X chromosome inactivation (XIC) is the pre-eminent model for formation of facultative heterochromatin in mammalian cells, a process central to genome regulation in early embryos, and relevant to its abrogation in cancer. In pluripotent stem cells, a remarkable, large non-coding RNA from the X-linked XIST gene accumulates and spreads across one female X chromosome, thereby initiating a process which transforms the euchromatic chromosome into a dense, heterochromatic Barr Body. While much has been learned about histone modifications which mark heterochromatin, it is still not understood what specifically silences transcription. Further, it remains a mystery how XIST RNA localizes to and paints the structure of its parent chromosome, and how this leads to the cascade of biochemical, higher-order structural, and transcriptional changes of a whole chromosome. The fundamental principles are directly relevant to all chromosomes, since autosomes can be silenced by XIST. Although studies of the silencing process have been largely restricted to mouse ES/transgenic cells, we recently demonstrated successful creation of the first tractable, robust model that fully emulates human chromosome silencing. This advantageous new system, in the pluripotent cell context, provides a strong foundation for this project, and a needed resource to the field. We will examine novel concepts that inter-relate non-coding RNA, chromosome architecture, and genomic sequence, at progressive steps during the chromosome remodeling cascade. Our studies have the potential to change the existing view that interspersed repetitive DNA, comprising almost half the human genome, is unimportant for genome function. Our recently published findings demonstrate a new class of chromosomal RNA, rich in repeat sequences and associated with euchromatin. They further suggest that abundant, widely distributed chromosomal RNAs in active chromosomes serve to promote open chromatin state. The research strategy integrates a range of cutting-edge approaches, from in situ analysis of RNAs or DNAs within nuclear structure, to sophisticated gene targeting, high through-put screening, in vitro RNA biochemistry, and bioinformatics.
Aim 1 is focused on dissecting the inter-relationships between key events in the chromosome silencing process, in a new system to initiate human chromosome silencing in patient iPS cells Aim 2 will define the chromosomal anchor proteins and domains of human XIST RNA required for its localization and function, and test a novel hypothesis regarding competition between chromosomal RNAs.
Aim3 investigates the role of chromosome sequence context, particularly different repeat families, in XIST RNA function, and its escape. Research proposed has high potential at multiple levels to advance understanding of the fundamental structure of chromosomes, the DNA sequences involved, and the RNAs/proteins integral to their higher-order structure.

Public Health Relevance

This project uses one of the most powerful biological systems to understand how DNA within the human genome is regulated in different cells during development, which has direct relevance for understanding how it is mis-regulated in diseases, such as cancer. As we recently demonstrated, understanding the basic mechanisms whereby a whole human chromosome is regulated or 'silenced' has implications for clinically important areas of human stem cell biology, and the understanding and potential therapeutics for chromosomal disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053234-18
Application #
9208133
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1996-09-30
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
18
Fiscal Year
2017
Total Cost
$322,995
Indirect Cost
$128,595
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Hall, Lisa L; Byron, Meg; Carone, Dawn M et al. (2017) Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bodies. Cell Rep 18:2943-2956
Wang, Feng; McCannell, Kurtis N; BoŇ°kovi?, Ana et al. (2017) Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs. Cell Rep 21:3691-3699
Hall, Lisa L; Lawrence, Jeanne B (2016) RNA as a fundamental component of interphase chromosomes: could repeats prove key? Curr Opin Genet Dev 37:137-147
Wang, Feng; Shin, JongDae; Shea, Jeremy M et al. (2016) Regulation of X-linked gene expression during early mouse development by Rlim. Elife 5:
Kolpa, Heather J; Fackelmayer, Frank O; Lawrence, Jeanne B (2016) SAF-A Requirement in Anchoring XIST RNA to Chromatin Varies in Transformed and Primary Cells. Dev Cell 39:9-10
Harada, Akihito; Mallappa, Chandrashekara; Okada, Seiji et al. (2015) Spatial re-organization of myogenic regulatory sequences temporally controls gene expression. Nucleic Acids Res 43:2008-21
Hall, Lisa L; Carone, Dawn M; Gomez, Alvin V et al. (2014) Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes. Cell 156:907-19
Shin, JongDae; Wallingford, Mary C; Gallant, Judith et al. (2014) RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast. Nature 511:86-9
Carone, Dawn M; Lawrence, Jeanne B (2013) Heterochromatin instability in cancer: from the Barr body to satellites and the nuclear periphery. Semin Cancer Biol 23:99-108
Swanson, Eric C; Manning, Benjamin; Zhang, Hong et al. (2013) Higher-order unfolding of satellite heterochromatin is a consistent and early event in cell senescence. J Cell Biol 203:929-42

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