PROJECT SUMMARY Thrombocytopenia (low platelet count) is a significant problem that can cause life-threatening hemorrhage. Platelets are produced by megakaryocytes (MKs), which differentiate from hematopoietic stem cells (HSCs). Due to a lack of understanding of the fundamental processes driving MK differentiation, therapeutic strategies for thrombocytopenia remain limited. MicroRNAs are small noncoding RNAs that repress gene expression by directing silencing machinery to mRNA targets. Dozens of microRNAs have been implicated in hematopoiesis, or the production of mature blood cells from HSCs. Most microRNAs are long-lived in cells (up to several days), but some are actively degraded in a process termed target-directed microRNA degradation (TDMD), which is mediated by the protein ZSWIM8. TDMD rapidly depletes cells of specific microRNAs, thus relieving repression on their target mRNAs. Homozygous knockout (KO) of Zswim8 in mice leads to 100% perinatal lethality and increased expression >50 microRNAs (ZSWIM8-sensitive microRNAs) in at least one embryonic tissue examined, highlighting ZSWIM8’s physiologic importance. Several of these ZSWIM8-sensitive microRNAs have been previously implicated in hematopoiesis. Limited published data suggest a particularly important role for microRNA decay mechanisms in MK differentiation, as numerous microRNAs are reported to be downregulated during MK maturation. Further, preliminary results from Zswim8 KO mouse embryos showed that out of all cell lineages assessed, the largest effect was in the MK lineage. Hematopoiesis is a stepwise process that requires sequential shifts in the HSC’s gene expression profile as it differentiates into progressively restricted cell types, which may necessitate active degradation of microRNAs that would otherwise persist in the cell for several days. Therefore, the central hypothesis of this proposal is that ZSWIM8- mediated TDMD remodels the cellular microRNA environment to promote gene expression shifts during hematopoietic differentiation. Currently, there is no published information on the role of ZSWIM8-mediated TDMD in hematopoiesis. Aim 1 seeks to characterize the effect of ZSWIM8-mediated TDMD on mammalian hematopoiesis in the fetal and adult stages, as well as during reconstitution of the hematopoietic stem cell niche after bone marrow transplant. Aim 2 focuses on the apparent role of ZSWIM8-mediated TDMD in MK differentiation. MKs will be derived from murine primary cell cultures. Small-RNA sequencing of these cultured MKs will identify microRNAs that are degraded by ZSWIM8-mediated TDMD. Overexpression experiments will pinpoint the microRNAs able to explain the ZSWIM8-dependent MK differentiation phenotype. Pairing these analyses with bulk RNA sequencing will implicate downstream gene regulatory networks impacted by ZSWIM8. Altogether, this work will elucidate the role of ZSWIM8-mediated TDMD on cell fate specialization and uncover novel roles for microRNA regulation in m...