| Estrogens, growth factors, and other mitogens induce proliferation of MCF-7 human breast cancer cells (Dickson RB 1988; Dickson RB 1991; Dickson RB 1995; Ethier 1995), and 17β-estradiol (E2) induced responses are accompanied by activation of the cell cycle and important metabolic genes. The classical mechanism of hormone-dependent gene activation involves binding of the liganded hormone receptor to hormone responsive elements (HRE) in promoter regions of target genes. The bound receptor in turn recruits coactivators, chromatin remodelling proteins, and the transcriptional machinery necessary for gene expression. Our lab has elucidated an alternative DNA binding-independent mechanism of estrogen receptor (ER) action in which transactivation occurs by an interaction of the ligand-bound ERα with the transcription factor Sp1 bound to G or GC-rich motifs in promoters of E2 responsive genes (Krishnan, Wang et al. 1994; Porter, Wang et al. 1996; Duan, Porter et al. 1998).; DNA polymerase α is the major replicase in eukaryotic cells (Alama, Meazza et al. 1993; Burgers 1998) and thus an essential gene for their survival. Preliminary data indicates that DNA polymerase α is upregulated by E2 in MCF-7 breast cancer cells. We hypothesize that DNA polymerase α is activated by ERα in a DNA independent manner and propose to examine the mechanism of hormonal regulation of this gene in breast cancer cells, and to identify the cis-elements in the promoter responsible for this activity.; We propose to study the interactions of the liganded ERα and Sp proteins in the context of these promoters using a novel in vitro footprinting technique that uses the viral CpG methylase Sssl instead of nucleases to map binding of proteins to DNA (Kladde MP 1996; Kladde MP 1996). In addition, the chromatin immunoprecipitation (ChIP) assay will also be developed for investigating the assembly of proteins bound to a given gene promoter at a given point in time. Preliminary results show the importance of cell context, coactivator expression, and rapid exchange of transcription factors in protein binding to E2 responsive promoter regions. |
