! PROJECT SUMMARY/ABSTRACT Malaria is the mosquito-borne disease responsible for the greatest morbidity and mortality world-wide. After significant reductions over the past two decades, malaria incidence and mortality have plateaued over the last five years highlighting the need for additional interventions. Malaria transmission requires parasites to differentiate from the replicating asexual blood stages to non-replicating male and female gametocytes necessary to infect mosquitos. The early stages of gametocyte development are a key period during which parasites not only need to initiate the gametocyte-specific transcriptional program, but must also block expression of genes that lead to cell-cycle entry associated with asexual replication. Our long-term goal is to block transmission and replication of malaria parasites by interfering with the regulatory mechanisms that govern this essential point of control in the parasite lifecycle. Recent studies reported specific up-regulation of chromatin-modifying enzymes in sexually committed parasites, suggesting that chromatin re-organization plays a crucial role during the asexual to gametocyte transition. While technical barriers have thwarted studying chromatin organization and gene expression in these critical early stages of gametocyte development, a recently published study observed specific expansion of subtelomeric heterochromatin domains in more mature gametocytes. This proposal aims to test the hypothesis that restructuring of the parasite chromatin landscape in sexually committed parasites and early gametocytes is critical for the developmental switch from asexual replication to the formation of transmissible forms. New approaches, including single-cell transcriptomics and cell-type specific chromatin analysis, now allow researchers to define gene expression and chromatin organization during early gametocyte development for the first time (Aim 1) and study the function of genes critical for regulation of chromatin specifically during gametocyte development. Experiments in Aim 2 will define the critical role of putative histone demethylases in gametocyte development. While these enzymes appear individually dispensable for asexual growth in vitro, they are notably upregulated in gametocytes, and early gametocytes are highly susceptible to inhibitors of these enzymes, with susceptibility peaking in gametorings and Stage I gametocytes. Lastly, Aim 3 focuses on defining the role of a newly identified DNA- binding protein in heterochromatin mediated gene-silencing during gametocyte development. Preliminary experiments show that loss of expression results in aberrant gametocyte development, upregulation of heterochromatin-silenced genes, and alterations in chromatin organization. The proposed experiments will offer a first window into the critical early stages of gametocyte development and provide new targets for transmission blocking strategies. !