Project Summary/Abstract The Y chromosome is widely believed to be the sole source of sex differences in mammals. Emerging work, including from our group, however, also implicates X-linked genes in inducing sexually dimorphic gene expression. Although most X-linked genes are expressed equally between females and males through the transcriptional inactivation of one of the two X chromosomes in females, some escape X-inactivation and are subject to higher expression in females. Differences in the levels of expression of X-linked genes between the sexes may thus contribute to sex differences. The long-term goal of this proposal is to elucidate mechanisms of sex-biased transcriptional regulation. In the prior round of funding, we found that the higher expression in females of the X-inactivation escapee Kdm5c induces X-inactivation selectively in females and not males in an evolutionarily conserved. We have also accumulated preliminary data suggesting that whereas at a higher expression dose KDM5C induces X-inactivation, at a lower dose it paradoxically first prevents inactivation of the active X chromosome in males as well as in females. We further found that KDM5C also regulates autosomal genes in a sex-biased manner. The overall objective of this proposal, which constitute the next steps toward attaining our long-term goal, is to determine mechanistically how KDM5C functions in a sex- biased manner. Our central hypothesis is that KDM5C regulates gene expression in a sex-biased manner on the X chromosomes as well as autosomes through dose-dependent and locus-specific H3K4me2/3 demethylation. The rationale for this proposal is that the mechanistic dissection of KDM5C function will inform the hormone-independent regulation of sexually dimorphic gene expression. The proposal's central hypothesis will be tested by the following two specific aims: 1) Determine how KDM5C orchestrates the selection of the active and the inactive X chromosome in mouse embryos and stem cells; and, 2) Mechanistically dissect the functions of KDM5C in hormone-independent regulation of sex-biased gene expression in mouse embryos and differentiating embryonic stem cells. The Aims will be tested by ablating or tuning the expression of KDM5C protein and connecting changes in histone H3K4me1/2/3 modifications and their downstream effectors to changes in the expression of the KDM5C target loci genome-wide. The proposed research is innovative because it promises a novel mechanism underlying mammalian sex differences, including in the divergent transcriptional fates of the X chromosomes, using cutting-edge chromatin and single-cell transcriptional profiling technologies. The significance of the proposal is that it will identify novel dose-dependent genetic and epigenetic mechanisms that contribute to sex differences independently of hormones.