I use live fluorescence imaging to probe how molecular interactions regulate transcription. I recently developed proximity-assisted photoactivation (PAPA), which detects protein-protein interactions by using excitation of a “sender” fluorophore to reactivate a “receiver” fluorophore from a photochemical dark state. PAPA has made it possible to visualize specific endogenous protein complexes at single-molecule resolution in live cells. My long-term goal is to combine PAPA, high-throughput single-molecule tracking, other fluorescence imaging methods, and genetic approaches to understand how networks of interactions between proteins, DNA, and RNA regulate the transcription of specific genes.
I will launch my own lab in the Johns Hopkins Department of Biophysics in July, 2025, as a member of the Epigenome Sciences Cluster. Prospective students and postdocs are welcome to contact me by email!
PAPA papers:
Graham, T. G. W., Ferrie, J. J., Dailey, G. M., Tjian, R., & Darzacq, X. (2022). Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA). eLife, 11, e76870. https://doi.org/10.7554/eLife.76870. The ur-text of PAPA.
Graham, T.G.W., et al. (2024) Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes. https://www.biorxiv.org/content/10.1101/2024.06.25.600644v1. First application of PAPA to endogenous protein complexes that regulate the transcription elongation factor P-TEFb.
Dahal, L., Graham, T. G.W., Dailey, G. M., Heckert, A., Tjian, R., & Darzacq, X. (2023). Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging. https://doi.org/10.1101/2023.09.16.558083. Application of PAPA to Type II nuclear receptors.
PAPA perspectives:
Katsnelson A. (2024). Red light, green light: flickering fluorophores reveal biochemistry in cells.
Nature, https://www.nature.com/articles/d41586-024-02964-8
Watch this, PAPA! (2022) eLife, https://elifesciences.org/digests/76870/watch-this-papa
Review about single-molecule tracking:
Dahal, L., Walther, N., Tjian, R., Darzacq, X., & Graham, T. G. W. (2023). Single-molecule tracking (SMT): a window into live-cell transcription biochemistry. Biochemical Society transactions, 51(2), 557–569. https://doi.org/10.1042/BST20221242
GitHub: https://github.com/tgwgraham. GitLab: https://gitlab.com/tgwgraham PAPA/SMT analysis code, 3D printing files, and plasmid sequences.
LinkedIn: https://www.linkedin.com/in/tgwgraham/
More bibliographic blobbity blah…
First postdoc in the lab of Vanessa Ruta, The Rockefeller University:
Handler, A., Graham, T. G. W., Cohn, R., Morantte, I., Siliciano, A. F., Zeng, J., Li, Y., & Ruta, V. (2019). Distinct Dopamine Receptor Pathways Underlie the Temporal Sensitivity of Associative Learning. Cell, 178(1), 60–75.e19. https://doi.org/10.1016/j.cell.2019.05.040. Built an automated system to study learning and memory in flies by presenting odor stimuli while optogenetically activating or inhibiting neurons with red and green light pulses. (I guess I like red and green light pulses.)
Graduate work in the labs of Joe Loparo and Johannes Walter, Harvard Med School:
Non-homologous end joining (NHEJ)
Graham, T. G., Walter, J. C., & Loparo, J. J. (2016). Two-Stage Synapsis of DNA Ends during Non-homologous End Joining. Molecular Cell, 61(6), 850–858. https://doi.org/10.1016/j.molcel.2016.02.010. Developed an in vitro single-molecule FRET (smFRET) assay in Xenopus egg extracts that allowed us to discover two intermediates in double-strand break repair by non-homologous end joining (NHEJ).
Graham, T. G. W., Carney, S. M., Walter, J. C., & Loparo, J. J. (2018). A single XLF dimer bridges DNA ends during nonhomologous end joining. Nature Structural & Molecular Biology, 25(9), 877–884. https://doi.org/10.1038/s41594-018-0120-y. Showed that XLF is present as a single dimer, in contrast to the prevailing model in the field.
Graham, T. G. W., Walter, J. C., & Loparo, J. J. (2017). Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts. Methods in enzymology, 591, 233–270. https://doi.org/10.1016/bs.mie.2017.03.020
DNA-bridging by ParB proteins.
Graham, T. G., Wang, X., Song, D., Etson, C. M., van Oijen, A. M., Rudner, D. Z., & Loparo, J. J. (2014). ParB spreading requires DNA bridging. Genes & development, 28(11), 1228–1238. https://doi.org/10.1101/gad.242206.114. Showed that bacterial centromeric ParB proteins can bridge DNA.
MPhil work in the group of Robert Best, University of Cambridge:
Graham, T. G., & Best, R. B. (2011). Force-induced change in protein unfolding mechanism: discrete or continuous switch?. The journal of physical chemistry. B, 115(6), 1546–1561. https://doi.org/10.1021/jp110738m
Undergrad work with Ilaria Rebay and Aaron Dinner, UChicago:
Graham, T. G., Tabei, S. M., Dinner, A. R., & Rebay, I. (2010). Modeling bistable cell-fate choices in the Drosophila eye: qualitative and quantitative perspectives. Development (Cambridge, England), 137(14), 2265–2278. https://doi.org/10.1242/dev.044826
Zhang, J., Graham, T. G., Vivekanand, P., Cote, L., Cetera, M., & Rebay, I. (2010). Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan. Molecular and cellular biology, 30(5), 1158–1170. https://doi.org/10.1128/MCB.01225-09
Other stuff I’ve contributed to:
Mammalian cell imaging:
Ferrie, J. J., Karr, J. P., Graham, T. G. W., Dailey, G. M., Zhang, G., Tjian, R., & Darzacq, X. (2024). p300 is an obligate integrator of combinatorial transcription factor inputs. Molecular Cell, 84(2), 234–243.e4. https://doi.org/10.1016/j.molcel.2023.12.004
Chong, S., Graham, T. G. W., Dugast-Darzacq, C., Dailey, G. M., Darzacq, X., & Tjian, R. (2022). Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription. Molecular Cell, 82(11), 2084–2097.e5. https://doi.org/10.1016/j.molcel.2022.04.007
Drosophila:
Versluis, P., Graham, T. G. W., Eng, V., Ebenezer, J., Darzacq, X., Zipfel, W. R., & Lis, J. T. (2024). Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation. Molecular cell, 84(15), 2856–2869.e9.
https://doi.org/10.1016/j.molcel.2024.07.009
Schlomann et al. Spatial microenvironments tune immune response dynamics in the Drosophila larval fat body. https://www.biorxiv.org/content/10.1101/2024.09.12.612587v1
In vitro single-molecule imaging:
Song, D., Rodrigues, K., Graham, T. G. W., & Loparo, J. J. (2017). A network of cis and trans interactions is required for ParB spreading. Nucleic acids research, 45(12), 7106–7117.
https://doi.org/10.1093/nar/gkx271
Song, D., Graham, T. G., & Loparo, J. J. (2016). A general approach to visualize protein binding and DNA conformation without protein labelling. Nature communications, 7, 10976.
https://doi.org/10.1038/ncomms10976. This was based on another accidental photophysical observation.
Price, A. C., Pilkiewicz, K. R., Graham, T. G. W., Song, D., Eaves, J. D., & Loparo, J. J. (2015). DNA motion capture reveals the mechanical properties of DNA at the mesoscale. Biophysical journal, 108(10), 2532–2540. https://doi.org/10.1016/j.bpj.2015.04.022
DNA replication in Xenopus egg extract (a.k.a. magic frog juice):
Vrtis, K. B., Dewar, J. M., Chistol, G., Wu, R. A., Graham, T. G. W., & Walter, J. C. (2021). Single-strand DNA breaks cause replisome disassembly. Molecular cell, 81(6), 1309–1318.e6. https://doi.org/10.1016/j.molcel.2020.12.039
Budzowska, M., Graham, T. G., Sobeck, A., Waga, S., & Walter, J. C. (2015). Regulation of the Rev1-pol ζ complex during bypass of a DNA interstrand cross-link. The EMBO journal, 34(14), 1971–1985. https://doi.org/10.15252/embj.201490878
Protein folding:
Dudko, O. K., Graham, T. G., & Best, R. B. (2011). Locating the barrier for folding of single molecules under an external force. Physical review letters, 107(20), 208301.
https://doi.org/10.1103/PhysRevLett.107.208301
COVID:
Graham, T. G. W., Dugast-Darzacq, C., Dailey, G. M., Darzacq, X., & Tjian, R. (2021). Simple, Inexpensive RNA Isolation and One-Step RT-qPCR Methods for SARS-CoV-2 Detection and General Use. Current protocols, 1(4), e130. https://doi.org/10.1002/cpz1.130. Useful DIY protocols for purifying Taq polymerase and reverse transcriptase and preparing master mixes for RT-qPCR.
Graham, T. G. W., Dugast-Darzacq, C., Dailey, G. M., Nguyenla, X. H., Van Dis, E., Esbin, M. N., Abidi, A., Stanley, S. A., Darzacq, X., & Tjian, R. (2021). Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection. PloS one, 16(2), e0246647. https://doi.org/10.1371/journal.pone.0246647
Nguyenla, X., Wehri, E., Van Dis, E., Biering, S. B., Yamashiro, L. H., Zhu, C., Stroumza, J., Dugast-Darzacq, C., Graham, T. G. W., Wang, X., Jockusch, S., Tao, C., Chien, M., Xie, W., Patel, D. J., Meyer, C., Garzia, A., Tuschl, T., Russo, J. J., Ju, J., … Schaletzky, J. (2022). Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells. Scientific reports, 12(1), 18506. https://doi.org/10.1038/s41598-022-21034-5
Biering, S. B., Van Dis, E., Wehri, E., Yamashiro, L. H., Nguyenla, X., Dugast-Darzacq, C., Graham, T. G. W., Stroumza, J. R., Golovkine, G. R., Roberts, A. W., Fines, D. M., Spradlin, J. N., Ward, C. C., Bajaj, T., Dovala, D., Schulze-Gamen, U., Bajaj, R., Fox, D. M., Ott, M., Murthy, N., … Stanley, S. A. (2021). Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2. ACS infectious diseases, 7(8), 2337–2351. https://doi.org/10.1021/acsinfecdis.1c00017
IGI Testing Consortium (2020). Blueprint for a pop-up SARS-CoV-2 testing lab. Nature biotechnology, 38(7), 791–797. https://doi.org/10.1038/s41587-020-0583-3