Reciprocal regulation of DNA binding of the paired domain and homedomain of Pax3
Bibliographic record
Abstract
Pax3 is a gene that encodes for a transcription factor involved in embryogenesis. Pax3 heterozygote mutants in mice (Splotch) and humans (Waardenburg Syndrome) exhibit pigmentary disturbances. Also, neural tube defects (spina bifida) develop in homozygous splotch mice. Pax3 has 2 DNA binding domains, a paired Domain (PD), and a paired-type homeodomain (HD). We have used site-specific modification of single Cys Pax3 mutants (in a Cys-less backbone) to probe structure: function relationships. N-Ethyl maleimide (NEM) modification of a single Cys in the PD (C82) abrogates DNA binding by the PD but also by the HD. Likewise, NEM modification of V263C in the HD abrogates DNA binding by both the HD and the PD. These results demonstrate that the PD and HD do not function as independent DNA binding modules in Pax3, but are functionally interdependent. Pax3 was also modified by insertion of Factor Xa protease sites in either the PD (Xa100) or the HD (Xa216). The effect of DNA binding by the PD and HD on conformational changes in the other domain was monitored by accessibility of the Factor Xa site. Binding by the PD causes a structural change not only in the PD but also in the HD; similarly, DNA binding by the HD causes a conformational change in the PD. These results provide a structural basis for the functional interdependence of the PD and HD in Pax3. Site-specific modification of single Cys mutants was further used to identify a cluster of solvent exposed residues in the beta-hairpin structure and in the first 2 helices of the PD, which when modified (with NEM) impair DNA binding by the HD. Three-dimensional modeling shows that these residues form a hydrophobic pocket possibly defining the site of interaction of the HD. Pax3 double cysteine mutants were also generated and used with bifunctional cysteine specific reagents to monitor proximity relationships in Pax3. We have observed that cysteines placed in the N-terminal portion of the HD can be crosslinked with cysteines placed in the putative HD interaction pocket in the PD, suggesting proximity of the two segments. Taken together, these results suggest that residues in or close to the PD helix 2 and those in or close to the N-terminal arm of the HD undergo direct physical interaction, which we postulate is responsible of the functional interdependence we observe between the PD and HD of Pax3.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.001 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".