Our manuscript is available as a preprint here: https://www.biorxiv.org/content/10.1101/2024.03.12.584681
Lambourne L, Mattioli K, Santoso C, Sheynkman G, Inukai S, Kaundal B, Berenson A, Spirohn-Fitzgerald K, Bhattacharjee A, Rothman E, Shrestha S, Laval F, Yang Z, Bisht D, Sewell JA, Li G, Prasad A, Phanor S, Lane R, Campbell DM, Hunt T, Balcha D, Gebbia M, Twizere JC, Hao T, Frankish A, Riback J, Salomonis N, Calderwood MA, Hill DE, Sahni N, Vidal M, Bulyk ML, Fuxman Bass JI. Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms. bioRxiv. 2024 p. 2024.03.12.584681
There are ~1,600 human transcription factor (TFs) genes which play crucial roles in development and disease. However, through alternative splicing, alternative transcription start sites, and alternative polyadenylation, most TF genes encode at least one alternative protein isoform, resulting in a total of at least 4,000 different TF isoforms. Outside of a few well-studied examples, very little is known about the molecular functions of the vast majority of these alternative TF isoforms. To address this, we systematically cloned and tested the molecular properties of hundreds of TF isoforms. Our overall findings are presented in our manuscript and we have built this web portal to allow anyone interested to browse our data for every individual TF.
We used a PCR-based cloning strategy on human fetal and adult heart, brain, and liver tissues. After extensive quality filtering, the resulting TFiso1.0 clone collection contains 693 isoforms of 246 TF genes.
PDIs were assessed with the enhanced yeast one-hybrid (eY1H) assay. Data quality was confirmed by testing a large random sample in a luciferase PDI assay. We also (see the preprint for more details).
PPIs were assessed with the yeast two-hybrid (Y2H) assay. Our PPI data was confirmed to be high-quality through testing of a large random sample in the mamalian nanoluc two-hybrid (mN2H) PPI assay, and comparing to positive and negative controls.
Was assessed in the M1H assay where full-length TF isoforms are tethered to a Gal4 DBD and transcriptional activity is then measured by co-transfecting with a plasmid containing four arrayed Gal4 upstream activation sequences (UAS) upstream of the firefly luciferase gene.
Were generated with AlphaFold 2.3.
We expressed monomeric, enhanced green fluorescent protein (mEGFP)-tagged isoforms in HEK293T and U2OS cells and evaluated both their subcellular localization and ability to form condensates using high-throughput confocal fluorescence microscopy.
Is short-read data from GTEx data re-mapped to include the novel isoforms we cloned discovered.
Is short-read data from Cardoso-Moreira et al. Nature 2019 re-mapped to include the novel isoforms we cloned discovered.
The data generated in TFisoDB was primarily supported by NCI U01CA232161 and NHGRI U24HG011451
We used of PPIs from the Human Reference Interactome (HuRI) and effector domains from TFRegDB.