Our molecular, biochemical, genetic and microscopic analyses of differential gene regulation during hematopoiesis have yielded novel and surprising results. For example, we made the unanticipated finding that, contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, ?- globin gene expression initiates at the nuclear periphery prior to relocalization of the gene more centrally where high level expression occurs. Moreover, we showed that the ?-globin locus control region (LCR) is necessary for this relocation. Our work also revealed genes that are co-regulated in the erythroid or myeloid lineages tend to be clustered in the genome, and that in each lineage, distinct chromosomes tend to associate on the basis of the chromosomal distribution of co-regulated genes. We also discovered that MLL5, a member of the mammalian Trithorax group of proteins, is essential for erythroid differentiation in an in vitro model. We now propose experiments using single locus, genomic, proteomic, genetic and high-resolution microscopy approaches to investigate the relationships among nuclear localization, initiation and maintenance of transcription state, and the mechanism by which MLL5 regulates erythroid differentiation. Specifically, we propose to: 1. Determine the relationships between peripheral localization and transcriptional activity of gene loci during differentiation. To accomplish this, we will use a combination of high-resolution microscopy, mutagenesis of the native ?-globin locus, and tethering of wildtype (WT) and mutant loci to nuclear pore complexes (NPC) and lamina during murine erythropoiesis. We will also determine the genome-wide alterations in peripheral localization and expression state during erythroid differentiation. 2. Determine the molecular basis of cellular memory. We will use the mouse Toll like receptor 4 (Tlr4) model of monoallelic expression to identify cis-sequences and trans-factors that specify positioning of the inactive and active alleles in different nuclear compartments. Using live cell imaging, we will test the hypothesis that maintenance of the alleles in distinct compartments is involved in propagation of transcription state in the absence of the signals responsible for establishing that state, so called "cellular memory". 3. Determine the composition and function of MLL5-containing complexes during differentiation. To test the hypothesis that MLL5 functions are mediated via interactions with different protein partners, we will identify MLL5-interacting proteins during erythroid differentiation in vitro and in vivo, and, using genomic approaches, determine the function of MLL5 containing complexes during erythropoiesis. We also will perform genetic and biochemical screens in Drosophila melanogaster to identify evolutionarily conserved dMLL5 interacting proteins and regulated pathways to complement and inform our analysis of MLL5 functions in mice. In combination, these experiments will lead to a greater understanding of gene activation and silencing in the hematopoietic lineage.

Public Health Relevance

Localization of genes in different compartments the nucleus is a mechanism by which genes are activated and silenced, and various cellular processes, including differentiation, are regulated. Importantly, several malignancies, including Burkitt lymphoma, acute myeloid leukemia (AML) and breast cancer are thought to arise from alterations in nuclear organization. These data highlight the importance of understanding the relationships between gene regulation and nuclear organization.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065440-13
Application #
8255598
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Qasba, Pankaj
Project Start
2000-09-15
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
13
Fiscal Year
2012
Total Cost
$905,478
Indirect Cost
$391,002
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
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