Project Summary / Abstract
The goal of this proposal is to define the role of the long non-coding RNA (lncRNA) XACT (X-active
coating transcript). XACT is a primate-specific and X-chromosome-linked lncRNA expressed exclusively in
pluripotent stem cells. In naïve human embryonic stem cells (hESCs), which model the pluripotent state of the
pre-implantation embryo, the XACT RNA forms two clouds in female and one cloud in male cells over the
respective X-chromosome territories. At this stage of development, female and male cells express X-linked
genes at comparable levels despite both female X-chromosomes being transcriptionally active. This gene
dosage compensation mechanism, known as X-chromosome dampening (XCD), is unique to human
development. Upon implantation, female pluripotent cells switch to complete silencing of one X-chromosome via
X-chromosome inactivation (XCI), a process mediated by the well-studied chromatin-associated lncRNA XIST.
In primed hESCs, which capture the pluripotent state of the post-implantation embryo, XACT expression is
silenced on the inactive X-chromosome (Xi) of female cells, but is retained on the active X-chromosome in female
and male cells. Considering that many lncRNAs function as transcriptional regulators, we hypothesize that XACT
modulates the naïve and primed pluripotent states in early human development, potentially by regulating X-
chromosome dosage compensation. Intriguingly, preliminary findings in our lab have shown that XIST also
controls X-chromosome dampening in naïve pluripotent cells, but does not induce complete silencing in this
developmental state. One possible function of XACT may be to counteract the silencing capacity of XIST in naïve
pluripotent cells, in which XCD is characterized by concurrent expression of XACT and XIST from the X-
chromosome. We posit that XACT antagonizes the activity of XIST during XCD through competitive chromatin
association, thus preventing premature XCI prior to implantation. To unveil the role of XACT in pluripotent states
and in the regulation of X-chromosome dosage compensation, we will: 1) delete XACT in naïve and primed
hESCs and analyze the effect on cell states and transcriptomes; and 2) define its molecular mechanism of action
as a chromatin-associated lncRNA. Taken together, these approaches will elucidate the contribution of XACT to
the control of pluripotent states and its role in X-chromosome gene dosage regulation. By exploring the link
between XACT function and the modulation of XIST in early human development, we may uncover a novel,
primate-specific strategy for gene dosage compensation and define fundamental features of human pluripotency.