Project Summary/ Abstract Humans vary considerably in the process of red blood cell production (erythropoiesis). Such variation contributes to diseases, including numerous forms of anemia. Much of this variation is attributable to genetic polymorphisms found in the population. However, as with most other complex diseases and traits, how such variation impacts the process of erythropoiesis remains poorly understood. We have made tremendous progress in beginning to decipher how human genetic variation impacts erythropoiesis during the first five years of this grant from the NIDDK. We have pioneered the use of massively parallel reporter assays as an approach to systematically dissect how non-coding genetic variation revealed from genome-wide association studies (GWAS) functionally impacts transcriptional regulatory elements. We have also utilized CRISPR/Cas9 genome editing to bolster these findings in an endogenous context. In this competitive renewal application, we aim to invigorate these studies and directly address how human genetic variation shapes the non-coding genomic landscape of erythropoiesis. We will build upon our prior studies and use innovative new approaches to ask these questions in primary human erythroid cells. We have recently conducted one of the largest GWAS of erythroid traits and have identified over 1,800 distinct associated loci. We propose to utilize a number of cutting-edge genomic tools that will allow us to gain important insights from these emerging large-scale genetic studies. First, we aim to define putative causal variants underlying these associations and their functional effects on the linked transcriptional regulatory elements. Second, we will systematically identify target genes that may be affected by the regulatory elements harboring trait-associated variation. Finally, we will dissect mechanisms of action by which a subset of these variants impact target genes and alter human erythropoiesis. Not only will the proposed studies over the next five years significantly advance our understanding of human erythropoiesis and related diseases, but this work will also serve as a paradigm for the systematic dissection of other complex diseases and traits.