Species-specific differences exist in human and mouse with respect to X chromosome inactivation (XCI): a developmentally regulated process in all mammals for achieving dosage compensation of X- linked genes. The long non-coding RNA Xist is crucial in establishing XCI as cells of the inner cell mass (ICM) of the pre-implantation blastocyst progress to differentiation. In mouse, Xist is not expressed in the nave pluripotent cells of the ICM: it only becomes induced at the start of differentiation and silences the X chromosome from which it is expressed for the life of the cell. Contrary to this, human XIST is expressed from all X chromosomes of the nave ICM cells without conferring silencing. During differentiation, only one of the two X chromosomes of female human cells retains XIST expression and becomes silenced, similar to mouse. Here we propose to decipher how functions of XIST are changed in early human development. Specifically, we will address how human XIST can induce silencing in differentiated and primed pluripotent (post-XCI) but not in nave pluripotent (pre-XCI) cells, and decipher the molecular mechanisms that enable human XIST to silence the X chromosome. We hypothesize that different RNA species of XIST are expressed in pre- and post-XCI cells due to alternative splicing and/or differential enhancer/promoter usage. We will test this by examining the RNA species expressed in pre- and post- XCI environments using PCR, RNA-seq, RAP-seq, and 5'-RACE. Moreover, the ability of XIST to silence may depend on the presence of certain protein factors differentially expressed in pre- and post- XCI cells. To this end we will perform genome-scale knockout library screen using CRISPR/Cas9 editing to find proteins that, when disrupted, interfere with XIST's ability to initiate silencing. Our work on the X chromosome in the human system will shed light on how fundamental epigenetic processes have changed in evolution.

Public Health Relevance

Female mammals inactivate one of their two X chromosomes for the life of the organism, and this process is initiated by the long non-coding RNA XIST. Most of our current understanding of X chromosome inactivation comes from mouse studies, but it is clear that key species- specific differences exist in mouse versus human. This proposal will examine the molecular mechanisms specific to human XIST-mediated X chromosome silencing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31GM115122-01
Application #
8910055
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sledjeski, Darren D
Project Start
2015-09-01
Project End
2017-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
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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Patel, Sanjeet; Bonora, Giancarlo; Sahakyan, Anna et al. (2017) Human Embryonic Stem Cells Do Not Change Their X Inactivation Status during Differentiation. Cell Rep 18:54-67
Gu, Wen; Gaeta, Xavier; Sahakyan, Anna et al. (2016) Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State. Cell Stem Cell 19:476-490
Pastor, William A; Chen, Di; Liu, Wanlu et al. (2016) Naive Human Pluripotent Cells Feature a Methylation Landscape Devoid of Blastocyst or Germline Memory. Cell Stem Cell 18:323-329
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