Structural and functional analysis of domains within the E2F1 transcription factor

Kelly Lynn Jordan, Thomas Jefferson University

Abstract

The E2F1 transcription factor has been shown to play a pivotal role in regulating progression from G1 into S phase of the cell cycle. This function of E2F1 is dependent on its ability to (1) interact with a number of nuclear regulatory proteins and (2) bind a consensus DNA sequence in target promoters. Two domains were, therefore, analyzed as to their contribution to E2F1 function: the DNA binding domain and the proline-alanine rich amino terminus, the latter a site for protein:protein interaction. The DNA binding domain of E2F1 was shown to be distinct from previously identified DNA binding motifs of the basic helix-loop-helix class, in that it contains a proline-glycine pair in the center of its basic region. This is likely to preclude $\alpha$-helix formation which is the common structure found in many DNA binding motifs including basic helix-loop-helix proteins. Thus, E2F1 appears to contain a novel structure for DNA binding. Adjacent to the DNA binding domain is the proline-alanine rich amino terminus. Previous studies from this laboratory have shown that cell lines expressing an E2F1 mutant lacking the amino terminus (E2F1d87) display several characteristics which are distinct from the parental cell lines. E2F1d87 expressing cells exhibit an increase in S phase duration, increased sensitivity to S-phase specific toxins, an increase in E2F target promoter expression, loss of contact inhibition, and alteration in cellular morphology characterized by disassembly of the cytoskeleton. Here, the change in cell shape and altered gene expression were analyzed more thoroughly. The striking change in shape observed when cells expressing E2F1d87 were cultured in the presence of serum growth factors was found to be due, at least in part, to reduced fibronectin expression at the transcriptional level. However, reduced fibronectin is not responsible for the increase in S phase duration seen in these cells. The ability of a mutant E2F1 in combination with serum growth factors to alter cellular morphology by reducing fibronectin expression suggests a mechanism whereby wild type E2F1 activity may affect cell shape during proliferation. Since the amino terminus contains a motif for interaction with cyclin A:cdk2, it is a potential site for regulation by other interacting proteins. To determine if this was so, genetic screens were employed to identify proteins which interact with the amino terminal domain. Two cDNA's were identified by the dihybrid yeast screen that encode proteins which associate with the amino terminus. The proteins encoded by the isolated cDNA's are novel and interact with the amino terminus of E2F1 in the yeast dihybrid system and on E2F1 affinity columns in vitro. When the E2F1 binding proteins (EBP's) are expressed in vivo. They alter E2F1 mediated gene expression. One of the isolated genes, EBP2, is expressed at higher levels in growing and transformed fibroblasts than in quiescent fibroblasts indicating that its expression is growth regulated. Thus, E2F1 activity is regulated by its interaction with several factors in the amino terminal domain, thereby altering its ability to regulate gene expression and cell cycle progression.

Subject Area

Biochemistry|Molecular biology

Recommended Citation

Jordan, Kelly Lynn, "Structural and functional analysis of domains within the E2F1 transcription factor" (1996). ETD Collection for Thomas Jefferson University. AAI9633538.
https://jdc.jefferson.edu/dissertations/AAI9633538

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