The goal of this proposal is to examine a novel mechanism of X-chromosome Inactivation (XCI) specific to developing female B cells, and to determine the transcriptional impact that this dynamic maintenance of XCI has on the inactive X chromosome (Xi). XCI is an essential epigenetic process used by mammals to equalize the dosage of X-linked genes between XX females and XY males. Each cell in the developing female embryo selects one X-chromosome for transcriptional silencing, generating an epigenetically distinct inactive Xi. This silencing is initiated by expression of the long noncoding RNA Xist in cis, and Xist transcripts recruit proteins necessary for heterochromatin formation. Importantly, continuous silencing of the Xi in adult somatic cells is achieved through constant Xist expression and Xi heterochromatin, as both are required for maintaining the appropriate expression of X-linked genes after development. We have recently made the remarkable discovery that developing female B-lymphocytes, unlike all other adult somatic cell types, maintain XCI through a novel mechanism. Nave B cells have unusual patterns of Xist RNA localization and lack Xi heterochromatin formation, yet the mechanisms underlying this dynamic XCI maintenance, and how it affects transcription from the Xi, are completely unknown. We will test the hypothesis that colocalization of Yy1 protein and Xist RNA is required for transcriptional silencing as B lymphocytes develop from hematopoietic stem cells into mature B cells.
In aim 1, we will examine the mechanism of XCI maintenance by determining the role of the transcription factor Yy1 in recruiting Xist RNA to the Xi in B cell development. Conditional Yy1 deletion experiments will determine if Yy1 is required for Xist RNA localization to the Xi. In the second aim, we will determine the amount of transcription from the Xi during B cell development using a powerful allele specific mouse model. High throughput RNA-sequencing in F1 hybrid mice with two different X-chromosomes (XmusXcas) and non-random XCI will allow us to distinguish transcription from the active (mus) and inactive (cas) X chromosomes in B lineage cells with both canonical and absent Xist RNA localization on the Xi. Together, these innovative approaches will enhance knowledge of X-linked gene regulation and mechanisms of XCI maintenance, providing insights into the origins of female-biased diseases where X-linked genes are uniquely overexpressed.

Public Health Relevance

X-chromosome inactivation (XCI) equalizes the dosage of X-linked genes in somatic cells between males and females from embryonic development through adulthood. Aberrant differences in X-linked gene expression have been implicated in sex-specific susceptibility to a wide range of diseases including metabolic, cognitive, cardiovascular, and autoimmune disorders. To understand how XCI can impact X-linked gene expression, it is imperative to elucidate novel mechanisms of XCI maintenance in somatic cell lineages not yet investigated, as will be studied in this proposal.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Sledjeski, Darren D
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University of Pennsylvania
Schools of Medicine
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Le Coz, Carole; Trofa, Melissa; Syrett, Camille M et al. (2018) CD40LG duplication-associated autoimmune disease is silenced by nonrandom X-chromosome inactivation. J Allergy Clin Immunol 141:2308-2311.e7
Syrett, Camille M; Sindhava, Vishal; Hodawadekar, Suchita et al. (2017) Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells. PLoS Genet 13:e1007050