We are interested in how the temporal and tissue specific expression of a gene is brought about during development. The orderly activation and repression of individual members of the human globin gene family is a prominent example of this phenomenon. We are studying the changes in chromatin structure that these genes undergo when they are activated. We have used DNAse I footprinting and the electrophoretic mobility shift assay to detect, in vitro, interactions between nuclear proteins from erythroid and non-erythroid cells and DNA sequences from regulatory regions both 5' and 3' to the human epsilon-globin gene. At least one of the interactions we observed was erythroid specific and corresponded to binding of EryF1/NF-E1/GF-1 to two sites in the promoter region. This protein also has strong binding sites in the region 3' to the gene. Other interactions are in the process of being analyzed. To investigate the functional significance of these protein-DNA interactions transient assays in K562 cells and HeLa cells are being employed in which transcription from the epsilon-globin promoter is monitored via a chloramphenicol acetyltransferase reporter gene. An episomal vector system is being developed in order to study in vivo the effect of these protein DNA interactions on higher order chromatin structure and transcription of the epsilon-globin gene. Preliminary studies suggest that an EB virus-based vector can be maintained as an amplified episome in K562 cells. A marked epsilon-globin gene is being inserted into the vector and its regulation will be studied.

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Lee, Jongjoo; Krivega, Ivan; Dale, Ryan K et al. (2017) The LDB1 Complex Co-opts CTCF for Erythroid Lineage-Specific Long-Range Enhancer Interactions. Cell Rep 19:2490-2502
Krivega, Ivan; Dean, Ann (2016) Chromatin looping as a target for altering erythroid gene expression. Ann N Y Acad Sci 1368:31-9
Deng, Wulan; Rupon, Jeremy W; Krivega, Ivan et al. (2014) Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158:849-860
Song, Sang-Hyun; Hou, Chunhui; Dean, Ann (2007) A positive role for NLI/Ldb1 in long-range beta-globin locus control region function. Mol Cell 28:810-22
Zhao, Hui; Kim, Aeri; Song, Sang-Hyun et al. (2006) Enhancer blocking by chicken beta-globin 5'-HS4: role of enhancer strength and insulator nucleosome depletion. J Biol Chem 281:30573-80
Dean, Ann (2006) On a chromosome far, far away: LCRs and gene expression. Trends Genet 22:38-45
Zhao, Hui; Dean, Ann (2005) Organizing the genome: enhancers and insulators. Biochem Cell Biol 83:516-24
Dean, Ann (2004) Chromatin remodelling and the interaction between enhancers and promoters in the beta-globin locus. Brief Funct Genomic Proteomic 2:344-54
Kim, AeRi; Dean, Ann (2004) Developmental stage differences in chromatin subdomains of the beta-globin locus. Proc Natl Acad Sci U S A 101:7028-33
Zhao, Hui; Dean, Ann (2004) An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene. Nucleic Acids Res 32:4903-19

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