Polymerization of ZBTB transcription factors

Abstract

The regulation of gene expression at the level of transcription is essential and underlies critical processes such as maintaining cell identity and orchestrating development. Transcription factors (TFs) play critical roles in this process and therefore themselves are subject to complex regulatory processes. Many TF families form complex higher-order structures, ranging from simple dimers to large multi-protein complexes, to regulate gene expression with greater efficiency and precision. The broad-complex, tramtrack and bric-à-brac (BTB) family of TFs serve as important regulators of hematopoietic development. Their characteristic BTB domain mediates protein-protein interactions, such recruitment of co-repressors, as well as homomeric assemblies, including dimers, tetramers, pentamers, and hexamers. BCL6, a master TF of germinal center B cells, forms a homodimer through its BTB domain to mediate transcriptional repression by recruiting its co-repressor proteins. Recently, we reported that BCL6 forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6’s BTB domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, but their propensity to self-assemble into higher-order structures has not been established. In my thesis work, we survey 189 human BTB proteins with a cellular fluorescent reporter assay and uncover a novel phenomenon in which 18 zinc finger and broad-complex, tramtrack and bric-à-brac (ZBTB) TFs undergo polymerization under normal physiologic conditions. Furthermore, we present structural and biochemical data demonstrating that three ZBTB TFs – ZBTB3, ZBTB5, and ZBTB9 – polymerize into filaments in vitro. Through ChIP-Seq and RNA-Seq analyses, we identify that wild-type ZBTB polymers bind DNA better than their non-polymerizing mutant counterparts. Importantly, these polymers selectively bind homotypic clusters of repeated TF binding sites, thereby enhancing the transcriptional repressive function of these transcription factors. This systematic study of ZBTB TF polymerization provides a novel framework to better understand regulation of this important class of TFs. Currently, instances of human TFs polymerizing in the absence of DNA are scarce. Our finding of ZBTB TF polymerization implies that this phenomenon could be more widespread, highlighting a potentially common and functionally significant aspect of transcription factor behavior.