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Abstract

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape to regulate transcription is unclear. TFs also differ in their ability to associate with mitotic chromosomes, which has been shown to be mediated largely by non-specific binding. The TFs identified so far for their ability to bind mitotic chromatin are known cell fate regulators and often have been shown to have pioneering activity (i.e. they are able to bind and open closed chromatin regions). However, the number of TFs tested for mitotic chromosome binding (MCB) and the knowledge about properties allowing TFs to bind to mitotic chromatin are limited. Thus, we performed a large-scale quantification MCB by TFs. Out of 502 quantified TFs, we observed that 22% of TFs were enriched on mitotic chromosomes, 24% were depleted and 54% were neither enriched nor depleted, and these behaviors were largely cell-type non-specific. As TF-DNA interactions are mediated by their DNA-binding domains, we investigated their influence on MCB. However, despite the fact that TFs bearing certain types of DNA-binding domains were more likely to be enriched or depleted from mitotic chromosomes, it was not sufficient to explain differential MCB. Amino acid sequence-based analysis using machine learning showed that electrostatic properties impact the propensity of TFs to bind mitotic chromosomes. This confirms that co-localization of TFs with mitotic chromosome is affected by non-specific electrostatic protein-DNA interactions. Therefore, we next investigated whether the MCB reflected properties of TFs in interphase. We observed a clear correlation of MCB with DNA co-localization in interphase fibroblasts and with mobility in both interphase and mitosis. As mobility is influenced by both TF diffusion and binding to DNA, we excluded that differences in diffusion, caused by TF sizes, could explain the variability in mobility rates, and tested whether TFs differed in their association with specific DNA sites. Single molecule imaging of DNA binding in live cells showed that differences in MCB were correlated with differences in relative TF on-rates, but not with TFs residence times. To test whether this affected target site occupancy, genome-wide mapping of TF binding was performed, showing that TFs associating to mitotic chromosomes also have a higher interphase genome occupancy and greater ability to find their specific sites in the genome. Lastly, we investigated the correlation between the abilities to bind mitotic chromatin and to modify chromatin accessibility and observed that the broader impact of TFs with a high MCB on chromatin accessibility can be explained by their high number of bound target sites, and not by a higher potency to alter chromatin accessibility. Altogether, our data suggests that TFs differ broadly in their non-specific DNA binding properties, which regulate their search efficiency and thereby their occupancy of specific sites in the genome. We suggest that measuring mitotic chromosome association of TFs by fluorescence microscopy can be used as a metric of non-specific DNA binding.

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