Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains

Abstract

The intricate organization of chromatin within the nucleus of eukaryotic cells is fundamental to regulating gene expression. Within active genetic regions, the precise spatial arrangement of DNA and associated proteins is often facilitated by dynamic interactions, particularly between promoter-promoter and enhancer-promoter elements. These crucial interaction sites are notably enriched in specific epigenetic marks, primarily histone acetylation. Histone acetylation serves as a key signal recognized by a specialized class of proteins known as bromodomains. Bromodomains function as epigenetic “readers,” acting as molecular sensors that specifically recognize and bind to acetylated lysine residues on histone tails. It is common for bromodomains to occur in tandem within a single protein or to be associated with other types of reader domains, suggesting a more complex and cooperative mode of action.

Previous research has indicated that the cellular knockdown of proteins belonging to the bromodomain and extraterminal domain (BET) family leads to significant disruptions in chromatin organization. However, the precise molecular mechanisms through which BET proteins actively preserve and maintain this vital chromatin structure have largely remained enigmatic. Our central hypothesis posits that BET proteins contribute to the overall maintenance of chromatin structure by leveraging their tandem bromodomains to multivalently scaffold acetylated nucleosomes. This scaffolding could occur in either an intranucleosomal manner, where the bromodomains bind to multiple acetylation sites on the same nucleosome, or in an internucleosomal manner, where they bridge and connect different acetylated nucleosomes.

To rigorously test this hypothesis from a biophysical perspective, we employed a suite of advanced experimental techniques. Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) were utilized in conjunction with Rosetta protein modeling. Our findings from these integrated approaches demonstrated that a flexible, disordered linker region separates the tandem bromodomain acetylation binding sites within BET proteins. This linker provides a significant range of spatial separation, from approximately 15 to 157 Ångströms (Å). Notably, a considerable portion of these modeled distances, specifically those exceeding 57 Å, are sufficient to span the typical length of a single nucleosome.

Focusing specifically on BRD4, a prominent member of the BET protein family, we utilized bioluminescence resonance energy transfer (BRET) and isothermal titration calorimetry (ITC) to investigate the nature of its interaction with histone tails. Our experiments revealed that the binding of BRD4 bromodomains to multiple acetylation sites on a single histone tail does not result in an increased affinity between BRD4 and the histone tail. This particular finding suggests that an intranucleosomal binding mechanism for BET bromodomains may not be the primary or most biologically relevant mode of action within the cellular context.

Shifting our focus to internucleosomal interactions, we employed sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays. Through these sophisticated techniques, we successfully provided the first direct biophysical evidence demonstrating that BET bromodomains possess the inherent capacity to scaffold multiple acetylated nucleosomes. This crucial observation indicates a direct physical bridging capability between separate nucleosomes.

Taken together, our comprehensive results unequivocally demonstrate that BET bromodomains are capable of engaging in multivalent internucleosome scaffolding under in vitro conditions. This new knowledge has significant implications for understanding the fundamental mechanisms by which BET bromodomain-mediated acetylated internucleosome scaffolding may contribute to the maintenance of critical cellular chromatin interactions, GNE-987 particularly within highly active genetic regions of the genome. The insights gained from this study thus advance our understanding of epigenetic regulation and chromatin dynamics.