Analysis of expressed SNPs identifies variable extents of expression from the human inactive X chromosome

Background

X-chromosome inactivation (XCI) results in the silencing of most genes on one X chromosome, yielding mono-allelic expression in individual cells. However, random XCI results in expression of both alleles in most females. Allelic imbalances have been used genome-wide to detect mono-allelically expressed genes. Analysis of X-linked allelic imbalance in females with skewed XCI offers the opportunity to identify genes that escape XCI with bi-allelic expression in contrast to those with mono-allelic expression and which are therefore subject to XCI.

Results

We determine XCI status for 409 genes, all of which have at least five informative females in our dataset. The majority of genes are subject to XCI and genes that escape from XCI show a continuum of expression from the inactive X. Inactive X expression corresponds to differences in the level of histone modification detected by allelic imbalance after chromatin immunoprecipitation. Differences in XCI between populations and between cell lines derived from different tissues are observed.

Conclusions

We demonstrate that allelic imbalance can be used to determine an inactivation status for X-linked genes, even without completely non-random XCI. There is a range of expression from the inactive X. Genes escaping XCI, including those that do so in only a subset of females, cluster together, demonstrating that XCI and location on the X chromosome are related. In addition to revealing mechanisms involved in cis-gene regulation, determining which genes escape XCI can expand our understanding of the contributions of X-linked genes to sexual dimorphism.     

Bayesian modeling of skewed X inactivation in genetically diverse mice identifies a novel Xce allele associated with copy number changes

Female mammals are functional mosaics of their parental X-linked gene expression due to X chromosome inactivation (XCI). This process inactivates one copy of the X chromosome in each cell during embryogenesis and that state is maintained clonally through mitosis. In mice, the choice of which parental X chromosome remains active is determined by the X chromosome controlling element (Xce), which has been mapped to a 176-kb candidate interval. A series of functional Xce alleles has been characterized or inferred for classical inbred strains based on biased, or skewed, inactivation of the parental X chromosomes in crosses between strains. To further explore the function structure basis and location of the Xce, we measured allele-specific expression of X-linked genes in a large population of F1 females generated from Collaborative Cross (CC) strains. Using published sequence data and applying a Bayesian “Pólya urn” model of XCI skew, we report two major findings. First, inter-individual variability in XCI suggests mouse epiblasts contain on average 20–30 cells contributing to brain. Second, CC founder strain NOD/ShiLtJ has a novel and unique functional allele, Xce^g, that is the weakest in the Xce allelic series. Despite phylogenetic analysis confirming that NOD/ShiLtJ carries a haplotype almost identical to the well-characterized C57BL/6J (Xce^b), we observed unexpected patterns of XCI skewing in females carrying the NOD/ShiLtJ haplotype within the Xce. Copy number variation is common at the Xce locus and we conclude that the observed allelic series is a product of independent and recurring duplications shared between weak Xce alleles.     

Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

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.     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

Spread of X-chromosome inactivation into chromosome 15 is associated with Prader–Willi syndrome phenotype in a boy with a t(X;15)(p21.1;q11.2) translocation

X-chromosome inactivation (XCI) is an essential mechanism in females that compensates for the genome imbalance between females and males. It is known that XCI can spread into an autosome of patients with X;autosome translocations. The subject was a 5-year-old boy with Prader?CWilli syndrome (PWS)-like features including hypotonia, hypo-genitalism, hypo-pigmentation, and developmental delay. G-banding, fluorescent in situ hybridization, BrdU-incorporated replication, human androgen receptor gene locus assay, SNP microarrays, ChIP-on-chip assay, bisulfite sequencing, and real-time RT-PCR were performed. Cytogenetic analyses revealed that the karyotype was 46,XY,der(X)t(X;15)(p21.1;q11.2),?15. In the derivative chromosome, the X and half of the chromosome 15 segments showed late replication. The X segment was maternal, and the chromosome 15 region was paternal, indicating its post-zygotic origin. The two chromosome 15s had a biparental origin. The DNA methylation level was relatively high in the region proximal from the breakpoint, and the level decreased toward the middle of the chromosome 15 region; however, scattered areas of hypermethylation were found in the distal region. The promoter regions of the imprinted SNRPN and the non-imprinted OCA2 genes were completely and half methylated, respectively. However, no methylation was found in the adjacent imprinted gene UBE3A, which contained a lower density of LINE1 repeats. Our findings suggest that XCI spread into the paternal chromosome 15 led to the aberrant hypermethylation of SNRPN and OCA2 and their decreased expression, which contributes to the PWS-like features and hypo-pigmentation of the patient. To our knowledge, this is the first chromosome-wide methylation study in which the DNA methylation level is demonstrated in an autosome subject to XCI.     

Evidence for Local Regulatory Control of Escape from Imprinted X Chromosome Inactivation

X chromosome inactivation (XCI) is an epigenetic process that almost completely inactivates one of two X chromosomes in somatic cells of mammalian females. A few genes are known to escape XCI and the mechanism for this escape remains unclear. Here, using mouse trophoblast stem (TS) cells, we address whether particular chromosomal interactions facilitate escape from imprinted XCI. We demonstrate that promoters of genes escaping XCI do not congregate to any particular region of the genome in TS cells. Further, the escape status of a gene was uncorrelated with the types of genomic features and gene activity located in contacted regions. Our results suggest that genes escaping imprinted XCI do so by using the same regulatory sequences as their expressed alleles on the active X chromosome. We suggest a model where regulatory control of escape from imprinted XCI is mediated by genomic elements located in close linear proximity to escaping genes.     

Escape from X Inactivation Varies in Mouse Tissues

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.     

High-resolution chromosomal microarray analysis of early-stage human embryonic stem cells reveals an association between X chromosome instability and skewed X inactivation

X chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell via a random process. Skewed XCI is relevant to many diseases, but the mechanism leading to it remains unclear. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of blastocyst-stage embryos have provided an excellent model system for understanding XCI initiation and maintenance. Here, we derived hESC lines with random or skewed XCI patterns from poor-quality embryos and investigated the genome-wide copy number variation (CNV) and loss of heterozygosity (LOH) patterns at the early passages of these two groups of hESC lines. It was found that the average size of CNVs on the X chromosomes in the skewed group is twice as much as that in the random group. Moreover, the LOH regions of the skewed group covered the gene locus of either XIST or XACT, which are master long non-coding RNA (lncRNA) effectors of XCI in human pluripotent stem cells. In conclusion, our work has established an experimentally tractable hESC model for study of skewed XCI and revealed an association between X chromosome instability and skewed XCI.     

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xce^a < Xce^b < Xce^c < Xce^d. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xce^a allele, and Mus musculus castaneus EiJ, which carries the Xce^c allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Bipartite structure of the inactive mouse X chromosome

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0728-8) contains supplementary material, which is available to authorized users.