We propose to study the involvement of coactivators in the function of the beta-globin locus control region (LCR), a genetic element that regulates the chromatin structure, transcription, and replication of the beta-globin genes. Multiple hematopoietic and ubiquitous transcription factors are important for transactivation by the LCR. A common theme among transcriptional regulatory proteins is their involvement in protein-protein interactions with coactivators . Coactivators mediate activation by covalent modification of chromatin or engagement in protein-protein interactions that facilitate assembly of RNA polymerase II preinitiation complexes. To date, no coactivators have been identified that mediate LCR function. Because the LCR disrupts chromatin over long distances (greater than 50-kb), we hypothesize that the LCR recruits chromatin modifying enzymes, specifically histone acetylases (HATs), and a ubiquitin ligase, which acts as coactivators to catalyze a chromatin changes necessary for long-range activation.
In Specific Aim 1, we will assess the roles of the HATs, CBP/p300, P/CAF, and GCN5 in long- range activation by the LCR. To test the hypothesis that HATs mediate transactivation of globin genes through interactions with the LCR, the influence of wild-type HATs and acetylase-deficient mutants on LCR function will be studied in transfection assays.
In Specific Aim 2, we will determine whether the ubiquitin ligase WWP1 and related WW domain proteins influence LCR function. Based on physical and functional interactions between WW domains and the LCR binding protein, NF-E2, we will test the hypothesis that such proteins are important for LCR function.
In Specific Aim 3, we will test whether amino acid sequences of NF-E2 important for CBP/p300 and WW domain binding are required for long-range transactivation by the LCR. We have delineated distinct sequences within the activation domain of NF-E2 necessary for CBP/p300 and WW domain binding and will determine whether these interactions are required for NF-E2 mediated transactivation. These studies represent the first phase of our long-term goal to understand the role of coactivators in LCR function, to test whether hematopoietic signaling pathways modulate their activity, and to ascertain their role in hematopoiesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK055700-02
Application #
6177458
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1999-05-01
Project End
2003-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
2
Fiscal Year
2000
Total Cost
$208,610
Indirect Cost
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Wozniak, Ryan J; Bresnick, Emery H (2008) Epigenetic control of complex loci during erythropoiesis. Curr Top Dev Biol 82:55-83
Kim, S-I; Bresnick, E H (2007) Transcriptional control of erythropoiesis: emerging mechanisms and principles. Oncogene 26:6777-94
Johnson, Kirby D; Boyer, Meghan E; Kang, Jeong-Ah et al. (2007) Friend of GATA-1-independent transcriptional repression: a novel mode of GATA-1 function. Blood 109:5230-3
Kim, Shin-Il; Bultman, Scott J; Jing, Huie et al. (2007) Dissecting molecular steps in chromatin domain activation during hematopoietic differentiation. Mol Cell Biol 27:4551-65
Martowicz, Melissa L; Grass, Jeffrey A; Bresnick, Emery H (2006) GATA-1-mediated transcriptional repression yields persistent transcription factor IIB-chromatin complexes. J Biol Chem 281:37345-52
Johnson, Kirby D; Kim, Shin-Il; Bresnick, Emery H (2006) Differential sensitivities of transcription factor target genes underlie cell type-specific gene expression profiles. Proc Natl Acad Sci U S A 103:15939-44
Bresnick, Emery H; Johnson, Kirby D; Kim, Shin-Il et al. (2006) Establishment and regulation of chromatin domains: mechanistic insights from studies of hemoglobin synthesis. Prog Nucleic Acid Res Mol Biol 81:435-71
Bresnick, Emery H; Martowicz, Melissa L; Pal, Saumen et al. (2005) Developmental control via GATA factor interplay at chromatin domains. J Cell Physiol 205:1-9
Im, Hogune; Grass, Jeffrey A; Johnson, Kirby D et al. (2005) Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region. Proc Natl Acad Sci U S A 102:17065-70
Pal, Saumen; Nemeth, Michael J; Bodine, David et al. (2004) Neurokinin-B transcription in erythroid cells: direct activation by the hematopoietic transcription factor GATA-1. J Biol Chem 279:31348-56

Showing the most recent 10 out of 20 publications