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)
Type
Research Project (R01)
Project #
2R01DK052356-19
Application #
8830790
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Bishop, Terry Rogers
Project Start
Project End
Budget Start
Budget End
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
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 14:103-14
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; 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
Stees, Jared R; Hossain, Mir A; Sunose, Tomoki et al. (2015) High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation. Mol Cell Biol 36:238-50
Barrow, Joeva J; Li, Ying; Hossain, Mir et al. (2014) Dissecting the function of the adult β-globin downstream promoter region using an artificial zinc finger DNA-binding domain. Nucleic Acids Res 42:4363-74
Rosenberg, Michael; Fan, Alex Xiucheng; Lin, I-Ju et al. (2013) Cell-cycle specific association of transcription factors and RNA polymerase ii with the human β-globin gene locus. J Cell Biochem 114:1997-2006
Deng, Changwang; Li, Ying; Liang, Shermi et al. (2013) USF1 and hSET1A mediated epigenetic modifications regulate lineage differentiation and HoxB4 transcription. PLoS Genet 9:e1003524
Stees, Jared S; Varn, Fred; Huang, Suming et al. (2012) Recruitment of transcription complexes to enhancers and the role of enhancer transcription. Biology (Basel) 1:778-93
Yang, Tao; Jian, Wei; Luo, Yi et al. (2012) Acetylation of histone deacetylase 1 regulates NuRD corepressor complex activity. J Biol Chem 287:40279-91
Barrow, Joeva J; Masannat, Jude; Bungert, Jörg (2012) Neutralizing the function of a β-globin-associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain. Proc Natl Acad Sci U S A 109:17948-53

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