Enhanced Reprogramming of Xist by Induced Upregulation of Tsix and Dnmt3a

Abstract

Reactivation of Oct4 gene expression occurs within 2 days of fusion of somatic cells with pluripotent stem cells and within 9 days of postinfection of four transcription factors. We sought to determine whether somatic genome reprogramming is completed by the onset of Oct4 reactivation. The complex regulation of the reactivation of inactive X chromosome (Xi) serves as a model for studying reprogramming of chromatin domains. A time-course analysis of the DNA methylation, gene expression, and X inactivation-specific transcript (Xist)/Tsix RNA fluorescence in situ hybridization revealed that expression of pluripotency-and tissue-specific marker genes was reset to the level of pluripotent stem cells within 2 days of fusion, whereas reprogramming of Xist/ reactivation of Xi took at least 9 days. We found that trichostatin A, which normally activates gene expression, results in downregulation of Xist. This is due to activation of Dnmt3a and Tsix, two negative regulators of Xist. Moreover, delayed reprogramming of Xist/reactivation of inactive X chromosome after cell fusion was accelerated by DNA methylation and histone deacetylation of Xist, which follow upregulation of Dnmt3a and Tsix.

Chromatin dynamics during cellular reprogramming

“…It is further important to mention that the reprogramming of hybrids is not complete upon activation of somatic pluripotency genes 2 days post fusion. Activation of the silenced X chromosome in female hybrids takes several days, which is reminiscent of the delayed X reactivation observed during Xenopus SCNT 95 . Consistently, transcriptome-wide analysis of hybrid formation documented that some silenced ESC-associated genes are activated more rapidly than others 96 .…”

Conversion of genomic imprinting by reprogramming and redifferentiation

“…Xist RNA was detected with a Cy3-labeled Xist RNA FISH probe spanning the entire Xist cDNA. Xist RNA FISH was performed as previously described (Do et al, 2008). Images were obtained using an applied spectral imaging (ASI) camera and analyzed with FishView software (Applied Spectral Imaging; ASI GmBH).…”

The emerging role of pseudogene expressed non-coding RNAs in cellular functions

“…HOTAIR and it’s associated complex appears to be a bimodal complex consisting of a lncRNA and several proteins exhibiting opposing functions such as the repressive PRC2 and the activating LSD1, all tethered in one complex (Khalil, Guttman et al 2009; Gupta, Shah et al 2010; Tsai, Manor et al 2010; Chu, Qu et al 2011)(Figure 1B). Many other lncRNAs are known to also function in trans such as Tsix, an antisense transcript that evolved from a pseudogenization of the XIST locus (Duret, Chureau et al 2006; Do, Han et al 2008), and ANRIL of the INK4b/ARF/INK4a locus (Yap, Li et al 2010). …”