The broad long-term objective of this proposal is to elucidate the mechanism of globin gene regulation by the erythroid transcription factor GATA-1.
The specific aims are to identify amino acid residues encoding DNA binding specificity and activation of transcription, clarify the role of binding affinity in the function of specific GATA-1 sites, and characterize the effect of interactions of GATA-1 with other transcription factors on promoter activity and developmental specificity in erythroid cells. We will continue site-directed mutagenesis studies to identify the amino acid residues directing sequence specificity and stability of DNA binding. Fusion of GATA-1 domains to the DNA binding domain of GAL will be tested in erythroid cell lines for the ability to activate transcription and interact with other transcription factors. Mutants of GATA-1 will be used to reconstitute erythropoiesis derived from an ES cell line lacking a functional GATA-1 gene. GATA sites selected from the human beta-globin locus will be assayed for binding affinity, and sites with differing affinities will be compared for activity and developmental specificity. We will construct minimal erythroid promoters from GATA-1, CACCC, and AP- 1/NFE-2 binding sites, and assay their activity and developmental specificity in erythroid cell lines. Cis-elements binding other transcription factors will be linked to GATA-1 sites in minimal promoters and tested for interaction in transient assays. Erythroid """"""""minigenes"""""""" will be constructed from oligonucleotides encoding GATA-1 sites and interacting cis-elements flanked by matrix attachment regions (MARs), and driving expression of LacZ. These will be tested in transgenic mice for developmental stage specificity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048790-02
Application #
3367944
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-08-01
Project End
1996-07-30
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
Bigas, A; Martin, D I; Milner, L A (1998) Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines. Mol Cell Biol 18:2324-33
Sutherland, H G; Martin, D I; Whitelaw, E (1997) A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene. Mol Cell Biol 17:1607-14
Walters, M; Andrews, N C; Magis, W et al. (1996) Erythroid AP-1/NF-E2 elements vary in their response to NF-E2. Exp Hematol 24:445-52
Martin, D I; Whitelaw, E (1996) The vagaries of variegating transgenes. Bioessays 18:919-23
Magis, W; Martin, D I (1995) HMG-I binds to GATA motifs: implications for an HPFH syndrome. Biochem Biophys Res Commun 214:927-33
Fiering, S; Epner, E; Robinson, K et al. (1995) Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev 9:2203-13
Walters, M; Martin, D I (1992) Functional erythroid promoters created by interaction of the transcription factor GATA-1 with CACCC and AP-1/NFE-2 elements. Proc Natl Acad Sci U S A 89:10444-8