Deletion of CRH Expression in the Placenta

Cincinnati Childrens Hosp Med Ctr, Cincinnati OH

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Abstract

PROJECT SUMMARY The fundamental molecular signals that initiate human parturition at term gestation, or lead to preterm birth, are unknown. The long-term goals of our research efforts are to elucidate the pathways that determine human birth timing. Our published work has demonstrated that anthropoid primate pregnancy has been subjected to evolutionary selective pressures that have left genomic signatures in parturition-related genes related to this divergence from other species. We have exploited these differences in regulation of birth timing between humans and other mammals by applying comparative and functional genomics to reveal primate and human adaptations that initiate labor and delivery. One compelling molecular target in the regulation of human parturition is corticotropin-releasing hormone (CRH). Relative maternal plasma CRH concentration, derived from placental synthesis and secretion, predicts the likelihood of preterm, term, and post-term birth. The specific functional role of CRH in the control of birth timing, or other aspects of pregnancy, has not been determined. CRH is only expressed in anthropoid placenta, and rodents do not express the CRH binding protein in their peripheral blood, making traditional model organisms for study of limited utility. With the advent of CRISPR/Cas9 technology, we are no longer limited to ?traditional? model organisms for study, and have the potential for using non-human primates for genome editing and physiological analysis. Using comparative genomics, we computationally tested the hypothesis that anthropoids acquired a new regulatory element after the divergence from prosimians that confers the unique capacity of their placentae to make CRH. Surprisingly, we found a simian-specific THE1B retroviral long-terminal repeat (LTR) insertion approximately 2.5 kb 5' of the usual CRH transcriptional start site in anthropoid primates. We have generated 2 founder lines of transgenic mice that harbor a 180 kb bacterial artificial chromosome (BAC) containing the human CRH locus and its extended flanking regions. Both of these lines have been found to express human CRH mRNA in the placenta and other sites of normal CRH expression demonstrating the sufficiency of human cis-acting sequences to drive expression. Most recently, using CRISPR/Cas9 genome editing, we deleted the THE1B from the BAC transgenic mice, and proved this abolishes placental but not hypothalamic expression. Given this compelling information, we are uniquely poised to dissect the role of placental CRH by deleting the CRH THE1B element in a non-human primate, rhesus, in collaboration with the California National Primate Research Center. Our Specific Aim will be to establish germline modification of the rhesus genome to generate animals that lose placental CRH expression without loss of CRH expression in other tissues. The outcome from this R21 application, to develop a novel, tractable, non-human primate system will provide the foundation for future investigation of the function and epigenetic regulation of placental CRH in primate pregnancy.