Evolution of Mammalian Sex Chromosome Dosage Compensation

Abstract Mammalian dosage compensation equalizes X-linked gene expression between XX females and XY males. Defective dosage compensation can cause cell and organismal lethality. Current models posit that mammalian dosage compensation requires X-inactivation and that X-inactivation requires the X-linked Xist

long noncoding RNA. Despite much work, however, whether Xist and X-inactivation are essential for dosage compensation remains unclear. The goal of this proposal is to define how mammalian dosage compensation originated and evolved. The overall objectives of this proposal are to (i) determine the temporal and tissue-

specific requirement of Xist RNA and X-inactivation in dosage compensation; and, to (ii) test the contributions of ancestral and evolutionarily conserved X chromosome-encoded factors in dosage compensation in the mouse model system. The central hypothesis of the proposal is that mammalian dosage compensation

originated independently of Xist and X-inactivation and that ancestral X chromosome-encoded factors equalized X-linked gene expression between the sexes. The rationale for this proposal is that it provides insights into how cells manage differences in their sex chromosome complements specifically and how cells

equalize gene expression despite differences in chromosome copy number more generally. The proposal's central hypothesis will be tested through the following approaches: 1) determine the requirements of Xist RNA and X-inactivation in dosage compensation in the epiblast lineage, which generates all somatic tissues; and, 2)

test the contributions of two ancestral X chromosome-encoded genes in dosage compensation in the absence Xist and X-inactivation. Dosage compensation will first be analyzed in homozygous Xist-null (XistD/D) female mouse embryos, embryonic stem cell (ESC)-derived epiblast-like cells (EpiLCs) and neural progenitor cells

(NPCs), and tissues of XistD/D female mice, which we have found to be viable and fertile. Next, X chromosome dosage compensation will be tested in embryos, EpiLCs, NPCs, and tissues of adult mice that lack Xist and also one or both alleles of the candidate ancestral dosage compensation factors. The proposed research is

innovative and potentially transformative because it defines novel and Xist- and X inactivation-independent mechanisms of mammalian dosage compensation using advanced transcriptomic and chromatin profiling techniques. In the absence of Xist and X-inactivation, the proposal's central hypothesis predicts that the

ancestral candidate genes execute dosage compensation either through random monoallelism or by simultaneously diminishing expression from both alleles of X-linked genes in XX female cells. The significance of the proposal is that it will define when, where, and which genes Xist RNA silences during development and

delineate the functions of ancestral X-linked genes in novel Xist- and X inactivation-independent modes of dosage compensation. Overall, the proposal promises to mechanistically define how mammalian X chromosome dosage compensation originated and has evolved. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page