To ensure appropriate dosage compensation, female cells selectively inactivate one of their two X chromosomes in a process called X chromosome inactivation (XCI), a form of epigenetic regulation. In early female mouse embryos, somatic cells of the inner cell mass inactivate their paternal or maternal X chromosomes with equal probability. Thus, cells in somatic female tissues display a random XCI pattern. The mammary gland represents a prime object to investigate genetic pathways that control the specification, proliferation, differentiation, survival and death of cells. Cellular survival and cell death decisons are critically regulated by the ubiquitin proteasome system (UPS). In particular, RING finger ubiquitin ligases act as key components in cell death signaling cascades. The functions of these enzymes for cell survival/death often rely on their RING finger-dependent ubiquitin ligase activity as well as their cellular localizations. We have generated a conditional KO mouse model for the RING finger ubiquitin ligase RLIM which is encoded by the X-linked gene Rnf12. Our preliminary results indicate that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy. We find that alveolar cells lacking RLIM undergo apoptosis as they differentiate from mammary epithelia. Our results suggest further that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom XCI in mammary epithelial cells which primarily silence their maternal X chromosomes. We hypothesize 1) that RLIM is crucially involved in alveolar cell survival pathways and is key for triggering involution, and 2) sex-specific epigenetic regulation of mammary gland biology by the paternal X chromosome. The proposed project will test these hypotheses and identify underlying molecular mechanisms in three specific aims.
Aim 1 will establish RLIM as triggering weaning-induced alveolar cell death/involution and test the involvement of the UPS system.
Aim 2 will connect RLIMs survival functions with nucleo- cytoplasmic shuttling, while Aim 3 establishes and examines mechanisms to establish the non-random XCI pattern in mammary precursor cells. Combined our results will establish RLIM as a novel paradigm in regulating alveolar cell survival/death pathways. Moreover, the results of our research will show that mammary gland biology is decisively controlled by sex-specific epigenetic mechanisms with profound implications for development, differentiation, evolution and disease.
Our preliminary results provide strong evidence that paternal Rnf12/RLIM is required for alveolar morphogenesis in the mouse mammary gland, serves as a critical regulator of alveolar cell survival/cell death decision and likely triggers involution. Th proposed project will establish these functions in vivo and expected results will unravel the underlying molecular, cellular and epigenetic mechanisms for the sex-specific Rnf12/RLIM functions in mammary glands.
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