Molecular Self-Titration as a Mechanism of Gene Regulation

Abstract

In systems biology, molecular titration is a mechanism for introducing sensitivity of genetic networks to their regulatory proteins. Since most eukaryotic transcription factors lack chemical control by low-molecular-weight hormones or metabolites, transcription factors have evolved various strategies, such as cooperative DNA binding and sequestering partner proteins, to generate sensitivity in the expression of the genes that they regulate. The ETS superfamily of transcription factors, which are broadly distributed in the animal kingdom, regulate target genes in an active monomeric state. On the one hand, many ETS members, such as the master transcription factor PU.1, are involved in critical cell-fate decisions and signaling pathways that require highly sensitive output responses, yet no coherent mechanism exists for generating the required sensitivity. On the other hand, dimerization of ETS proteins in both DNA-bound and free states via their DNA-binding domain has been reported, but its purpose and biophysical nature remain obscure. We propose that dimerization serves as a sensitivity-generating mechanism by molecular self-titration: self-association sequesters free ETS proteins in inactive dimers, while dimerization in the DNA-bound state inhibits transactivation by the active protein/DNA complex. With the positive auto-regulation of the PU.1 (Spi1) gene generating self-amplification of PU.1, homodimerization would provide the minimal negative feedback needed to prevent explosion and maintain stable dynamics of PU.1 target genes in the absence of additional interactions. We are defining the thermodynamic and structural parameters of the PU.1 homodimer in the free and DNA-bound states. The data show that dimerization involves previously unexplored molecular surfaces that are distinct from the “orthosteric” protein/DNA contact interface and offer insight into how molecular self-titration would participate in the dynamics of PU.1-dependent gene expression.