The goal of this proposal is to reveal mechanisms by which the long non-coding (lnc) RNAs Xist and Tsix find their genomic target sites and control gene expression at these sites during the process of X chromosome inactivation (XCI). XCI is a remarkable paradigm of lncRNA-mediated chromatin regulation, controlled by the oppositely transcribed RNAs Xist and Tsix, that transcriptionally silence one of the two X chromosomes in female mammalian cells. While Xist induces silencing of the X chromosome, Tsix negatively regulates Xist early in development to ensure that Xist only becomes active on one X chromosome. We already know several functions of Xist, including spreading in cis from its site of transcription to cover the entire X chromosome, excluding RNA Polymerase II, triggering the accumulation of repressive chromatin marks, and inducing a chromosome-wide compaction, demonstrating the remarkable versatility of this lncRNA. The mechanisms by which Xist carries out these functions remain poorly understood. In particular, with few exceptions, we still do not know domains of Xist or interacting proteins that mediate these various roles. However, our preliminary data yield new insight into the roles of Xist and Tsix. Specifically, we have been able to define three domains of Xist as mediators of distinct functions, consistent with the idea that distinct RNA domains carry out different functions. We hypothesize that these domains are required for the efficient association of Xist with the X, ensuring its function in cis; the targetng of Xist to active genes, which is a prerequisite for their subsequent silencing; and for changes in chromosome conformation, respectively. To further define the mechanisms by which these domains act, we have also identified a critical protein interaction partner of one of these domains. Moreover, our recent work on Xist spreading suggests an intriguing link between Xist and genome organization, that led to the hypothesis that Xist utilizes long-range chromatin interactions to spread across the X chromosome, and, upon tethering to distal sites acts as a nuclear organization factor that creates a new 3D topology of the chromosome to induce gene silencing. Finally, by comparing the RNA localization of Tsix and Xist on the X chromosome, we found that Tsix can spread along the X, but in contrast to Xist, only spreads locally around its site of transcription. We speculate that specific RNA sequences in Xist and Tsix direct these distinct localization patterns. Based on our findings, we are well positioned to unveil regulatory mechanisms by which Xist and Tsix control XCI, with these Aims: 1) To understand how Xist RNA mediates its various functions through specific RNA domains. 2) To investigate the link between 3D chromosome structure and Xist. 3) To characterize the function and localization of Tsix.
Each aim will be supported by outstanding collaborators. Together, our proposed studies will provide novel insights into XCI, and should significantly advance our understanding of the role of nuclear architecture on nuclear function. Exploring the differences between Xist and Tsix will also yield a better understanding of how lncRNAs locate to their genomic targets.

Public Health Relevance

The regulation of gene expression is of fundamental importance to establish and maintain different types of cells and to control cell physiology, and mis-regulation underlies numerous diseases including cancer. We propose to study the molecular mechanisms of a particular aspect of regulation of gene expression by long non-coding RNA molecules, specifically the regulation of gene expression of the X chromosome, controlled by the two long non-coding RNAs Xist and Tsix. We hope that our work will provide significant insights into how these RNAs mediate the silencing of an entire chromosome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM115233-01A1
Application #
8988214
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Carter, Anthony D
Project Start
2015-08-01
Project End
2019-06-30
Budget Start
2015-08-01
Budget End
2016-06-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Sahakyan, Anna; Yang, Yihao; Plath, Kathrin (2018) The Role of Xist in X-Chromosome Dosage Compensation. Trends Cell Biol 28:999-1013
Sahakyan, Anna; Plath, Kathrin (2016) Transcriptome Encyclopedia of Early Human Development. Cell 165:777-9
Pandya-Jones, Amy; Plath, Kathrin (2016) The ""lnc"" between 3D chromatin structure and X chromosome inactivation. Semin Cell Dev Biol 56:35-47
McHugh, Colleen A; Chen, Chun-Kan; Chow, Amy et al. (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521:232-6
Pasque, Vincent; Plath, Kathrin (2015) X chromosome reactivation in reprogramming and in development. Curr Opin Cell Biol 37:75-83
Minkovsky, Alissa; Sahakyan, Anna; Bonora, Giancarlo et al. (2015) A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2'-dC upon inhibition of ribonucleotide reductase. Epigenetics Chromatin 8:42