Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Of the inherited anemias, the hemoglobinopathies, particularly sickle cell disease (SCD) and ?-thalassemia, stand out due to their prevalence and severity. The ability to postnatally elevate fetal hemoglobin (HbF) levels in SCD and ?-thalassemia via co-inheritance of positive genetic modifiers of HbF production or hydroxyurea (HU) treatment can significantly alleviate disease severity. HU, however, has serious side effects and may even be carcinogenic. Novel therapies aimed at elevating HbF expression in adults are, therefore, desperately needed. Three major loci, including BCL11A, modify HbF expression in humans. Together, however, they account for only ~50% of the variation in HbF levels. Hence, significant gaps in knowledge remain regarding the genetic control of HbF production. We will take advantage of two powerful mouse resources to identify novel regulators of ?-like globin switching, the mouse mutant Nan (neonatal anemia) and the newly developed high resolution Diversity Outbred (DO) mapping resource. In Nan a single amino acid change in the second zinc finger of KLF1 (erythroid Krppel-like factor, EKLF) causes sequence selective disruption of binding to a subset of its target genes resulting in severe anemia and a striking failure of hemoglobin switching. Expression of embryonic ?h1 globin is upregulated 100-fold in adult Nan spleen vs. wild type via a BCL11A independent mechanism that is not secondary to stress erythropoiesis. In highly genetically diverse DO mice, expression of ?h1 globin in adults varies substantially from individual to individual. Thus, Nan and DO mice are ideal tools with which to detect novel regulators of ?-like globin switching using powerful, unbiased genetic QTL (quantitative trait locus/loci) mapping integrated with global genomic and proteomic strategies. The overall goal of this proposal is to identify novel genes regulating ?-like globin switching. To accomplish this goal, the specific aims are to (1) map modifiers of embryonic globin expression in Nan F2 intercrosses and in DO mice to identify KLF1-dependent and independent loci, respectively; (2) perform ChIP-seq in erythroid populations to compare DNA targets differentially bound by wild type (WT) and mutant (Nan) KLF1; (3) obtain erythroid global transcriptome (RNA-seq, miR-seq) and phospho-proteome profiles to identify gene expression and post-transcriptional differences; and (4) analyze and integrate all data to identify, prioritize, and initiate functional analysis of the most compelling candidate genes. Identification of genetic loci regulating ?-like globin switching, differences i Nan- vs. WT-KLF1 DNA targets, and differences in the transcriptome and proteome of Nan and WT erythroid cells will converge to identify novel regulators of ?-like globin switching, thereby providing important new therapeutic targets for hemoglobinopathies.

Public Health Relevance

Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Notably, anemias share common etiologies in mice and man. Thus, mice are excellent model organisms in which to study mechanisms of anemia and potential therapies. Of the inherited anemias, the hemoglobinopathies, particularly sickle cell disease and ?-thalassemia, stand out due to their prevalence and severity. Elevation of fetal hemoglobin (HbF), which is normally switched off at birth, is an effective means to alleviate disease severity in these disorders. Our aim is to identiy gene variants and genetic pathways that lead to natural increases in HbF postnatally, thus identifying novel targets for the development of new therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK100692-04
Application #
9261519
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2013-09-16
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
4
Fiscal Year
2017
Total Cost
$354,375
Indirect Cost
$151,875
Name
Jackson Laboratory
Department
Type
Research Institutes
DUNS #
042140483
City
Bar Harbor
State
ME
Country
United States
Zip Code
04609
Planutis, Antanas; Xue, Li; Trainor, Cecelia D et al. (2017) Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development. Development 144:430-440
Gillinder, Kevin R; Ilsley, Melissa D; NĂ©bor, Danitza et al. (2016) Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability. Nucleic Acids Res :
Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M Inmaculada et al. (2015) PPAR-? and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal. Nature 522:474-7