Molecular Regulation of Myeloid Cell Differentiation

Sakamoto, Kathleen

Abstract

This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION: Myeloid leukemias result from the dysregulation of proliferation and differentiation. Hematopoietic growth factors and their receptors play a critical role in lineage commitment and differentiation of myeloid progenitor cells. Egr-1, or early growth response gene-1, is an immediate early response gene which is required for terminal differentiation of myeloid cells. In response to differentiating agents, the induction of egr-1 is rapid, transient, and not dependent on protein synthesis. Egr-1 is transcriptionally regulated in response Granulocyte-Colony Stimulating Factor (G-CSF) or 12-0- tetradecanoylphorbol-13-acetate (TPA) in myeloid leukemic cell lines. The overall goal of this First Award proposal is to use a molecular and biochemical approach to define the transcriptional regulation of egr-1 by specific DNA-binding proteins activated by myeloid-specific differentiating agents. Myeloid cell lines will be used as a tool to study the transcriptional regulation of egr-1 by growth factors and agents which induce differentiation. The myeloid leukemic cell line, 32Dc13, differentiates but does not proliferate in the presence of G-CSF. TF-1, a human myeloid leukemic cell line, differentiates to macrophages in response to TPA.
The specific aims of this proposal are to: 1) define egr-1 promoter sequences critical for signal transduction by myeloid-specific differentiating agents; 2) isolate and purify proteins that interact with egr-1 promoter sequences identified in Aim 1; and finally 3) characterize the role of these proteins in signaling pathways activated by myeloid-specific inducers of differentiation. In this manner, the signaling pathways mediating G-CSF or TPA response will be traced in reverse. First, recombinant constructs containing regions of the egr-1 promoter will be transiently transfected into 32Dc13 or TF-1 cells and stimulated with G-CSF or TPA, respectively. Critical egr-1 promoter sequences will be identified by deletion analysis and site-directed mutagenesis. Second, the proteins binding to these sequences will be characterized by Dnase I footprinting (for novel transcription factor binding sites) and simultaneously, electromobility shift assays with antibodies to proteins binding to known recognition sequences will be performed. The ultimate goal of this proposal is to determine the post-translational modification of transcription factors activated by myeloid-specific differentiating growth factors such as G-CSF.