bioRxiv preprint doi: https://doi.org/10.1101/699967; this version posted July 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Nucleosome scaffolding by Brd4 tandem bromodomains in acetylation-dependent chromatin compartmentalization Michael D. Olpa, Vaughn Jacksona, and Brian C. Smitha aDepartment of Biochemistry, Medical College of Wisconsin, 8701 W Watertown Plank Rd., Milwaukee, WI 53226 1 Bromodomain binding of acetyl-lysine residues is a crucial step in chromatin organization that allows enhancers and promoters 20 2 many epigenetic mechanisms governing transcription. Nearly half of to communicate within the nuclear space (7). However, the 21 3 human bromodomains exist in tandem with at least one other bro- specific protein-histone interactions that lead to formation, 22 4 modomain on a single protein. The Bromodomain and ExtraTerminal maintenance, and disassembly of these critical 3D chromatin 23 5 domain (BET) family of proteins (BrdT, Brd2, Brd3 and Brd4) each structures remains largely unknown. 24 6 encode two bromodomains at their N-termini and are important reg- Which chromatin regions interact depends on the histone 25 7 ulators of acetylation-dependent transcription in homeostasis and post-translational modifications (PTMs) at the sites of inter- 26 8 disease. Previous efforts have focused on identifying protein acety- action. The functional consequences of histone PTMs were 27 9 lation sites bound by individual bromodomains. However, the mech- first appreciated in the context of lysine acetylation, in which 28 10 anisms through which tandem bromodomains act cooperatively on histone hyperacetylation is associated with increased DNA 29 11 chromatin are largely unknown. Here, we first used small angle x- accessibility (8) and active transcription (9, 10). While this 30 12 ray scattering combined with Rosetta ab initio modeling to explore correlation was originally attributed to decreased electrostatic 31 13 conformational space available to BET tandem bromodomains. For attraction between DNA and histones upon charge neutraliza- 32 14 Brd4, the flexible tandem bromodomain linker allows for distances tion of lysine residues by acetylation (11), the functional conse- 33 15 between the two acetyl-lysine binding sites ranging from 15 to 157 quences of acetylation are now known to be more complicated. 34 16 Å. Using a bioluminescence resonance energy transfer assay, we Discovery of proteins that deposit, remove and specifically 35 17 show a clear distance dependence for Brd4 tandem bromodomain bi- bind lysine acetylation, as well as other histone PTMs, has led 36 18 valent binding of multiply acetylated histone H4 peptides. However, to the histone language hypothesis in which histone PTMs act 37 19 isothermal titration calorimetry studies revealed Brd4 binding affin- in combination to trigger downstream functions in a context- 38 20 ity toward multiply acetylated peptides does not correlate with the specific manner (12). Histone lysine acetylation is bound by 39 21 potential for bivalent binding. We used sucrose gradient assays to bromodomains, evolutionarily conserved ~110 amino-acid pro- 40 22 provide direct evidence in vitro that Brd4 tandem bromodomains can tein modules (13). Recognition of acetylated nucleosomes by 41 23 simultaneously bind and scaffold multiple acetylated nucleosomes. the 46 human bromodomain-containing proteins is involved 42 24 Intriguingly, our bioinformatic analysis of deposited chromatin im- in diverse transcriptional processes across cell-types in both 43 25 munoprecipitation sequencing data indicates that Brd4 colocalizes homeostasis and disease (14–17). 44 26 with subsets of histone acetyl-lysine sites across transcriptionally Anti-proliferative and anti-inflammatory effects associated 45 27 active chromatin compartments. These findings support our hypoth- with bromodomain inhibition and genetic knockdown have 46 28 esis that scaffolding of acetylated nucleosomes by Brd4 tandem bro- driven recent drug discovery efforts (18, 19). Selective in- 47 29 modomains contributes to higher-order chromatin architecture. hibitors of bromodomain and extraterminal domain (BET) 48 family of bromodomains (BrdT, Brd2, Brd3 and Brd4) were 49 DRAFTdiscovered in 2010 (20, 21) and since have entered clinical trials 50 as potential treatment for cancer (22–27) and inflammation- 51 1 Histone-DNA interactions act in a multiscale manner and driven disease (28, 29). However, the molecular mechanisms 52 2 form the basis for epigenetic processes that dictate transcrip- underlying the potential therapeutic effects of bromodomain 53 3 tional output over time and space without modifying the inhibitors are poorly understood. As isolated bromodomains 54 4 underlying genetic code. DNA is organized with eukaryotic harbor relatively weak affinity toward monoacetylated histone 55 5 nuclei by histone octamers that contain two copies each of tail peptides in vitro (K d = 10 µM - 1 mM) (13), multivalency 56 6 H2A, H2B, H3 and H4 (1). At the atomic scale, 145-147 is emerging as an increasingly crucial concept in bromodomain 57 7 base pairs (bp) of DNA are wrapped around histone octamers binding of modified chromatin (30, 31). One category of po- 58 8 to form nucleosomes (2) that control accessibility to DNA tential multivalent interactions is the recognition of multiple 59 9 replication, repair and transcriptional processes (3, 4). On a sites on one histone tail by an individual bromodomain. For 60 10 larger scale, the nucleosome forms the functional unit of chro- instance, the N-terminal bromodomains (BD1) of Brd4 and 61 11 matin that organizes eukaryotic DNA in three dimensions (3D) BrdT cooperatively bind two adjacent acetyl-lysine residues 62 12 within the nucleus (5). The degree of chromatin compaction (i.e. lysines 5 and 8) on the histone H4 tail (13, 32) with 3- 63 13 partitions eukaryotic genomes between transcriptionally active to 20-fold tighter affinity compared to either acetylation in 64 14 euchromatin and transcriptionally repressed heterochromatin. isolation (33, 34) suggesting that multiple neighboring acety- 65 15 These higher-order 3D chromatin structures necessarily bring lation sites are necessary to recruit these bromodomains to 66 16 distant genetic loci into physical proximity, governing com- 17 plex transcriptional networks (6). For example, enhancers 18 are often located hundreds of kilobases from the promoters 19 they control, and enhancer-promotor looping is critical for 3D bioRxiv preprint doi: https://doi.org/10.1101/699967; this version posted July 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 chromatin. Our bioinformatic analysis of deposited chromatin immuno- 128 precipitation sequencing (ChIP-seq) data indicates that Brd4 129 68 Another potential category of multivalent bromodomain- colocalizes with subsets of histone acetyl-lysine sites across 130 69 nucleosome interactions is possible through the encoding of annotated topologically associated chromatin domains. We 131 70 proteins containing multiple (tandem) bromodomains in a also provide the first direct evidence for physical scaffolding 132 71 single polypeptide, a feature observed in yeast to mammalian of multiple acetylated nucleosomes by Brd4 tandem bromod- 133 72 genomes including 11 human proteins. Early studies of tandem omains using in vitro sucrose gradient binding assays. These 134 73 bromodomain proteins in yeast demonstrated that subunit 4 observations allow us to hypothesize a role for Brd4-mediated 135 74 of the chromatin structure-remodeling complex (Rsc4) and nucleosome scaffolding in the maintenance of higher-order 136 75 bromodomain-containing factor 1 (Bdf1) each require their chromatin structure. 137 76 tandem bromodomains for full function (35, 36). The first 77 crystallographic study of a tandem bromodomain proposed 78 that the bromodomains of human transcription initiation fac- Results 138 79 tor TFIID subunit 1 (Taf1) can bind multiple acetylation sites BET bromodomains are separated by flexible linkers that af- 139 80 on the same histone H4 tail (37). Recently, studies of human ford a high degree of conformational freedom. We hypothe- 140 81 polybromo-1 (Pbrm1) that contains six bromodomains in tan- size that the range and relative occupancy of intersite dis- 141 82 dem have led to a similar hypothesized model in which subsets tances between tandem bromodomain acetyl-lysine binding 142 83 of the Pbrm1 bromodomains work in concert to bind and sites dictate the types of multivalent nucleosome interactions 143 84 position multiply acetylated nucleosomes (38–40). Similarly, accessible to tandem bromodomains. These multivalent in- 144 85 Brd4 tandem bromodomains associate with nucleosomes hy- teractions can broadly be classified into two possibilities that 145 86 peracetylated on histones H3 (lysines 9, 14, 18, 23 and 27) and require very different distances between acetyl-lysine binding 146 87 H4 (lysines 5, 8, 12, 16 and 20) with tighter affinity compared sites: 1) within one or between two histone tails in one nucle- 147 88 to nucleosomes hyperacetylated at either H3 or H4 in isolation osome (intranucleosome) or 2) between histone tails