An X-linked human collagen transgene escapes X inactivation in a subset of cells.

Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.     

Mapping of Replication Origins in the X Inactivation Center of Vole Microtus levis Reveals Extended Replication Initiation Zone

DNA replication initiates at specific positions termed replication origins. Genome-wide studies of human replication origins have shown that origins are organized into replication initiation zones. However, only few replication initiation zones have been described so far. Moreover, few origins were mapped in other mammalian species besides human and mouse. Here we analyzed pattern of short nascent strands in the X inactivation center (XIC) of vole Microtus levis in fibroblasts, trophoblast stem cells, and extraembryonic endoderm stem cells and confirmed origins locations by ChIP approach. We found that replication could be initiated in a significant part of XIC. We also analyzed state of XIC chromatin in these cell types. We compared origin localization in the mouse and vole XIC. Interestingly, origins associated with gene promoters are conserved in these species. The data obtained allow us to suggest that the X inactivation center of M. levis is one extended replication initiation zone.     

Small marker X chromosomes lack the X inactivation center: implications for karyotype/phenotype correlations.

The abnormal phenotype and/or mental retardation seen in persons with small marker X (mar(X)) chromosomes has been hypothesized to be due to the loss of the X inactivation center (XIC) at Xq13.2, resulting in two active copies of genes in the pericentromeric region. In order to define precisely the DNA content of mar(X) chromosomes and to correlate phenotype with karyotype, we studied small mar(X) chromosomes, using FISH with probes in the juxtacentromeric region. One of the probes was a 40-kb genomic cosmid for the XIST gene, which maps to the smallest interval known to contain the XIC and is thought to be involved in X inactivation. Our findings reveal that small mar(X) chromosomes do not include the XIC and therefore cannot be subject to X inactivation, supporting the premise that abnormal dosage of expressed genes in the pericentromeric region of the X generates the aberrant phenotype seen in patients with small mar(X) chromosomes.     

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.     

Chromosomal localization and molecular organization of the human genomic fragment containing the TNF/LT locus in transgenic mice

The molecular organization, copy number, and chromosome location of the human TNF/LT transgenes were studied in the genomes of two transgenic mouse strains. One strain proved to carry two transgene copies arranged head-to-tail and detected on chromosome 8 by karyotyping. The other strain had one transgene copy observed on chromosome 5. The strains provide a model for studying the physiological functions of the tumor necrosis factor and lymphotoxin.     

Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies

X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.     

X chromosome inactivation revealed by the X-linked lacZ transgene activity in periimplantation mouse embryos

Using H253 mouse stock harboring X-linked HMG-lacZ transgene, we examined X chromosome inactivation patterns in sectioned early female embryos. X-gal staining patterns were generally consistent with the paternal X inactivation in the trophectoderm and the primitive endoderm cell lineages and random inactivation in the epiblast lineages. The occurrence of embryonic visceral endoderm cells apparently at variance with the paternal X chromosome inactivation in 7.5 dpc embryos was explained by the replacement of visceral endoderm cells with cells of epiblast origin. The frequency of cells negative for X-gal staining in 4.5-5.5 dpc XmXp* embryos fluctuated considerably especially in the extraembryonic ectoderm and the primitive endoderm, whereas it was less variable in the embryonic ectoderm. We could not, however, determine whether it is a normal phenomenon revealed for the first time by the use of HMG-lacZ transgene or an abnormality caused by the multicopy transgene.     

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.     

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.     

Identifying and genotyping transgene integration loci

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.     

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.     

Influence of a nonfragile FHIT transgene on murine tumor susceptibility

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.     

X-chromosome inactivation: molecular mechanisms from the human perspective

X-chromosome inactivation is an epigenetic process whereby one X chromosome is silenced in mammalian female cells. Since it was first proposed by Lyon in 1961, mouse models have been valuable tools to uncover the molecular mechanisms underlying X inactivation. However, there are also inherent differences between mouse and human X inactivation, ranging from sequence content of the X inactivation center to the phenotypic outcomes of X-chromosome abnormalities. X-linked gene dosage in males, females, and individuals with X aneuploidies and X/autosome translocations has demonstrated that many human genes escape X inactivation, implicating cis-regulatory elements in the spread of silencing. We discuss the potential nature of these elements and also review the elements in the X inactivation center involved in the early events in X-chromosome inactivation.