SUMMARY Cell-restricted transcriptional modulators play critical roles in the process of selective gene regulation during hematopoiesis. We have been investigating the molecular and biological function of Erythroid Krüppel-like Factor (EKLF; KLF1). EKLF is a cell-restricted transcription factor that is a global regulator of genes essential for the erythroid program. Our proposal builds on unanticipated novel observations made during the previous funding cycle that extend this long-term study towards innovative directions: We found that EKLF is modified at arginine residues by mono- and di-methylation. The experiments of Aim 1 will address the context for these alterations and their function, and identify the enzymes responsible. We found that the metabolomes of the mouse Nan-EKLF and human CDA-KLF1 mutant erythroid cells are altered; this includes a metabolite involved in chromatin-associated changes in histone modification. The experiments of Aim 2 will examine the global nature of these modifications in mouse primary cells and in our newly-developed CDA patient-derived cell lines. We find that chromatin accessibility is altered in EKLF KO and in the Nan/+ cell. The experiments of Aim 3 will examine these changes in the context of the sets of decreased and ectopically expressed genes in these genetic backgrounds, an analysis that will be expanded to include CDA patient-derived cells. We found that the metabolome of the EKLF KO cell is altered, and leads to changes in DNA epigenetics. The experiments of Aim 4 will globally analyze these changes in the context of the altered RNA expression and chromatin accessibility observed in the absence of EKLF. These studies will be aided by the use of molecular, metabolic, and biochemical analyses, genetic approaches, and use of primary or minimally manipulated cells that have been newly established. Elucidating EKLF’s role in regulatory phenomena will continue to illuminate novel aspects of erythroid biology and the essential mechanisms by which a cell-restricted transcription factor can exert varied yet highly controlled influences on genetic expression, metabolism, and epigenetics.