Epigenetic transcriptional regulation is required for a myriad of developmental processes as well as being a significant contributor to human diseases. X-chromosome inactivation provides an experimentally tractable system for the dissection of epigenetic inheritance. X-inactivation results in the mitotically-heritable transcriptional inactivation of one X-chromosome in female mammals, thereby equalizing X-linked gene dosage between males and females. X-inactivation requires the Xist long non-coding RNA that is expressed only from the inactive X-chromosome. Current models posit that Xist RNA induction and coating of the X- chromosome in cis triggers a series of epigenetic events that culminates in X-inactivation. Notably, how Xist is selectively induced in females and not at all in males and how it triggers silencing are still unresolved questions. The objective of this proposal is to address how Xist expression and X-linked gene silencing are triggered during X-inactivation. Our central hypothesis, based on our published work and preliminary data, is that genes that escape X-inactivation function as dose-sensitive factors that induce Xist and, separately, potentiate X-linked gene silencing selectively in females. X-inactivation escapees are expressed from both X- chromosomes in XX females, including from the otherwise inactivated X-chromosome; hence, their expression is higher in females compared to XY males. We developed and employed a custom allele-specific RNA- sequencing pipeline to compile a list of the escape genes in mouse epiblast stem cells, which harbor an inactive-X. We then prioritized validated escapees that are predicted to be evolutionarily conserved and which function as transcriptional/chromatin regulators for a role in inducing X-inactivation. In this proposal, we systematically test the dose-dependent and biochemical activities of two of these escape genes in triggering Xist expression and X-linked gene silencing using unbiased and integrated high-throughput approaches. The results are expected to inform how genes along the length of the X-chromosome are silenced and why females undergo X-inactivation and males do not. The epigenetic factors and mechanisms that execute X-inactivation are known to overlap with those that regulate embryonic development and disease progression. Thus, understanding the cascade of epigenetic events that characterizes X-inactivation offers a window into identifying the common factors and mechanisms that establish epigenetic expression patterns broadly.

Public Health Relevance

X-chromosome inactivation has provided essential insights into epigenetic regulation, since the same or similar epigenetic control systems are known to orchestrate normal development and disease progression. X- inactivation is a paradigm of epigenetic inheritance, since within a shared nucleoplasm one X of an identical pair becomes inactivated while the other remains active, and that replicated copies of the inactive and active X- chromosomes faithfully maintain their transcriptional states through many cell division cycles. Here we propose to test a novel hypothesis that genes which escape X-inactivation trigger X-inactivation in a dose- dependent manner.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM124571-04
Application #
9978593
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Carter, Anthony D
Project Start
2017-09-08
Project End
2021-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
4
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Genetics
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Cloutier, Marissa; Harris, Clair; Gayen, Srimonta et al. (2018) Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation. Methods Mol Biol 1861:177-203
Gayen, Srimonta; Kalantry, Sundeep (2017) Chromatin-enriched lncRNAs: a novel class of enhancer RNAs. Nat Struct Mol Biol 24:556-557