Investigating the cellular response to folate depletion

Abstract

Folate (folic acid, vitamin B9) is an essential nutrient required for many cellular processes, including nucleotide synthesis, cellular methylation, and redox balance. The series of metabolic reactions involving folate are collectively referred to as one-carbon (1C) metabolism. This is due to folate’s role in regulating the transfer of one-carbon units from metabolic donors, such as serine, to metabolic acceptors, such as nucleotide synthesis intermediates. 1C metabolism has major roles in the prevention of birth defects and megaloblastic anemia, as well as an importance as a target in cancer and anti-autoimmune diseases. However, at the cellular level, little is known about adaptation to and sensing of folate nutrient stress. In this thesis, we characterize the metabolic changes associated with adaptation to mild folate depletion. We find that both folate depletion and 1C metabolism inhibition induce erythroid differentiation in erythroid lineage leukemia, as well as in primary murine erythroid progenitor cells. This induction of differentiation can occur even in the presence of growth factors that typically maintain a stem-cell state and is blocked by supplementation with exogenous nucleosides. These findings suggest that purine levels are sensed in erythroid precursors, and that depletion of purines activates premature erythroid differentiation. In addition, we performed a loss-of-function CRISPR/Cas9-based genomic screen with the goal of identifying genes that are important or detrimental for cell survival in folate depleted nutrient conditions. We provide evidence for previously unexplored metabolic, and nonmetabolic connections, with 1C metabolism. Together, this work identifies new pathways that aid in adaptation to folate depletion and increases our understanding of this important branch of metabolism.