Polymerization of BTB transcription factors regulates chromatin occupancy [RNA-Seq]

The broad-complex, tramtrack and bric-à-brac (BTB) family of transcription factors (TFs) serve as important regulators of hematopoietic development. The BTB domains of these TFs mediate co-repressor recruitment and dimerization, but their propensity to self-assemble into higher-order structures has not been established. Here, we survey 189 human BTB proteins and identify 18 BTB TFs that form punctate nuclear foci. We demonstrate that these foci-forming BTB TFs polymerize into filaments and present the cryo-EM structures of ZBTB5BTB and ZBTB9BTB filaments. Finally, we find that polymerization in BTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites. This increased chromatin occupancy in turn leads to the repression of target genes. Taken together, our results reveal a novel mechanism to regulate BTB TFs, and suggest an underappreciated role for polymerization in regulating TF function. Overall design: RNA sequencing (RNA-seq) was performed for ZBTB3 and ZBTB9 in HEK 293T cells modified to knock out endogenous ZBTB3 and ZBTB9 respectively. In each case, sequencing was performed across each of otherwise-unaltered-cells treated with doxycycline/DMSO (control) and cells with a doxycycline inducable over expression of wild-type (WT) ZBTB3/9 and non-polymerizing mutant (K166A for ZBTB3, W128A for ZBTB9). Among cells with the doxycycline system, treatment ranged across DMSO 24h/48h (control), dosage of 0.01, 0.1, and 1 ug/mL doxycycline for 24h and one treatment group of 1 ug/mL doxycycline for 48h. Each condition was prepared and sequenced in triplicate.