Characterizing the functional effects of transcription factor mutations using a high-throughput microfluidic platform
Abstract/Contents
- Abstract
- Transcription factors (TFs) are DNA binding proteins that regulate gene expression in cells, enabling cellular processes and organismal development. Mutations in TFs and regulatory DNA sequences are also implicated in developmental disease and cancer. While various technologies have enabled high-throughput studies of mutations to regulatory DNA sequences, far less is known about how TF mutations alter DNA binding, largely due to technical hurdles associated with expressing and purifying proteins at scale. To remedy this, we developed STAMMP (Simultaneous Transcription Factor Affinity Measurements via Microfluidic Protein arrays), a microfluidic platform that enables quantitative characterization of hundreds of TF mutants simultaneously. As proof-of-concept, we applied STAMMP to measure binding affinities for ~210 mutants of Pho4, a model TF from yeast, interacting with 9 DNA sequences (> 1800 Kds). Our work reveals that Pho4 is highly sensitive to protein mutations, even at poorly conserved protein regions, and that mutations can both strengthen and weaken binding, suggesting a selective pressure for moderate affinity binding. Using biochemical double-mutant cycles, we further identify novel residues that contribute to Pho4 DNA binding specificity. More recently, we have applied STAMMP to study MAX, a TF from humans mutated in cancer, yielding biophysical functional characterizations for a wide variety of variants of unknown significance. In the future, STAMMP will enable rapid characterization of disease variants of TFs to accelerate functional characterization of clinically relevant TF mutants and assist therapeutic development. More broadly, STAMMP will enable deciphering of rules and biophysical principles governing TF function, providing a strong basis to study and engineer genetic regulation.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Aditham, Arjun Krishna |
|---|---|
| Degree supervisor | Fordyce, Polly |
| Thesis advisor | Fordyce, Polly |
| Thesis advisor | Bintu, Lacramioara |
| Thesis advisor | Kundaje, Anshul, 1980- |
| Degree committee member | Bintu, Lacramioara |
| Degree committee member | Kundaje, Anshul, 1980- |
| Associated with | Stanford University, Department of Bioengineering |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Arjun K. Aditham. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/jj953hz0196 |
Access conditions
- Copyright
- © 2021 by Arjun Krishna Aditham
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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