Lab Mission

To understand how noncoding RNAs (Tsix, Xist) regulate X-chromosome silencing.
To establish a paradigm for antisense regulation (Tsix).
To identify factors controlling X-chromosome counting and choice.
To delineate the evolutionary relationship between imprinted and random X-inactivation.


Dissecting the Pathway of X-Chromosome Inactivation

The evolution of unequally sized X/Y sex chromosomes posed a dosage problem for mammals. While females have two X-chromosomes (XX), males have one X- and one Y-chromosome (XY). Because the Y contains just a small fraction of the 2000 genes on the X, this system of sex determination endows the female with nearly twice the number of sex chromosome genes as the male. As first described by Dr. Mary Lyon 40 years ago, X-chromosome inactivation (XCI) evolved to compensate for this dosage imbalance by silencing one whole X-chromosome in XX individuals. Dosage compensation is essential for proper embryonic development, as embryos which fail to achieve it differentiate poorly and die at the time of uterine implantation. This presented a challenging problem for the mammalian cell, because it required that the cell be able to epigenetically mark two chromosomes that otherwise share a nucleoplasm and that all genes on one X be inactivated without affecting genes on the other X. Since the earliest days of its discovery, XCI was known to be a strictly cis-limited process and to be controlled by a master activity switch on the X. To understand how the process is controlled, our laboratory has taken a genetic approach in mice, coupling a whole organism model with an embryonic stem cell model, which is capable of recapitulating X-inactivation in culture.

Two forms of XCI have been described. In stochastic XCI, either the mother's or the father's X can be silenced. Just after implantation, cells which form the embryo proper count the number of X-chromosomes and will undergo XCI only if there is more than one X-chromosome. In XX cells, a choice mechanism designates one active and one inactive X, with each X having an approximately equal chance of being inactivated. In contrast, during imprinted XCI, the father's X is inactivated in all cells. This form is believed to have evolved first and is still found in extant marsupials, such as the kangaroo, and in the extraembryonic tissues of some placental mammals such as the mouse. In contrast to random XCI, the imprinted process bypasses counting and choice so that the only chromosomes capable of being silenced are those inherited from the father.

Both processes are controlled by an X-linked inactivation center (Xic). Transgenic studies show that various steps of XCI (counting, choice, and silencing) can be recapitulated by fragments of the Xic ranging from 80 - 450 kb. This region includes two genes, Xist and its antisense partner, Tsix. The identification of Xist/XIST 10 years ago by Dr. Hunt Willard's group represented a major breakthrough in the field. Xist encodes a 17 kb untranslated nuclear RNA and is required for initiating the silencing step once a chromosome has been chosen. On the chromosome which will become the future inactive X, Xist is expressed at high levels and the RNA accumulates along the chromosome which transcribes the RNA. It is believed that Xist RNA recruits silencing factors as it spreads along the X.

Our laboratory first found evidence for an antisense transcript when studying a transgene with an Xist promoter truncation but which nonetheless appeared to have ample Xist expression. Analysis using strand-specific techniques revealed that Tsix initiates 15 kb downstream of Xist and completely transverses the Xist gene. Prior to XCI when Xist is expressed at basal levels, Tsix expression is found on all X-chromosomes in male and female cells. At the onset of XCI, Tsix RNA disappears on the future inactive X, the chromosome on which Xist expression is to be upregulated to high levels. At the same time, Tsix remains expressed on the future active X where Xist expression becomes repressed. Interestingly, Tsix is imprinted in the extraembryonic tissues of mice so that it is expressed only from the maternal allele. This expression pattern contrasts with Xist's, which shows exclusive paternal expression in extraembryonic tissues.

We have now shown that Tsix is a repressor of Xist expression. Knocking out the 5' end of the gene results in distinct phenotypes for stochastic and imprinted XCI. In epiblast-derived XX cells (e.g., mouse embryonic stem cells) that normally undergo stochastic XCI, loss of one Tsix copy leads to near exclusive inactivation of the chromosome which bears the mutation (nonrandom XCI). This primary effect on the randomness of XCI led us to conclude that Tsix plays a role in X-chromosome choice. Furthermore, the data support a role for Tsix in repressing Xist action. Interestingly, however, the loss of Tsix in XY cells results in no deleterious effects. This suggests that some other factor, presumably present in XX but not XY cells, is required to initiate silencing.

In cells that ordinarily undergo imprinted XCI, deleting one copy of Tsix creates a phenotype only when the deletion is maternally inherited, consistent with its maternal-specific expression pattern. In both XX and XY mutants, maternal transmission of a Tsix deletion results in derepression of the normally silent maternal Xist allele. Thus, XX mutants show inactivation of both X's, while XY mutants show inactivation of their only X-chromosome. These results showed that Tsix is a protective factor that blocks inactivation of the maternal X in the extraembryonic tissues of mice. We proposed that Tsix harbors an imprinting center on which a mark is placed by mother and father.

We envision that Tsix could block Xist in several ways. First, antisense transcription per se may block the advancement of an Xist transcription complex. Second, Tsix could produce a functional antisense RNA that basepairs with Xist RNA. Duplex formation could either diminish Xist RNA half-life or block access of Xist RNA to silencing proteins. Finally, Tsix RNA may be incidental to a role of specific DNA elements in Tsix. Recent evidence suggests that Tsix works in several ways.

To test the role of Tsix transcription, we generated an allele which overexpresses Tsix and extends the window of expression. We find that Tsix hypertranscription is sufficient to block Xist RNA accumulation in a cis-limited manner. We therefore propose that Tsix transcription is necessary to restrict Xist activity on the future active X and, conversely, that Tsix repression is required for Xist RNA accumulation on the future inactive X. Interestingly, however, Tsix hypertranscription does not affect X-chromosome choice. Thus, choice is mediated by elements within Tsix that are most likely independent of transcription.

Indeed, DNA sequences at the 5' end of Tsix may mediate the choice decision. We have now identified the insulator and transcription factor, CTCF, as a candidate trans-acting factor for X-chromosome selection. The genetically defined choice/imprinting center contains tandem CTCF binding sites which function in an enhancer-blocking assay. In vitro binding is reduced by CpG methylation and abolished by including non-CpG methylation. We postulate that Tsix and CTCF together establish a regulatable epigenetic switch for X-inactivation. These findings provide tantalizing analogies to imprinting models proposed for H19/Igf2. At this locus, it is believed that methylation-sensitive CTCF binding upstream of H19 regulates access of enhancers to either gene. The identification of Tsix enhancers and differentially methylated regions is a main focus of ongoing studies.