The level of fetal hemoglobin (HbF) is a key modifier of the clinical severity of the ?-hemoglobin disorders sickle cell disease and ?-thalassemia. The goal of this proposal is to identify mechanisms that regulate HbF level and could serve as rational targets for efforts to re-induce HbF for the ?-hemoglobin disorders. Genome- wide association studies (GWASs) have identified BCL11A as a critical HbF-associated locus. Functional studies have demonstrated that BCL11A is a bona fide repressor of ?-globin transcription. Naturally occurring common genetic variation modulates an erythroid-specific enhancer of BCL11A. Our prior studies identified the GWAS-marked erythroid enhancer of BCL11A as the substrate for common genetic variation associated with HbF level. Genome editing has revealed critical sequences within this enhancer required for HbF repression in the adult stage. I hypothesize that these sequences recruit critical trans-acting factors, which themselves are required for suppressing HbF levels. In addition, I hypothesize that other critical sequences at the BCL11A and HBS1L-MYB loci are required to repress HbF in the adult-stage. To test these hypotheses, I will: (1) utilize a high-resolution, high-throughput genome editing approach to identify non-coding sequences at BCL11A and HBS1L-MYB associated with HbF level; and (2) define trans-acting factors that physically and functionally interact with critical on-coding sequences at BCL11A and HBS1L-MYB. I expect that accomplishment of these aims will not only deepen our current understanding of cis- and trans-acting determinants of developmental regulation of globin gene expression but also yield novel insights to guide therapeutic efforts for HbF re-induction.

Public Health Relevance

The ?-hemoglobin disorders sickle cell disease and ?-thalassemia are severe inherited blood diseases affecting millions of individuals worldwide. BCL11A and HBS1L-MYB are genetic loci that determine the level of fetal hemoglobin, which in turn can ameliorate the ?-hemoglobin disorders. In my previous work, I have identified novel mechanisms by which common genetic variation affects regulatory regions of the BCL11A gene. In this proposal, I seek to: (1) extend my previous work to identify additional critical regulatory sequences at BCL11A and HBS1L-MYB; and (2) characterize transcription factors that control these critical sequences. These results will be of vital relevance for the design of novel therapeutic strategies for the ?-hemoglobin disorders

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Small Research Grants (R03)
Project #
1R03DK109232-01
Application #
9092681
Study Section
Kidney, Urologic and Hematologic Diseases D Subcommittee (DDK-D)
Program Officer
Bishop, Terry Rogers
Project Start
2016-06-01
Project End
2018-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
1
Fiscal Year
2016
Total Cost
$88,500
Indirect Cost
$38,500
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Vinjamur, Divya S; Bauer, Daniel E (2018) Growing and Genetically Manipulating Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP) Cell Lines. Methods Mol Biol 1698:275-284
Liu, Nan; Hargreaves, Victoria V; Zhu, Qian et al. (2018) Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell 173:430-442.e17
Yamauchi, Takuji; Masuda, Takeshi; Canver, Matthew C et al. (2018) Genome-wide CRISPR-Cas9 Screen Identifies Leukemia-Specific Dependence on a Pre-mRNA Metabolic Pathway Regulated by DCPS. Cancer Cell 33:386-400.e5
Clement, Kendell; Farouni, Rick; Bauer, Daniel E et al. (2018) AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing. Bioinformatics 34:i202-i210
Schoonenberg, Vivien A C; Cole, Mitchel A; Yao, Qiuming et al. (2018) CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis. Genome Biol 19:169
Gundry, Michael C; Dever, Daniel P; Yudovich, David et al. (2017) Technical considerations for the use of CRISPR/Cas9 in hematology research. Exp Hematol 54:4-11
Lessard, Samuel; Gatof, Emily Stern; Beaudoin, Mélissa et al. (2017) An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility. J Clin Invest 127:3065-3074
Bauer, Daniel E; Brendel, Christian; Fitzhugh, Courtney D (2017) Curative approaches for sickle cell disease: A review of allogeneic and autologous strategies. Blood Cells Mol Dis 67:155-168
Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H (2017) Functional interrogation of non-coding DNA through CRISPR genome editing. Methods 121-122:118-129
Canver, Matthew C; Lessard, Samuel; Pinello, Luca et al. (2017) Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat Genet 49:625-634