DNA Occupancy of Polymerizing Transcription Factors: A Chemical Model of the ETS Family Factor Yan

Abstract

Transcription factors use both protein-DNA and protein-protein interactions to assemble appropriate complexes to regulate gene expression. Although most transcription factors operate as monomers or dimers, a few, including the E26 transformation-specific family repressors Drosophila melanogaster Yan and its human homolog TEL/ETV6, can polymerize. Although polymerization is required for both the normal and oncogenic function of Yan and TEL/ETV6, the mechanisms by which it influences the recruitment, organization, and stability of transcriptional complexes remain poorly understood. Further, a quantitative description of the DNA occupancy of a polymerizing transcription factor is lacking, and such a description would have broader applications to the conceptually related area of polymerizing chromatin regulators. To expand the theoretical basis for understanding how the oligomeric state of a transcriptional regulator influences its chromatin occupancy and function, we leveraged the extensive biochemical characterization of E26 transformation-specific factors to develop a mathematical model of Yan occupancy at chemical equilibrium. We find that spreading condensation from a specific binding site can take place in a path-independent manner given reasonable values of the free energies of specific and non-specific DNA binding and protein-protein cooperativity. Our calculations show that polymerization confers upon a transcription factor the unique ability to extend occupancy across DNA regions far from specific binding sites. In contrast, dimerization promotes recruitment to clustered binding sites and maximizes discrimination between specific and non-specific sites. We speculate that the association with non-specific DNA afforded by polymerization may enable regulatory behaviors that are well-suited to transcriptional repressors but perhaps incompatible with precise activation.

Figure 1

Lattice model for Yan binding at equilibrium. (A) Yan molecules (boxes) occupy discrete sites (bold black lines) at an element of DNA. The class of elements considered by the model contain one high-affinity specific binding site (ETS site) and n non-specific binding sites. Yan molecules participate in different types of interactions depending on their occupancy at the element, and the free energies of these interactions are given by the terms α, β, and γ. α represents the specific DNA binding free energy, β represents the non-specific DNA binding free energy, and γ represents the SAM-mediated protein-protein interaction free energy, contingent upon two molecules of Yan being adjacent to one another in the lattice. (B) Configurations of binding and free energy values for an element where n is equal to 2. The binary representation of the index of configurations (0,1,…,6,7) corresponds to the binding configuration itself. This provides a straightforward means of manipulating and scoring many microstates. Each microstate is shown with its free energy in terms of α, β, and γ. To see this figure in color, go online.

Figure 2

Yan occupancy spreads across the element at equilibrium and depends on both protein-DNA and protein-protein interactions. All plots show fractional occupancy of positions along a 24 site element as a function of concentration; insets plot the same data with concentration on a log scale. The ETS site is at position 1 in (A) and (C) and at position 13 in (B) and (D), as shown in the keys. (A) Site-by-site occupancy using the wild-type values for Yan binding parameters. Occupancy is highest at the ETS site (red) and progressively decreases at sites farther from the ETS, suggestive of a spreading profile. (B) Site-by-site occupancy for an element with the ETS site in the interior at position 13, using wild-type values for α, β, and γ. (C) Site-by-site occupancy when the specific DNA binding term is set equal to the wild-type value for non-specific binding, i.e., an element without any specific binding sites. Occupancy is highest in the center of the element (green) and decreases symmetrically from the center. (D) Site-by-site occupancy when the protein-protein interaction term (γ) is set to 0 kcal/mol. Significant occupancy is only observed at the ETS site for high concentrations (green curve at position 13). Note that identical lines are plotted on top of one another.

Figure 3

Occupancy of nucleated microstates depends on specific DNA binding of Yan. All graphs show fractional occupancy of types of Yan microstates as a function of concentration. Insets show the same data with concentration on a log scale (A and B) or a zoomed view of data at low fractional occupancies on a linear scale (C and D). (A) Fractional occupancy of nucleated microstates with two or more self-associated molecules versus that of non-nucleated microstates with two or more self-associated molecules, calculated for wild-type parameters. (B) Same as (A), but with specific DNA binding term set equal to the wild-type value for non-specific DNA binding. (C and D) Nucleated microstates with exactly x self-associated molecules. Values of x from 2 to 24 are shown. Colors progress through values of x, with red representing 2 and purple representing 24 (see key below the plots). (C) Fractional occupancy of nucleated microstates with exactly x self-associated molecules, for the wild-type parameters of Yan. (D) Same as (C), but with specific binding set equal to the wild-type value of non-specific binding. To see this figure in color, go online.

Figure 4

Exploration of parameter space for nucleated, self-associated microstates. All graphs are spectral heat maps plotting fractional occupancy of nucleated microstates with two or more self-associated molecules. Fractional occupancy of 0 is represented in black, and fractional occupancy increases moving through the visible color spectrum, ending with the fractional occupancy of 1 represented in white (see color key below). Values of the parameters α, β, and γ are plotted along the x or y axes from −3.5kcal/mol to −12.0 kcal/mol in 0.125 kcal/mol increments. All heat maps shown are calculated at a concentration of Yan of 0.1 nM. (A–A″) α versus β, with increasing values of γ shown from top to bottom (−4.0, −7.0, and −10.0 kcal/mol, respectively). (B–B″) α versus γ, with increasing values of β shown from top to bottom (−4.0, −6.0, and −9.0 kcal/mol, respectively). (C–C″) β versus γ, with increasing values of α shown from top to bottom (−5.0, −8.0, and −11.0 kcal/mol, respectively).

Figure 5

Clustering multiple ETS sites within an element increases occupancy at the sites. One ETS site is held fixed, whereas another ETS site is moved from the adjacent position (orange) to the most distal position (purple). The plotted fractional occupancies are measured at the fixed ETS site as a function of concentration. Dashed lines denote fractional occupancy at the fixed site without an additional ETS site in the system. (A) Fractional occupancy with the wild-type parameters of Yan. (B) Fractional occupancy when the protein-protein interaction term is set to 0 kcal/mol. Note that all lines are identical in fractional occupancy and plotted on top of one another.

Figure 6

Restricting Yan polymerization decreases occupancy at distal sites but maintains occupancy at specific sites, especially tandem ETS sites. Fractional occupancies are plotted as a function of concentration as in Fig. 2, with red curves representing the ETS site and purple curves representing the most distal site. Wild-type data from Fig. 2A are plotted as gray dashed lines. Wild-type parameters for α, β, and γ were used for all calculations. (A) Yan restricted to dimers (B) Yan restricted to trimers. (C) Yan restricted to tetramers. (D) Yan restricted to pentamers. (E and F) Fractional occupancy of Yan restricted to dimers, with multiple ETS sites. (E) Fractional occupancy at an element with two maximally separated ETS sites. Dashed lines are shown for some of positions 13–24 to show overlap of curves. (F) Fractional occupancy at an element with a tandem pair of ETS sites at positions 1 and 2.