Clonality of tuberous sclerosis harmatomas shown by non-random X-chromosome inactivation

Tuberous sclerosis (TSC) is an autosomal dominant condition characterised by tumour-like malformations (hamartomas) in the brain and other organs. A proportion of hamartomas from patients with TSC show loss of heterozygosity (LOH) for DNA markers in the region of either the TSCI gene on chromosome 9834 or the TSC2 gene on 16p13.3. This implies that these lesions are clonal. We have studied X-chromosome inactivation, as a marker of clonality, in 13 hamartomas from females with TSC. The hamartomas comprised five renal angiomyolipomas, three fibromas and seven other lesions. In previous studies, four of the lesions showed LOH. A polymerase chain reaction assay was used to analyse differential methylation of anHpalI restriction site adjacent to the androgen-receptor triplet-repeat polymorphism on Xg11-12. In 12 of the lesions, there was a skewed inactivation pattern with one X chromosome being fully methylated and the other unmethylated. Normal tissue showed a random pattern of inactivation. These data confirm that most TSC hamartomas are clonal in origin. This is an intriguing finding, since these lesions are composed of more than one cell type.     

The search for the mouse X-chromosome inactivation centre

The phenomenon of X-chromosome inactivation in female mammals, whereby one of the two X chromosome present in each cell of the female embryo is inactivated early in development, was first described by Mary Lyon in 1961. Nearly 30 years later, the mechanism of X-chromosome inactivation remains unknown. Strong evidence has accumulated over the years, however, for the involvement of a major switch or inactivation centre on the mouse X chromosome. Identification of the inactivation centre at the molecular level would be an important step in understanding the mechanism of X-inactivation. In this paper we review the evidence for the existence and location of the X-inactivation centre on the mouse X-chromosome, present data on the molecular genetic mapping of this region, and describe ongoing strategies we are using to attempt to identify the inactivation centre at the molecular level.     

Chromatin organization during mouse oocyte growth

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I–III, 10–40 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4–6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley-Liss, Inc.     

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.     

New lessons from random X-chromosome inactivation in the mouse

X-chromosome inactivation (XCI) ensures dosage compensation in mammals. Random XCI is a process where a single X chromosome is silenced in each cell of the epiblast of mouse female embryos. Operating at the level of an entire chromosome, XCI is a major paradigm for epigenetic processes. Here we review the most recent discoveries concerning the role of long noncoding RNAs, pluripotency factors, and chromosome structure in random XCI.     

Early development and X-chromosome inactivation in mouse parthenogenetic embryos.

Early development and X-chromosome inactivation were studied in ethanol-induced mouse parthenogenones. About 24% of oocytes transferred to 0.5-day pseudopregnant recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6- to 8-day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X-inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.     

Maternal imprinting during mouse oocyte growth in vivo and in vitro

Epigenetic regulation of gene expression is critical for oogenesis in mammals. In this study, a simple and efficient method was used to obtain the oocytes from cultured fetal mouse ovaries of 12.5 dpc. The methylation pattern of these oocytes was examined. The results showed that the establishment of imprinting of Igf2r and Peg3 in oocytes derived from cultured fetal mouse germ cells in vitro follows a slower time course than that of oocytes in vivo. However, oocytes in vitro and in vivo share similar methylation patterns. Igf2r was gradually de novo methylated, and the methylation covers 80% CpG sites in oocytes cultured for 28 days. However, only 45% of the CpG sites is methylated in Peg3 at the same stage. Furthermore, it demonstrated that the degree of DNA methylation is positively correlated with the size of oocytes in vitro and in vivo, indicating a progressive methylation process during oocyte growth.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

X-chromosome inactivation in differentiating mouse embryonic stem cells carrying X-linked GFP and lacZ transgenes

Three new female ES cell lines (GLM1, GLP1 and GLP2) were established from mouse embryos carrying GFP (green fluorescent protein) and HMG-lacZ transgenes on one of two X chromosomes in cis. Using these cell lines, we studied the temporal relationships among three events relevant to X-chromosome inactivation: replication asynchrony of the X chromosome, and quenching of GFP fluorescence and beta-galactosidase (beta-gal) activity, during cell differentiation induced by embryoid body (EB) formation and retinoic acid (RA) treatment. In embryoid bodies adhering to the bottom of culture dishes, GFP-negative cells appeared first in the peripheral outgrowths 4 days after the initiation of EB formation, followed about 24 hours later by the appearance of cells negative for beta-gal and those having a single allocyclic X chromosome. Although the frequency of cells with an allocyclic X chromosome could reach 80% in adherent embryoid bodies, it tended to remain low and variable in embryoid bodies maintained in suspension. In spite of apparently parallel extinction of GFP and lacZ in embryoid bodies, their concurrent occurrence did not always characterize RA-induced differentiation. Moreover, an allocyclic X chromosome was identified in not more than 20 percent of informative metaphase cells up to 10 days after initiation of RA treatment. These findings suggest that RA-induced differentiation of female ES cells does not always accompany X-inactivation.     

DNA damage response during mouse oocyte maturation

Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.     

X-chromosome inactivation: X marks the spot for BRCA1

X-chromosome inactivation equalizes the dosage of X-linked genes in XX females with that in XY males. Recent findings reveal that the BRCA1 breast cancer susceptibility gene has an important function in this epigenetic phenomenon.     

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.     

Subcellular localization of nardilysin during mouse oocyte maturation

We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with beta-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This finding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.