Anemia is a major source of morbidity and mortality worldwide, particularly among women and children. Much of this burden is attributable to defective red blood cell production (erythropoiesis). A greater understanding of erytkhropoiesis holds promise for the development of better therapies for various forms of anemia. Recent genome-wide association (GWA) studies have revealed over 75 loci associated with red blood cell traits. Furthermore, multiple bioinformatic analyses suggest that the majority of variation du to these common variants is intrinsic to red cell progenitors and precursors, allowing for directed studies on the effects of these genetic variants. Nevertheless, only a few studies of specific candidates have revealed new regulators of erythropoiesis. We and others have shown that many of the erythroid-trait associated loci (and variants in strong linkage disequilibrium (LD)) ar located in non-coding regions of the genome, enriched for empirically defined regions of open chromatin and erythroid enhancer elements marked by the erythroid transcription factors (TFs) GATA1, TAL1, KLF1, and NFE2. In select cases, we have shown that these variants are contained in and affect the activity of those enhancers and that they are dependent upon GATA1 activity. In this project, we propose to utilize a novel and innovate approach termed massively parallel reporter assay (MPRA) to systematically measure the activity of thousands of putative enhancer elements containing variants in strong LD with GWA study loci. Enhancers that show differential activity dependent upon the allele of the candidate variant contained will be prioritized for functional follow-up using cutting-edge CRISPR-Cas9 genome editing technology to confirm causality of that variant with respect to changes in proximal gene expression. Additionally, we will perform a second MPRA in GATA1-induced cells to examine whether each element is dependent upon the activity of the key hematopoietic TF GATA1. Increased differential enhancer activity will likely signal disruption of GATA1, co-factor, or nove TF binding sites, providing evidence of the underlying biological mechanism. Furthermore, each enhancer element of interest will be fully explicated per nucleotide using an innovative multiple mutagenesis approach in a similar MPRA, allowing for identification of TF binding site motifs linked to known or novel TFs involved in erythropoiesis. Successful execution of the proposed work will at a minimum identify multiple causal variants implicated in GWA studies of erythroid traits and create a public resource of erythroid-specific enhancer activity and important TF binding sites for future studies of erythropoiesis. Additionally, the proposed study establishes not just a new paradigm for high-throughput follow-up of GWA studies in troublesome non- coding regions of the genome, but creates a novel method for investigation of common transcriptional regulatory pathway disruption of cell-type or lineage-specific TFs and master regulators by common genetic variation which can be extended to many different cell types and human diseases.

Public Health Relevance

Numerous genome-wide association studies have identified thousands of disease-associated loci, yet the true causal variants and their underlying biological mechanisms remain unknown. Using a model of human erythropoiesis, we will utilize novel methodologies to (1) systematically dissect the function of 75 loci recently implicated in common erythroid trait variation using a massively parallel reporter assay with subsequent verification by state-of-the-art gene editing to implicate these loci in erythropoiesis, (2) describe novel erythroid- specific enhancer function and discover binding sites and their cognate transcription factors, and (3) determine the extent to which common transcriptional regulatory patterns dependent upon GATA1, a master regulator of erythropoiesis, are disrupted by common genetic variation. These studies have significant implications for improving our understanding of erythropoiesis generally, the role of genetic variation in this process, and the necessity of master regulator GATA1 and cofactors in this process;these findings may lead to improved therapies for numerous forms of anemia that are major sources of morbidity and mortality worldwide.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK103794-01
Application #
8797944
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2014-09-20
Project End
2019-07-31
Budget Start
2014-09-20
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
$395,625
Indirect Cost
$170,625
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Kim, Ah Ram; Sankaran, Vijay G (2018) Thrombopoietin: tickling the HSC's fancy. EMBO Mol Med 10:10-12
Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C et al. (2018) Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis. Cell 173:90-103.e19
Guo, Michael H; Nandakumar, Satish K; Ulirsch, Jacob C et al. (2017) Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms. Proc Natl Acad Sci U S A 114:E327-E336
Fiorini, Claudia; Abdulhay, Nour J; McFarland, Sean K et al. (2017) Developmentally-faithful and effective human erythropoiesis in immunodeficient and Kit mutant mice. Am J Hematol 92:E513-E519
Kim, Ah Ram; Ulirsch, Jacob C; Wilmes, Stephan et al. (2017) Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation. Cell 168:1053-1064.e15
Wakabayashi, Aoi; Sankaran, Vijay G (2016) Society for Pediatric Research 2015 Young Investigator Award: genetics of human hematopoiesis-what patients can teach us about blood cell production. Pediatr Res 79:366-70
Kim, Ah Ram; Sankaran, Vijay G (2016) Development of autologous blood cell therapies. Exp Hematol 44:887-94
Polfus, Linda M; Khajuria, Rajiv K; Schick, Ursula M et al. (2016) Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis. Am J Hum Genet 99:481-8
Wakabayashi, Aoi; Ulirsch, Jacob C; Ludwig, Leif S et al. (2016) Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders. Proc Natl Acad Sci U S A 113:4434-9
Ulirsch, Jacob C; Nandakumar, Satish K; Wang, Li et al. (2016) Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits. Cell 165:1530-1545

Showing the most recent 10 out of 20 publications