Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains. J Biol Chem 2025 Mar;301(3):108289
Date
02/13/2025Pubmed ID
39938804Pubmed Central ID
PMC11930079DOI
10.1016/j.jbc.2025.108289Scopus ID
2-s2.0-85219493438 (requires institutional sign-in at Scopus site) 2 CitationsAbstract
Promoter-promoter and enhancer-promoter interactions are enriched in histone acetylation and central to chromatin organization in active genetic regions. Bromodomains are epigenetic "readers" that recognize and bind histone acetylation. Bromodomains often exist in tandem or with other reader domains. Cellular knockdown of the bromodomain and extraterminal domain (BET) protein family disrupts chromatin organization, but the mechanisms through which BET proteins preserve chromatin structure are largely unknown. We hypothesize that BET proteins maintain overall chromatin structure by employing their tandem bromodomains to multivalently scaffold acetylated nucleosomes in an intranucleosomal or internucleosomal manner. To test this hypothesis biophysically, we used small-angle X-ray scattering, electron paramagnetic resonance, and Rosetta protein modeling to show that a disordered linker separates BET tandem bromodomain acetylation binding sites by 15 to 157 Å. Most of these modeled distances are sufficient to span the length of a nucleosome (>57 Å). Focusing on the BET family member BRD4, we employed bioluminescence resonance energy transfer and isothermal titration calorimetry to show that BRD4 bromodomain binding of multiple acetylation sites on a histone tail does not increase BRD4-histone tail affinity, suggesting that BET bromodomain intranucleosome binding is not biologically relevant. Using sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays, we provide the first direct biophysical evidence that BET bromodomains can scaffold multiple acetylated nucleosomes. Taken together, our results demonstrate that BET bromodomains are capable of multivalent internucleosome scaffolding in vitro. The knowledge gained provides implications for how BET bromodomain-mediated acetylated internucleosome scaffolding may maintain cellular chromatin interactions in active genetic regions.
Author List
Olp MD, Bursch KL, Wynia-Smith SL, Nuñez R, Goetz CJ, Jackson V, Smith BCAuthors
Brian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of WisconsinSarah L. Wynia Smith Research Scientist II in the Biochemistry department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
AcetylationCell Cycle Proteins
Histones
Humans
Nuclear Proteins
Nucleosomes
Protein Binding
Protein Domains
Transcription Factors
