Transcription Factors as Competitors in Gene Regulation and DNA Damage Repair

Transcription factors (TFs) bind genomic DNA to regulate gene expression. In the cell, the genome is decorated with numerous proteins, including nucleosomes and proteins involved in processes such as DNA repair and replication, which could compete with TFs. While the competition with nucleosomes is well studied, TFs can also compete with other DNA-binding proteins (e.g. other TFs, DNA repair enzymes, polymerases). The rules and the impact of such competition remain largely unknown. Here, we investigate how TFs compete with each other and with repair enzymes, and we reveal the significant role TFs play as competitors in multiple pathways.To capture the binding profiles of competing TFs, we designed a quantitative cell-free assay that we applied to study Cbf1-Pho4 competition in yeast and MYC-MAD competition in human. We found that TFs greatly influence each other’s occupancy, in a way that is dictated by the proteins’ divergence in DNA-binding specificity. Analyses of ChIP-seq data confirmed that the patterns of TF-TF competition, as observed in vitro, are preserved in the nuclear environment. Furthermore, gene expression data suggests that Cbf1-Pho4 competition plays a critical role in the specific activation of target genes in the cell. In the MYC-MAD system, we found that quantitative in vitro knowledge facilitates the interpretation of in vivo ChIP-seq data and reveals subtle signals in gene regulatory networks, demonstrating the advantage of combining in vitro quantification with in vivo detection. Next, we adapted our assay to study the competition between TFs and DNA repair enzymes. Recently (Afek et al. 2020) we showed that TFs bind with high affinity to mismatches, which can result from replication errors. We thus hypothesized that TFs can compete with TDG, the glycosylase that recognize T-G mismatch and initiates base excision repair, and MutS, the mismatch-binding enzyme that initiates mismatch repair. Our high-throughput competition assay showed that, as predicted, the binding of both repair enzymes to DNA decreases significantly in the presence of TFs. In addition, the magnitude of the decreases in repair enzyme binding correlates well with the TF binding levels, indicating specific competition. This suggests that, in the cell, TFs bound to mismatches may affect repair and lead to increased mutagenesis at regulatory sites. Overall, our study proposes an approach for studying competition between DNA-binding proteins in a quantitative and high-throughput manner, and highlights the significance of this competition not only for gene regulation (where TFs are known to play an important role), but also in DNA repair.