) and needs further investigation. Yet due to the restricted availability of human embryos and technical difficulties X-chromosome dynamics in human pre-implantation embryos remains elusive.

   Initiation of ‘X’ chromosome Inactivation

   Pluripotent factors, OCT4, SOX2, KFL2, MYC and REX1 (also known as ZFP42) (26–30) bind to TSIX promoter (DXPase34) as transcription factors to transcribe TSIX (31). REX1 is important for the activation as well as elongation of TSIX (31) Meanwhile, NANOG, SOX2 and OCT4 acts synergically by binding to the first intron of XIST preventing transcription of XIST (32). Knockout of NANOG will cause increase in expression of XIST while SOX2 and OCT4 remain bound but OCT4 knockouts will downregulate both SOX2 and NANOG leading to a loss in pluripotency of the cell (32).

   Once pluripotency is lost, cells begin to differentiate, X inactivation begins by transcriptional activation of XIST via RNA polymerase II. Once XIST is produced, it coats the X chromosome produced resulting gene silencing. Experimental evidence of fluorescence in situ hybridization (FISH) tagging of XIST RNA shows that the RNA never leaves the territory of X chromosome thereby restricting silencing of autosomal genes and remains attached to X inactive (Xi) throughout mitosis (33, 34). The interaction between CDKN1A- Interacting Protein (CIZ1) and XIST Repeat E motif is critical for stable association of XIST RNA with the inactive X chromosome (13, 35). Deletion of either CIZ1 or Repeat E or the loss of the heterochromatin mark H3K27me3 causes dispersal of XIST RNA throughout the nucleoplasm, from the inactive X chromosome (13). Recently it was shown that gene knockout of melanoma-associated antigen (Mageb1-3) affected H3K27me3 enrichment thereby affecting the spreading of XIST (36) hence Mageb3 decreases steeply during differentiation. Heterogeneous nuclear protein hnRNP U/SAF-A is required for XIST accumulation on to Xi (33, 37). Experimental evidence supports the fact that knockout of SAF-A causes detachment of XIST from Xi (38). Overexpressed SAF-A diminishes Mageb3 (36). Long interspersed elements (LINES) found repeatedly on X chromosome aids in XIST spreading over X chromosomes (39–42). LINES were observed more frequently near genes prone to escape X chromosome inactivation (XCL) leading to silencing of the genes. This was experimentally supported in models where LINES expressed in autosomes caused gene silencing in the autosomes (43). Special A-T binding protein 1 (SATB1) expressed during initiation of XCL aids in XIST localization over Xi genes moving genes away from transcription factor, RNA polymerase II while further aiding gene silencing acting as an anchor forming chromatin loops (44). Repeat A (RepA) region of XIST binds to PRC2 polycomb proteins and is recruited to the X chromosome to be inactivated (45, 46). These PRC2 specifically EZH2 and SUZ12 aids in histone trimethylation of lysine residues (47) leading to gene silencing and chromosome condensing and Barr body formation.

   At the active X chromosome site, the TSIX inhibits the interaction between RepA and PCR2 either by titrating RepA away from the chromatin or by preventing PCR2 catalysis (47, 48).

   Sensing one X chromosome to be active throughout

   The TSIX RNA a non-coding RNA found downstream to XIST transcribed by the XITE in the XIC makes sure one X chromosome is kept active throughout life (49–51). TSIX is antisense to XIST thereby inhibits XIST inactivation on the remaining X chromosome in eutherian mammals (52, 53). In mouse models, it was shown that before X inactivation the two XIC pair up at the X pairing region (Xpr), this along with Xist/Tsix content allows sensing and counting of X chromosome allowing only one X chromosome to be active (54). The X pairing does not occur in males (54).

   Another protein RNF12 aids in sensing mechanism and activation of XIST to induce X inactivation (55–57).

   RNF12 found upstream to XIST encodes for E3 ubiquitin ligase which causes degradation of REX1 (58, 59). RNF12 induces X chromosome inactivation only when present above a certain threshold, a condition fulfilled when at least two Xs are active (60–63). REX1 in optimal concentrations inhibits XIST transcription and activates TSIX, therefore, blocks XCI (64). REX1 breakdown is important for XCI initiation in females with two copies of RNF12. Once X chromosome is inactivated only one X chromosome will be active hence one copy of RNF12 is not sufficient to reduce REX1 down the optimal concentration thereby activates TSIX transcription causing termination of further initiation of XCI (63).

   A zinc finger protein, CTCF acts as in insulator (65,66) by binding to RS14 region present between TSIX and XIST. Binding of CTCF to RS14 (binding site upstream to XIST promoter) blocks transcription of XIST bycompeting with XIST promoter. RS14 is important for proper transcriptional initiation of XIST but deleting RS14 did not affect XIST silencing (1). Therefore, it was suggested that the binding of CTCF to RS14 acts as an insulator between TSIX and XIST hence deleting RS14 increased TSIX transcription and decreasing XIST (1). JPX a non-coding RNA acts as a positive regulator of XIST gene (67) is involved in activation of XIST transcription by removing CTCF (68). JPX isexpressed during cell differentiation and binds to CTCF moving CTCF away from XIST promoter thereby enhancing XIST transcription (51, 68, 69).

   JPX is expressed in two folds from both X chromosome therefore when one X chromosome inactivated JPX halves acting similar to that in males. JPX in one fold is not adequate to remove CTCF, therefore, CTCF remains bound to one X chromosome preventing XIST transcription hence remains active throughout cell division (68, 70). This way once the X chromosome inactivated the other X chromosome kept active.

   Maintenance of X chromosome inactivation

   Chromosome-wide transcriptional silencing on Xi is one of the most significant events that occur during XCI (71, 72). Immunofluorescence staining reveals that RNA polymerase II (PolII) is depleted from the inactive Xi hence is one of the earliest events of XCI, which cause transcriptional silencing on X (73).

   SHARP proteins are required for XIST-mediated transcriptional silencing (74) which is known to interact with the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) co-repressor that activates HDAC3 (75, 76). SHARP is not only essential for silencing but is also required for the exclusion of PolII from the inactive X (77). It was also shown that both SMRT and HDAC3 are required for XIST-mediated silencing and RNA polymerase II exclusion. Lamin B receptor, (LBR), is required for repositioning actively transcribed genes into the XIST-silenced compartment (76–79). XIST, through its interaction with LBR, a protein that is anchored in the inner nuclear membrane, effectively reposition XIST-coated DNA to the nuclear lamina, thereby changing the accessibility of other genes on the X-chromosome to enable XIST to spread to active genes across the entire chromosome to silence chromosome-wide transcription (79, 80). Hence evidence provides XIST can scaffold multiple proteins to orchestrate the complex functions required for the establishment of the inactive X-chromosome (81).

   Structural maintenance of chromosome hinge domain containing 1(SMCHD1) is a protein involve in hypermethylation of X linked transcriptional initiation sites (CpG islands) (82–86). Mutations of SMCHD1 cause reactivation of genes in Xi but not in the initiation of Xi (87). SMCHD1 associated with HBiX1 (a heterochromatin binding protein) causes compaction of Xi forming Barr bodies. This compaction is brought by linking Histone 3 trimethylated lysine 9 (H3K9Me3) domains and XIST- H3K27Me3 domains together (88) and requires XIST but not PRC2 of H3K27Me3.

   Another additional layer of epigenetic markers which involve SPOP and CULLIN3 (E3 ubiquitin ligase protein) further aid in maintaining a firm Xi compaction forming the Barr body. These proteins cause ubiquitination of polycomb proteins BMI1 and variant histone MacroH2A1 causing gene silencing (89). RNA1 knockout models of SPOP and CULLIN3 causes loss of MacroH2A1 from Xi resulting reactivation of Xi (90). Therefore, SPOP and CULLIN3 are actively involved in stabilizing Xi. Further, it was shown that PRC2 EED is important in maintaining Xi as mutations in EED causes loss of XIST thereby preventing inactivation (87).

   X inactivation in health and disease

   Surprisingly, X inactivation could occur in males in case of a presence of extra X chromosomes, this process is thought to involve X chromosome counting and choice. Analysis of XCI in tetraploid XXXX, XXXY, and XXYY embryonic stem cells had shown that X chromosome within a single nucleus has an independent probability to initiate XCI and suggest a stochastic mechanism involving XCI counting and choice. The X inactivation choice is directly proportional to X chromosome:ploidy ratio, indicating the presence of an X-encoded activator of XCI that itself is inactivated by the X inactivation process (114).

   However, in females, X inactivation occurs randomly meaning one cell might have the paternal X silent while the neighbouring cell has the maternal X chromosome silent and is passed to next cell line once imprinted during cell differentiation and are usually protected from diseases due to cellular mosaicism (94) but not in males (95).

   This means overall cells which contain healthy X linked gene expression carry out at least part of the function (96, 97), one such example is the fragile X syndrome (98, 99). Fragile X syndrome (FXS) is the most common form of inherited mental retardation in males caused by the increase CGG triplet repeats within the FMR1 (protein important for the regulation of translation of dendritic mRNAs in response to synaptic activation (100, 101), which leads to methylation of the CpG island and silencing of the FMR1 (102). In a case study of two sisters who were compound heterozygotes for a full mutation and a 53 repeat intermediate allele, one of them showing mental retardation and clinical features of an affected male (speech delay, hyperactivity, large ears, prominent jaw, gaze aversion), while the other was borderline normal (mild delay) was concluded that the observed phenotypic differences between both sisters though they were cytogenetically normal were due to extremely skewed X-chromosome inactivation (103). They were the only daughters of a nonconsanguineous married couple with normal cognitive abilities, interestingly the family of the mother has a history of mental retardation (103). Therefore, this indicates the diseased X chromosome was from the maternal X chromosome.

   The incidence of FXS varies among different ethnic groups. A study by Coffe et al, in 2009 showed 2.5 incidences per 10000 of Male Whites compared to African American with 1.9 per 10000 and Hispanics with 1.8 per 10000 of the US with full mutations for FMR1 (106). In comparatively when considering Asian males, Pakistanis showed a higher prevalence of FXS (2.8%) (107) compared to Sri Lankans (1.3%) (108) and Chinese (0.93%) (109) reported the least in the year 2015, indicating a racial or ethnic variation. Could this variation be due to lifestyle and environmental factors which ultimately results in control the balance between expression of the faulty or the protective genes? Interestingly in a study it was demonstrated that sex-differential developmental trajectories begin in utero and maintained 9 months of age, indicating long-term epigenetic reprogramming in relation to nutrition during pregnancy (104) hence there is a link between maternal nutrition and the methylome of the offspring (105) thereby maternal nutrition may influence the future health of both mother and baby. It is important to note environmental epigenetic factors is not always the nutrition, but includes behavioural and chemicals and industrial pollutants exposure of an individual (120). Environmental exposures may harm the fetus by tackling with the epigenome of the developing organism and hence modify disease risk later in life as epigenetic mechanisms are also implicated during development in utero and at the cellular levels (120).

   Sex chromosomes contains pseudoautososmal regions (PAR1 and PAR2) found at the tip of the telomere ends of both chromosomes X and Y and these pair up during meosis. PAR1 and PAR2 regions are not inherited via sex linked but act in an autosomal manner taking part in crossing over to bring diverse genetic variation (119) similar to other autosomal genes. PAR1 found at the tip of the short arm of the chromosome X and Y is important in recombination during meosis maintaining male sterility and contains at least 24 genes (118) while PAR2, located at the tip of the long arms of chromosome X and Y contains 4 genes discovered up to date (118). All the genes at the PAR escape x inactivation therefore these genes are equally expressed in both X and Y chromosomes of the male and avoids reverse dosage compensation in females which means females with single X chromosome will still have two copies of PAR in both X chromosomes (inactive X and the active X) (113, 118). Therefore interestingly in sex chromosome defects, such as the presence of extra X chromosome seen in male, a condition known as Klinefelter Syndrome (KS) (92, 93) shows most its phenotypes due to the extra dosage of PAR genes with more than one X chromosome. That is as PAR genes escape silencing dosage of these genes are abnormal compared to that of a normal individual with 2 PAR either in XX or XY. For instancethe SHOX gene, a gene found on PAR1 is likely causing the tall stature regularly seen in KS (116, 117) as PAR is expressed in abnormal levels due to extra X chromosome. Males do not express XIST RNA as XIST is only expressed in cells with more than one X chromosome (121) however, when considering KS, these individuals will have a small amount of XIST expressed from their extra X chromosomes.

   Analysis of X inactivation aids in discovering new therapeutic approaches for extra chromosome syndromes such as Klinefelter and Down’s Syndrome as well X linked disorders. Recently Jiang et al., (110), successfully silenced the trisomy Ch21 in pluripotent stem cells from Down’s syndrome using zinc finger-based genome editing by artificially planting the XIST transgene in one of the three chromosomes 21. Surprisingly the XIST RNA triggered stable heterochromatin modifications, DNA methylation, and chromosome-wide transcriptional silencing of the targeted chromosome 21 as mediated in X inactivation (110).

   All this sum up to the point that extensively analysis of X inactivation both its molecular contributions as well as environmental triggers such as nutrients on the epigenetic modification of X inactivation will aid in better understanding of X linked disorders and potentially provide better ways to treat most of the intellectual disabilities and would be a major first step towards potential development of ‘chromosome therapy’.

	CONCLUSION

   Once cells lose pluripotency that is during the late blastocyst stage of an embryo, X inactivation is initiated primarily by the transcription of XIST and with the recruitment of polycomb proteins PRC2 and PRC1 together causing epigenetic changes of X chromosome gene silencing. X inactivation occurs randomly in females causing cellular mosaicism providing protection from diseases. Interestingly X inactivation could occur in males as well in case of presence of an extra X chromosome due to the X counting mechanisms (X pairing).

   Studying X chromosome inactivation and reactivation (cell reprogramming) will allow paving the path to find treatments to X linked disorders.

	ACKNOWLEDGMENT

   Special thanks to Professor Ashwin Dalal, Head, Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, for his valuable time and guidance. The team Dr. Pulasthi Akalanka, Mrs. Dharshi Attanayake, Mr. Yoonus Imran of the Genetic Diagnostic Lab, University of Sri Jayewardenepura, Sri Lanka.

	CONFLICT OF INTEREST

   The authors declare that no conflicting interests exist.

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