Dosage compensation in the mammal results in transcriptional silencing of one X-chromosome in the female sex. Known as """"""""X-chromosome inactivation"""""""" (XCI), this process exemplifies epigenetic regulation and has served as a paradigm for understanding gene regulation by noncoding RNA (ncRNA) and chromatin-based mechanisms. XCI is controlled by the """"""""X-inactivation center"""""""" (Xic), an X- linked region known for an abundance of genes that produce ncRNA. The initiation of XCI is regulated by two such ncRNA loci, Xist and its antisense partner, Tsix. Expression of Tsix blocks initiation of silencing, whereas expression of Xist guides chromosome-wide inactivation. While our understanding of how Tsix opposes Xist has continued to improve over the past 10 years, how Xist induces XCI remains largely unsolved. In the past year, my laboratory has made significant progress towards understanding how silencing might be induced: (i) We have identified the first Xist RNA-interacting factor as Polycomb repressive complex 2 (PRC2), the complex of proteins responsible for trimethylating H3-lysine27 (H3- K27me3). We have shown that Ezh2, the catalytic subunit of PRC2, is the specific RNA-binding subunit. Xist RNA directly binds this subunit via a repeated stem-loop structure at the 5'end of the RNA known as """"""""Repeat A."""""""" (ii) We have also discovered that Repeat A is an independent transcription unit, which we named RepA. The 1.6 kb RepA RNA initially recruits PRC2 to the X and is required for induction of full-length Xist RNA, which in turn binds PRC2 also and spreads H3-K27 methylation along the whole X. (iii) The antisense Tsix RNA binds PRC2 as well. We propose that competition between RepA and Tsix for PRC2 determines if and when XCI will initiate. (iv) Curiously, Dicer-deficient cells show defects in Xist localization, PRC2 recruitment, and H3-K27 methylation, suggesting that the RNAi pathway may also play a hand in Polycomb recruitment and function. Taken together, these data implicate ncRNA at the interface between Polycomb proteins and their target genes, and suggest that ncRNA may be a general mechanism by which chromatin modifers - which often lack sequence- specific DNA-binding subunits - may be directed to specific loci. This research proposal is aimed at understanding mechanisms by which Polycomb complexes are targeted to genetic loci by ncRNA. We will examine how RepA, Tsix, and PRC2 interact to trigger the initiation of XCI, address how silencing spreads along the X through Xist RNA and PRC2, and then determine whether ncRNA plays a role in targeting PRC2 to other genomic regions.

Public Health Relevance

The proposed research to study ncRNA and Polycomb proteins is of significant public health relevance due to the growing awareness of their involvement in human disease. For example, a number of imprinting disorders (e.g., Beckwith-Wiedemann Syndrome, Prader Willi and Angelman Syndromes) can be traced to aberrant expression of large noncoding RNAs that control expression of a linked cluster of disease genes. The far-ranging effects of Polycomb proteins for human health can be illustrated in two situations: First, in the maintenance of pluripotency for embryonic stem (ES) cells;and second, in the development of cancer when expression of Polycomb proteins is abnormal (e.g., colon, bladder, endometrial, and lymphatic cancers). Because our research focuses on ncRNA at the interface between Polycomb proteins and specific genetic targets, we expect that the knowledge gained will enhance understanding of normal developmental processes as well as disease processes when ncRNA and interacting Polycomb proteins are not properly regulated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM090278-01
Application #
7787805
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Carter, Anthony D
Project Start
2010-03-01
Project End
2014-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
1
Fiscal Year
2010
Total Cost
$487,582
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Saltzman, Arneet L; Soo, Mark W; Aram, Reta et al. (2018) Multiple Histone Methyl-Lysine Readers Ensure Robust Development and Germline Immortality in Caenorhabditis elegans. Genetics 210:907-923
Kundu, Sharmistha; Ji, Fei; Sunwoo, Hongjae et al. (2018) Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation. Mol Cell 71:191
Wang, Chen-Yu; Jégu, Teddy; Chu, Hsueh-Ping et al. (2018) SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X. Cell 174:406-421.e25
Froberg, John E; Pinter, Stefan F; Kriz, Andrea J et al. (2018) Megadomains and superloops form dynamically but are dispensable for X-chromosome inactivation and gene escape. Nat Commun 9:5004
Rosenberg, Michael; Blum, Roy; Kesner, Barry et al. (2017) Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3' UTRs to Regulate mRNA Expression. Cell Syst 5:368-385.e15
Savol, Andrej J; Wang, Peggy I; Jeon, Yesu et al. (2017) Genome-wide identification of autosomal genes with allelic imbalance of chromatin state. PLoS One 12:e0182568
Sunwoo, Hongjae; Colognori, David; Froberg, John E et al. (2017) Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1). Proc Natl Acad Sci U S A 114:10654-10659
Wang, Chen-Yu; Froberg, John E; Blum, Roy et al. (2017) Comment on ""Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing"". Science 356:
Kundu, Sharmistha; Ji, Fei; Sunwoo, Hongjae et al. (2017) Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation. Mol Cell 65:432-446.e5
Zovoilis, Athanasios; Cifuentes-Rojas, Catherine; Chu, Hsueh-Ping et al. (2016) Destabilization of B2 RNA by EZH2 Activates the Stress Response. Cell 167:1788-1802.e13

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