The underlying goal of this proposal is to experimentally determine the mechanisms and molecules employed in the process of """"""""hemoglobin switching"""""""" in chickens, and to extend these studies into the homologous processes employed in globin gene regulation in mice and humans. During the past five years, many of the cis-regulatory sequences required for erythroid- specific transcriptional regulation of globin genes in several vertebrate organisms have been determined. Additionally, the identities of several of the protein factors used to accomplish these molecular switches have been revealed. During the next five years, we plan to define more precisely the cellular and molecular mechanisms which vertebrate organisms employ to elicit these erythroid cell lineage-specific changes in gene expression. We propose to test a number of central questions in erythropoiesis and in generation of the erythroid cell lineage. 1) Is the developmental process of hemoglobin switching homologous in chickens, mice and humans (are conceptually comparable mechanisms and trans-acting factors involved)? 2) Biochemically and mechanistically, how does the murine GATA-1 transcription factor activate erythroid-specific genes? 3) Do GATA transcription factor family members other than GATA-1 (all of which are found in erythroid cells) functionally contribute to erythroid cell differentiation and to hemoglobin switching? 4) Is hemoglobin switching determined in a single kind of progenitor cell, or do multiple erythroid progenitors exist during embryonic development? 5) How does transcription factor NF-E2 regulate cell lineage-specific transcriptional activation in chicken and mammalian erythroid differentiation? 6) Is NF-E4 a central factor involved in effecting the embryonic to adult beta-globin gene switch in chickens, and is the homologous factor playing a similar role in human globin gene switching? 7) Do the chicken and human beta-globin gene enhancers display a developmental stage-specific preference for the gene to be activated? 8) Is the sequential transcriptional activation and silencing of the human embryonic and fetal beta-globin genes due to developmentally regulated alternative utilization of globin gene distal and proximal regulatory elements during development? The answers to these questions, should these experiments prove to be successful, will provide unprecedented insight into this most fundamental developmental regulatory process, and coherent strategies for direct molecular intervention into altering human hemoglobin disease states could then be proposed.

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National Heart, Lung, and Blood Institute (NHLBI)
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Molecular Cytology Study Section (CTY)
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Northwestern University at Chicago
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Cui, Shuaiying; Tanabe, Osamu; Sierant, Michael et al. (2015) Compound loss of function of nuclear receptors Tr2 and Tr4 leads to induction of murine embryonic ?-type globin genes. Blood 125:1477-87
Cui, Shuaiying; Lim, Kim-Chew; Shi, Lihong et al. (2015) The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice. Blood 126:386-96
Shi, Lihong; Sierant, M C; Gurdziel, Katherine et al. (2014) Biased, non-equivalent gene-proximal and -distal binding motifs of orphan nuclear receptor TR4 in primary human erythroid cells. PLoS Genet 10:e1004339
Shi, Lihong; Lin, Yu-Hsuan; Sierant, M C et al. (2014) Developmental transcriptome analysis of human erythropoiesis. Hum Mol Genet 23:4528-42
Cui, Shuaiying; Tanabe, Osamu; Lim, Kim-Chew et al. (2014) PGC-1 coactivator activity is required for murine erythropoiesis. Mol Cell Biol 34:1956-65
Hosoya, Tomonori; Clifford, Mary; Losson, RĂ©gine et al. (2013) TRIM28 is essential for erythroblast differentiation in the mouse. Blood 122:3798-807
Suzuki, Mikiko; Yamazaki, Hiromi; Mukai, Harumi Y et al. (2013) Disruption of the Hbs1l-Myb locus causes hereditary persistence of fetal hemoglobin in a mouse model. Mol Cell Biol 33:1687-95
Shi, Lihong; Cui, Shuaiying; Engel, James D et al. (2013) Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction. Nat Med 19:291-4
Campbell, Andrew D; Cui, Shuaiying; Shi, Lihong et al. (2011) Forced TR2/TR4 expression in sickle cell disease mice confers enhanced fetal hemoglobin synthesis and alleviated disease phenotypes. Proc Natl Acad Sci U S A 108:18808-13
Cui, Shuaiying; Kolodziej, Katarzyna E; Obara, Naoshi et al. (2011) Nuclear receptors TR2 and TR4 recruit multiple epigenetic transcriptional corepressors that associate specifically with the embryonic ?-type globin promoters in differentiated adult erythroid cells. Mol Cell Biol 31:3298-311

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