The human -type globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) that is located far upstream of the genes. Mutations in the -globin locus are among the most common disease-causing mutations in the human population. These mutations lead to thalassemias, characterized by reduced or absent adult -globin gene expression, or sickle cell anemia. A possible therapy for these hemoglobinopathies is the reactivation of the normally repressed -globin genes in adult erythroid cells. We propose to continue our efforts to understand how the LCR regulates globin gene expression during development and differentiation and will develop new methodology for changing expression patterns in the - globin gene locus. We established a novel procedure utilizing small synthetic DNA binding domains to analyze and neutralize specific cis-regulatory DNA elements in the -globin gene locus. We will target these synthetic DNA binding domains to known repressor binding sites in the -globin gene promoters and to activator binding sites in the gene encoding for the -globin repressor Bcl11A. Targeting these sites with synthetic DNA binding proteins is expected to increase -globin expression in adult erythroid cells. We and others have shown previously that the LCR recruits transcription complexes which produce enhancer RNAs (eRNAs). Transcription factors NF-E2 and USF have been implicated in transcription complex recruitment to the LCR. We will utilize the synthetic DNA binding domains to analyze the contribution of cis-regulatory elements in the LCR that interact with transcription factors NF-E2 and USF. Furthermore, we will examine if LCR associated transcripts or the process of transcription contributes to LCR mediated activation of globin gene expression. The four aims of this proposal will focus on optimizing the DNA binding specificity and delivery methods for synthetic DNA binding proteins (aim 1), to use synthetic DNA binding proteins to analyze cis-regulatory elements in the -globin locus and to reactivate -globin expression in adult erythroid cells (aim 2), to identify all proteins associated with the human LCR in erythroid cells of transgenic mice (aim 3), and to analyze the mechanism(s) by which the LCR and LCR associated non-coding transcription activates the globin genes (aim 4).

Public Health Relevance

One goal of this proposal is to gain fundamental knowledge about the function of a powerful long distance regulatory element that activates expression of globin genes in erythroid cells. The other goal of this proposal is to establish procedures for changing gene expression patterns in the -globin gene locus using synthetic DNA binding domains. It is anticipated that the results of the proposed studies will contribute to improved therapies for hemoglobinopathies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK052356-19
Application #
8830790
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Bishop, Terry Rogers
Project Start
1997-06-01
Project End
2018-08-31
Budget Start
2014-09-15
Budget End
2015-08-31
Support Year
19
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Li, Biaoru; Zhu, Xingguo; Hossain, Mir A et al. (2018) Fetal hemoglobin induction in sickle erythroid progenitors using a synthetic zinc finger DNA-binding domain. Haematologica 103:e384-e387
Shen, Yong; Nar, Rukiye; Fan, Alex X et al. (2018) Functional interrelationship between TFII-I and E2F transcription factors at specific cell cycle gene loci. J Cell Biochem 119:712-722
Hossain, Mir A; Bungert, Jörg (2017) Genome Editing for Sickle Cell Disease: A Little BCL11A Goes a Long Way. Mol Ther 25:561-562
Hossain, Mir A; Knudson, Isaac J; Thakur, Shaleen et al. (2017) Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells. Methods Mol Biol 1654:361-375
Stees, Jared R; Hossain, Mir A; Sunose, Tomoki et al. (2016) High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range ?-Globin Gene Regulation. Mol Cell Biol 36:238-50
Li, Ying; Schulz, Vincent P; Deng, Changwang et al. (2016) Setd1a and NURF mediate chromatin dynamics and gene regulation during erythroid lineage commitment and differentiation. Nucleic Acids Res 44:7173-88
Hossain, Mir A; Shen, Yong; Knudson, Isaac et al. (2016) Activation of Fetal ?-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains. Mol Ther Nucleic Acids 5:e378
Hossain, Mir A; Shen, Yong; Knudson, Isaac et al. (2016) Activation of Fetal ?-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains. Mol Ther Nucleic Acids 5:e378
Deng, Changwang; Li, Ying; Zhou, Lei et al. (2016) HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development. Cell Rep 14:103-114
Hossain, Mir A; Barrow, Joeva J; Shen, Yong et al. (2015) Artificial zinc finger DNA binding domains: versatile tools for genome engineering and modulation of gene expression. J Cell Biochem 116:2435-44

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