Abstract
X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.
Highlights
In mammals, X chromosome inactivation (XCI) ensures dosage compensation of sex chromosomal genes between females (XX) and males (XY)[1,2]
Only two strategies have been described in which rat ES cells (rESCs) were triggered to differentiate into either cardiomyocytes or neuronal precursors and in these differentiation protocols MEK and GSK3β inhibitors, that are commonly used for embryonic stem cells (ESCs) culture, are always present in low concentrations in the differentiation media[74,75]
Several rESCs derived from different rat inbred strains were differentiated, including three pure Lewis lines (LEW) (A4p20, A9p20, A10p20), and two lines of a mixed background of dark agouti (DA) and Sprague-Dawley (SD) (135-7, 141-6)
Summary
X chromosome inactivation (XCI) ensures dosage compensation of sex chromosomal genes between females (XX) and males (XY)[1,2]. Specific culture conditions that allow studying dosage compensation in human ES cells (hESCs) have been established[52,53] These studies provided in vitro systems that resemble the pre-XCI state of human pre-implantation embryos, and showed that hESCs do not undergo random XCI upon differentiation. These observations suggest the presence of an epigenetic memory that may affect the faithful recapitulation of XCI as it occurs upon early human development, and highlight the need to further optimize cell culture conditions[53]. Our data has established the technical basis to study the dynamics of XCI in a different system from the mouse and suggests that specific aspects of XCI may be conserved in mouse and rat
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
