X inactive-specific transcript (Xist) expression and X chromosome inactivation in the preattachment bovine embryo.
Expression of the X inactive-specific transcript (Xist) is thought to be essential for the initiation of X chromosome inactivation and dosage compensation during female embryo development. In the present study, we analyzed the patterns of Xist transcription and the onset of X chromosome inactivation in bovine preattachment embryos. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of Xist transcripts in all adult female somatic tissues evaluated. In contrast, among the male tissues examined, Xist expression was detected only in testis. No evidence for Xist transcription was observed after a single round of RT-PCR from pools of in vitro-derived embryos at the 2- to 4-cell stage. Xist transcripts were detected as a faint amplicon at the 8-cell stage initially, and consistently thereafter in all stages examined up to and including the expanded blastocyst stage. Xist transcripts, however, were subsequently detected from the 2-cell stage onward after nested RT-PCR. Preferential [3H]thymidine labeling indicative of late replication of one of the X chromosomes was noted in female embryos of different developmental ages as follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts. These results suggest that Xist expression precedes the onset of late replication in the bovine embryo, in a pattern compatible with a possible role of bovine Xist in the initiation of X chromosome inactivation. (+info)
Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene.
The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function. (+info)
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. (+info)
Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain.
Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation. (+info)
A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation.
We have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation. (+info)
Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels.
The human telomerase RNA component (hTR) is present in normal somatic cells at lower levels than in cancer-derived cell lines. To understand the mechanisms regulating hTR levels in different cell types, we have compared the steady-state hTR levels in three groups of cells: (i) normal telomerase-negative human diploid cells; (ii) normal cells transfected with the human telomerase catalytic subunit, hTERT; and (iii) cells immortalized in vitro and cancer cells expressing their own endogenous hTERT. To account for the differences in steady-state hTR levels observed in these cell types, we compared the transcription rate and half-life of hTR in a subset of these cells. The half-life of hTR in telomerase-negative cells is about 5 days and is increased 1.6-fold in the presence of hTERT. The transcription rate of hTR is essentially unchanged in cells expressing exogenous hTERT, and the increased steady-state hTR level appears to be due to the increased half-life. However, the transcription rate of hTR is greatly increased in cells expressing endogenous hTERT, suggesting some overlap in transcriptional regulatory control. We conclude that the higher hTR level in cells expressing an endogenous telomerase can be a result of both increased transcription and a longer half-life and that the longer half-life might be partially a result of protection or stabilization by the telomerase catalytic subunit. The 4-week half-life of hTR in H1299 tumor cells is the longest half-life yet reported for any RNA. (+info)
The timing of XIST replication: dominance of the domain.
Contiguous replicons are coordinately replicated and may be organized in temporal-spatial domains with early replication domains containing expressed genes and late ones carrying silent genes. XIST is silent on the active, early replicating X chromosome and expressed from the inactive, late replicating homolog. These circumstances potentially deviate from the aforementioned generalization and make studies of replication timing for XIST of special interest. Although earlier investigations of XIST replication in fibroblasts based on analysis of extracted DNA from cells at different stages of the cell cycle suggested that the silent gene does replicate before the expressed allele, studies using FISH technology produced the opposite results. Because the FISH replication studies could not directly distinguish between the active and inactive X chromosomes in the same cell, we undertook a re-investigation of this question utilizing FISH analysis under conditions that allowed us to make that distinction using cells sorted into different cell cycle stages by flow cytometry. The findings reported here indicate that the silent XIST gene on the active X chromosome does replicate before the expressed allele on the inactive X, supporting the view that the time of a gene's replication is determined by the large, multi-replicon domain in which it is located and not necessarily its expression state. (+info)
The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.
Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent. (+info)