Cooperative Gsx2-DNA Binding Requires DNA Bending and a Novel Gsx2 Homeodomain Interface

Abstract

The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the question - how do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely seven base pairs apart. However, the mechanistic basis for Gsx DNA binding and cooperativity are poorly understood. Here, we used biochemical, biophysical, structural, and modeling approaches to (1) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation; (2) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions; (3) solve a high-resolution monomer/DNA structure that reveals Gsx2 induces a 20° bend in DNA; (4) identify a Gsx2 protein-protein interface required for cooperative DNA binding; and (5) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors, thereby providing a deeper understanding of HD specificity.

Keywords: Gsx; Homeodomain; cooperative; transcription factor.

Figure 1.. The homeodomain region of Gsx2 that cooperatively binds DNA is monomeric in solution.

(A) Schematic of Gsx2 with the conserved homeodomain colored orange, flanking sequences in purple, and the eh1 repression domain in cyan. Numbers denote amino acid positions in the M. musculus Gsx2 and D. melanogaster Ind homologs. (B) Sequence alignment of the murine Gsx2^167-305 protein with similar regions from its mouse paralog Gsx1, and its ortholog Ind from Drosophila. (C) The Gsx2 PWMs generated from HT-SELEX data revealed specific DNA monomer and DNA dimer sites^,. (D) Far UV CD spectra depicting the difference in spectra between Gsx2^HD, Gsx2^167-305, and buffer. Table generated by the CDSSTR program from the DicroWeb online server estimating the secondary structure predictions of the corresponding CD data. (E) DSF assays of Gsx2^HD and Gsx2^167-305 at 10 μM, highlighting a single melting peak averaging at 53.5°C and 51.1°C, respectively. (F) SV-AUC of Gsx2^HD and Gsx2^167-305 at increasing concentrations yields a sedimentation coefficient distribution showing a single peak for both constructs, which corresponds to a Gsx2 monomer.

Figure 2.. Gsx proteins bind a consensus DNA binding motif with high affinity.

Isotherms from isothermal titration calorimetry (ITC) depict the binding activity of Gsx2^HD, Gsx1^HD, and Gsx2^167-305 to both a -TAATTA- and -TAATGG- motif. (A) The Gsx2^HD shows an average 15 nM affinity for the -TAATTA- site and a 36 nM affinity for the -TAATGG- site. (B) The Gsx1^HD shows nearly identical affinities to Gsx2^HD, with an average 18 nM and 42 nM affinity for the -TAATTA- and -TAATGG- sites, respectively. (C) Conversely, the Gsx2^167-305 protein containing short N- and C-terminal flanking regions shows weaker affinity with 77 nM and 143 nM affinity for the -TAATTA- and -TAATGG- sites, respectively.

Figure 3.. Gsx2 forms a canonical HD fold with three major contacts within the major and minor DNA grooves.

X-ray crystal structure of Gsx2 203–264 (HD) bound to DNA containing the consensus motif TAATTA. (A) A single Gsx2^HD-DNA complex shows the classic HD three helix fold, with the third helix inserted into the major groove of DNA. Gsx2 is colored cyan while DNA is colored grey, except for the -TAATTA- motif, which is colored green. N- and C-terminal ends are labeled. (B) A water molecule mediates hydrogen bond interactions between Q252 (Q50) of the HD and two thymine bases within the major groove. (C) Highly conserved N253 (N51) makes two direct hydrogen bonds with an adenine in the major groove. (D) R207 (R5) makes two direct hydrogen bonds with a thymine and an adenine in the minor groove. (E) Schematic of all specific and nonspecific protein-DNA interactions.

Figure 4.. Modeling Gsx2^HD bound to a DNA dimer site reveals a potential protein-protein interface involved in cooperativity that is dependent on DNA bending.

(A) Top, side, and bottom views of the Gsx2^HD-DNA monomer structure, which shows significant bending of the DNA to an approximately 20° angle due to Gsx2^HD interactions. Gsx2^HD is shown in green, and DNA is shown in light grey. Blue lines parallel to the DNA represent the helical axis, while blue lines perpendicular to the DNA represent the degree and directionality of DNA bending. (B) Isolated view of the DNA from the structure without the Gsx2 protein, highlighting DNA bending. (C) Comparison views of an ideal B-form DNA duplex with an identical sequence to the DNA used in the Gsx2^HD-DNA structure. (D) Top and back view of a Gsx2^HD-DNA dimer model with optimal dimer sequence, 7bp spacer length, and orientation, but lacking any DNA bending. One Gsx2^HD protein is in yellow, the second Gsx2^HD protein is in green, and the DNA is light grey. No protein-protein contacts are observed in this model. (E) Comparison views of a Gsx2^HD-DNA dimer model based upon a dimer sequence with 7bp spacer length and 20° bend observed in monomer structure. Potential protein-protein interactions are observed between both Gsx2^HD proteins. (F) Comparison views of a Gsx2^HD-DNA dimer model with a dimer sequence with an 8bp spacer length and DNA bending shows a loss of direct contact between the two Gsx2^HD proteins.

Figure 5.. Gsx2 preferentially selects DNA dimer sequences with flexible A/T-rich spacers.

(A) Analysis of in vitro HT-SELEX data revealed significantly faster enrichment for dimer sequences with A/T-rich spacers compared to G/C-rich spacers. Note, the PWM generated from analyzing the sequences after the 4^th round of selection at top reveals a boxed spacer sequence of 5 nucleotides with strong overall A/T preference. Graph below depicts the percentage of sequences encoding A/T versus G/C rich spacers in the original library (cycle 0) and after each round of selection (cycles 1 through 4). (B) Analysis of in vivo CUT&RUN data showing that Gsx2 has a bias for DNA dimer sequences with A/T-rich spacers. At top is the Gsx2 dimer PWM from the top quartile of called peaks and the dashed line in the graph below indicates a perfect Gaussian curve if no bias was detected. (C) EMSAs of Gsx2^167-305 binding to DNA dimer duplexes with various A/T-rich or G/C-rich spacers show increased cooperative binding for all A/T-rich sequences compared to G/C-rich sequences. Note, each EMSA probe was tested in 5 lanes containing the following concentrations of purified Gsx2^167-305 protein (0, 50, 100, 200, and 400 nM) (D) The cooperativity of the EMSAs was quantified using the Tau factor, which reveals an approximately 6-fold increase for A/T-rich sequences versus G/C-rich sequences, consistent with our bioinformatic data highlighting Gsx2’s preference for more flexible A/T-rich spacer sequences. Each dot represents the Tau value from an individual binding reaction of either an A/T rich or G/C rich spacer probe (n = 12 for each group). Error bars denote standard deviation. Tau factors were compared with a two-sided unpaired student t-test.

Figure 6.. Gsx2^HD dimer model reveals a novel binding interface with residue conservation amongst the Gsx/Ind family.

(A) Expanded front and top view of the protein-protein interface observed in our 7bp spacer length dimer model. Four residues are primarily localized at the interface: S212, L216, L231, and I234. The two Gsx2^HD monomers are colored yellow and green, and the interface residues are shown in stick representation. (B) Sequence alignment of the HDs Gsx2^HD and Gsx1^HD, which have been shown to bind DNA cooperatively, with close relatives, none of which are expected to bind DNA cooperatively. Red triangles denote residues S212, L216, L231, and I234. (C) A representative EMSA and corresponding Tau factor calculations of WT^HD and I234E^HD binding to the 7bpS (7bp spacer, cooperative) and 8bpS (8bp spacer, non-cooperative) DNA dimer probes, demonstrating that the I234E^HD protein shows significantly reduced cooperative binding on the 7bpS probe compared to the WT^HD protein. Note, each EMSA probe was tested in 4 lanes containing the following concentrations of purified Gsx2^HD protein (0, 25, 100, and 400nM). Each dot represents the Tau value from for either the 7bp or 8bp spacer probe at each of the different protein concentrations. The mean Tau value for each probe:protein combination is noted and error bars denote standard deviation of the mean. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. (D) Replicate EMSA from A but instead showing WT^167-305 and I234E^167-305 binding to the 7bpS and 8bpS DNA dimer probes. Again, only WT^167-305 on the 7bpS probe is strongly cooperative, demonstrating that even in the presence of the flanking regions around the HD the I234E mutation is sufficient to greatly diminish Gsx2 cooperative DNA binding. Note, each EMSA probe was tested in 4 lanes containing the following concentrations of purified Gsx2^167-305 protein (0, 25, 100, and 400 nM). Each dot represents the Tau value from for either the 7bp or 8bp spacer probe at each of the different protein concentrations. Numbers beneath graph bars denote the mean Tau value for each probe:protein combination. Error bars denote standard deviation of the mean. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test.