Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation

Ha, Norbert; Lai, Lan-Tian; Chelliah, Rosi; Zhen, Yashu; Vanessa, Seet Pei Yi; Lai, Sarah; Hy, Li; Ludwig, Alexander; Sandin, Sara; Chen, Lingyi; Zhang, Lifeng

doi:10.1016/j.isci.2018.09.007

Cited by 24 publications

(25 citation statements)

References 34 publications

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“…The authors found that, although RRMs 2-4 are required to bind Xist, the SPOC domain is the essential mediator of gene silencing. As suggested by previously reported experiments 12 , forcing an interaction between Xist and the SPOC domain alone was enough to restore XCI in cells that lack SPEN.…”

supporting

confidence: 68%

How to silence an X chromosome

Trotman¹,

Calabrese²

2020

Nature

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supporting

confidence: 68%

How to silence an X chromosome

Trotman¹,

Calabrese²

2020

Nature

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“…Most notably, deletion of Repeat A from the endogenous Xist locus causes not only a failure of XCI but a dramatic reduction in the abundance of full-length, spliced Xist (7,20,29,31). Similar reductions in Xist RNA abundance have been observed upon Repeat A deletion in transgenic contexts (27,(32)(33)(34), seemingly due to a defect in the production of nascent Xist and not due to a reduction in RNA stability (33). Current evidence is at odds as to whether the loss of SPEN reduces or has no impact on Xist RNA abundance (25,27).…”

Section: Introductionmentioning

confidence: 70%

“…We speculate that an antiterminator function may be the major reason why deletion of Repeat A or its relocation further downstream can cause such a dramatic reduction in the abundance of full-length Xist (7,20,27,29,(31)(32)(33)(34)(35). Premature polyadenylation can repress transcription from nearby promoters as well as cause transcriptional attrition over the length of a gene (65,70).…”

Section: Discussionmentioning

confidence: 99%

Elements at the 5′ end ofXistharbor SPEN-independent transcriptional antiterminator activity

Trotman

Lee

Cherney

et al. 2020

Preprint

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The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a transgene comprising Xist's first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPENindependent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

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“…Here, we devised a different system for profiling the Xist interactome. We have previously shown that the Xist RNA can be efficiently tagged in live cells by the programmable RNA-sequence binding domain of the Pumilio protein (PUF: Pumiliohomology domain; PBS: PUF-binding site) (10). A transgenic cell line previously generated in the lab is a male mouse ES cell line carrying an X-linked single-copy inducible Xist transgene with PBSb sites fused to its 5'-ends ( Fig.…”

Section: Chu Et Al Identified 81 Candidate Xist-binding Proteins (4)mentioning

confidence: 99%

“…A transgenic cell line previously generated in the lab is a male mouse ES cell line carrying an X-linked single-copy inducible Xist transgene with PBSb sites fused to its 5'-ends ( Fig. 1A) (10). We further engineered the cell line to have it stably express a PUFb-FLAG fusion protein (i-FLAG-Xist, Fig.…”

Section: Chu Et Al Identified 81 Candidate Xist-binding Proteins (4)mentioning

confidence: 99%

The lupus autoantigen La is anXist-binding protein involved inXistfolding and cloud formation

Ding

et al. 2020

Preprint

Self Cite

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Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, the lupus autoantigen La (encoding gene Ssb) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female ES cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. La is a ubiquitous and versatile RNA-binding protein with RNA chaperone activity. Functional dissection of La shows that the RNA chaperon domain plays critical roles in XCI. In mutant cells, Xist transcripts are misfolded and unstable. These results show that La is involved in XCI as an RNA chaperone for Xist. 3 SIGNIFICANCE STATEMENTXist RNA functions as a scaffold to recruit and assemble a protein machinery to epigenetically silence genes along one X chromosome in each female mammalian cell. Here, we devised a FLAG-out system to profile the Xist interactome, and identified the lupus autoantigen La as an Xist-binding protein. The RNA chaperone activity of La is essential for X chromosome inactivation (XCI). When La is downregulated, the folding of Xist RNA is altered, leading to shorter half-life of Xist RNA and compromised Xist cloud formation, which in turn results in XCI defects, including chromatin compaction, chromosome-wide monoallelic gene expression pattern, and death of female ES cells during in vitro differentiation.

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Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation

Cited by 24 publications

References 34 publications

How to silence an X chromosome

How to silence an X chromosome

Elements at the 5′ end ofXistharbor SPEN-independent transcriptional antiterminator activity

The lupus autoantigen La is anXist-binding protein involved inXistfolding and cloud formation

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