Transcription Factor Binding Affinities and DNA Shape Readout

Abstract

An essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

Keywords: Biomolecules; Molecular Biology; Molecular Mechanism of Gene Regulation.

Graphical abstract

Figure 1

Experimental and Data Analysis Strategies (A) Sequence design and measurement of binding energies by HiP-FA. A consensus sequence is mutated with all possible mononucleotide and dinucleotide mutations. The individual TF-DNA binding energies are measured using a robotic system and an automated custom-modified fluorescence microscope; the titration binding curves are reconstructed and analyzed following the HiP-FA procedure (Jung et al., 2018, Jung et al., 2019). (B) Data analysis. After an off-target removal procedure (Transparent Methods), the binding energies are used to determine the 0^th order of binding (PWMs) and the first order of binding (DPWMs), as shown for the TF Bcd. The DPWMs exhibits the mutual information (MI, a metric similar to the information content IC but for dinucleotide representation), which is not included in the simple linear PWM. (C) Analysis of the DNA shape readout contribution. The sensitivity to DNA shape is analyzed following the subsequent steps: the DNA shape features are calculated using look-up tables (Chiu et al., 2017; Li et al., 2017; Zhou et al., 2013). The sensitivity to shape readout (termed shape readout value) is plotted per position against the binding energies (lower panel of c), and a robust linear regression is performed (Transparent Methods). Besides the fit (blue line), the steepest (gray dashed line) and the least steep fit (purple dashed line) are estimated using the confidence intervals provided by the robust linear regression. To make a conservative choice, the least steep slope is taken as the shape readout value. The shape readout values of all features and positions are depicted in the lower right panel for Bcd.

Figure 2

Overview of the PWMs and DPWMs for all the Investigated TFs In the DPWMs, the heights of the dinucleotide letters represent the mutual information (MI) between two positions for the first order of binding. The total information content (IC) and MI are indicated in the right hand side columns for the PWM and DPWMs, respectively. Homeodomain factors and zinc fingers are grouped by color. Average PWMs and DPWMs are shown when replicate measurements were performed.

Figure 3

Overview of DNA Shape Sensitivities for the investigated TFs The stacked shape readout values are plotted for each feature at each position (intra-base pair features) or between two positions (inter-base pair features). To facilitate the comparison with Figure 2, the positions are also labeled with their respective nucleobase at this position of the consensus sequence. The legend for the respective features is in the lower right corner. Homeodomain TFs (blue background color) and zinc finger TFs (green) are grouped together. The significance levels are indicated for each shape readout value bar with a hashing code indicated in the right bottom corner (see Transparent Methods for details). Average shape sensitivity plots are shown when replicate measurements were performed. Overall, there is a widespread use of the DNA shape readout by our TFs.

Figure 4

Correlation between DNA Shape Readout and Structural Information Homeodomains TFs. (A) Shape readout value profile for Bcd. Positions with strong shape readout highlighted with blue rectangles. (B) Residue contacts map for Bcd (obtained using the DNAproDB database (Sagendorf et al., 2019), details in Figure S4). Interaction between bases and positively charged residues highlighted in yellow. (C) Crystal structure of Bcd (pdb-ID: 1ZQ3) (Baird-Titus et al., 2006). Base contacts with the recognition helix in red, with the N-terminal tail in blue. The bluearrow points at the position where the binding domain contacts the narrowing minor groove. (D) Shape readout values for the three homeodomain TFs at position TAAT of the consensus sequence (position 4 of the corresponding PWMs in Figure 2). In addition to being very similar, all three homeodomains show a strong readout of the minor groove at this position. (E) MGW profile along the binding sequence for the consensus binding sequence used for the homeodomains. It exhibits a minimum value at position TAAT (red arrow). B-ZIP TF Gt. (F) Shape readout values for Gt. The black rectangle indicates positions with highly symmetrical shape readout values around the middle vertical axis (added to all three panels at the same position). (G) Crystal structure of a similar B-ZIP TF (pdb-ID: 1GD2) (Fujii et al., 2000) with the same core consensus sequence as Gt. The black box indicates the region of high mirror symmetry around the black axis. Base contacts highlighted in red. (H) Gt's PWM, the first position augmented with data from Jung et al. (Jung et al., 2018). The entire PWM is highly symmetrical. Zinc finger TF GATAe. (I) Shape readout values for GATAe. Positions with strong (1, blue) and weak (4, orange) shape readout values are indicated at the x axis. (J) Their corresponding positions in the protein structure of a similar GATA TF (pdb-ID: 3DFV) (Bates et al., 2008) at both sides. The perspective shows a position with pronounced contacts to the DNA's phosphate backbone and minor groove. Base contacts in red. All crystal structures were produced using the DNAproDB portal (Sagendorf et al., 2019).