TY - JOUR
T1 - Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
AU - Ha, Norbert
AU - Lai, Lan Tian
AU - Chelliah, Rosi
AU - Zhen, Yashu
AU - Yi Vanessa, Seet Pei
AU - Lai, Soak Kuan
AU - Li, Hoi Yeung
AU - Ludwig, Alexander
AU - Sandin, Sara
AU - Chen, Lingyi
AU - Zhang, Li Feng
N1 - Publisher Copyright:
© 2018 The Author(s)
PY - 2018/10/26
Y1 - 2018/10/26
N2 - We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist)to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES)cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen)in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.
AB - We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist)to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES)cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen)in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.
KW - Cell Biology
KW - Developmental Biology
KW - Genetics
KW - Molecular Biology
UR - http://www.scopus.com/inward/record.url?scp=85066289573&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85066289573&partnerID=8YFLogxK
U2 - 10.1016/j.isci.2018.09.007
DO - 10.1016/j.isci.2018.09.007
M3 - Article
AN - SCOPUS:85066289573
SN - 2589-0042
VL - 8
SP - 1
EP - 14
JO - iScience
JF - iScience
ER -