Nucleosome context regulates chromatin reader preference

Abstract

Chromatin is more than a simple genome packaging system but rather locally distinguished by histone post-translational modifications (PTMs) that can directly change nucleosome structure and/or be "read" by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to explain normal function and pathogenesis and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, though these cannot represent the full regulatory potential of nucleosome context. Here we apply a range of complementary and easily adoptable biochemical and genomic approaches to interrogate fully defined peptide and nucleosome targets with a diversity of mono- or multivalent chromatin readers. In the resulting data, nucleosome context consistently refined reader binding, and multivalent engagement was more often regulatory than simply additive. This included abrogating binding of the Polycomb group malignant brain tumor (MBT) protein L3MBTL1 to lysine methylated histone tails and confirmation that the CBX7 chromodomain and AT-hook-like motif (CD-ATL) tandem act as a functional unit to confer specificity for H3K27me3. These in vitro nucleosome preferences were confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that more representative chromatin substrates provide greater insight into biological mechanism and human disease.

Graphical Abstract

Figure 1.

Lysine-acetyl readers show refined PTM specificity in nucleosome versus histone peptide assays. (A) GST-BRD4 BD1 (query, concentrations as noted) interactions in histone-peptide based assays: EpiTriton array and Captify-Alpha. “Hits” are defined by signal-to-background (S/B) > 2 for peptide array and >100 for Captify-Alpha. (B) GST-BRD4 BD1 S/B in EpiTriton (2 μM) and Captify-Alpha (1 or 30 nM) for select H3 and H4 acetyl peptide targets. Each assay identifies the bromodomain preferentially interacting with various forms of H4 tail acetyl. (C) [Inset] GST-BRD4 BD1 query titration to potential nucleosome targets; dashed line represents EC₈₀^rel (5 nM) against ([H4K5acK8acK12acK16ac]₂) (aka. [H4tetra^ac]). [Main] GST-BRD4 BD1 (5 nM) discovery screen to indicated panel reveals strong preference for [H4tetra^ac] nucleosomes. (D) [Inset] GST-BRM BD titrations to potential nucleosome targets; dashed line represents EC₂₀^rel (30 nM) against ([H3K4acK9acK14acK18ac]₂) (aka. [H3tetra^ac]). The binding at near µM concentrations to unmodified nucleosomes is likely due to interactions with nucleosomal DNA [60] (Supplementary Fig. S4C). [Main] GST-BRM BD (30 nM) discovery screen to indicated panel identifies strong preference for [H3tetra^ac] nucleosomes. See Supplementary Figs S4–S6 for Captify-Alpha two-dimensional (2D) titrations with a range of lysine-acyl readers, and Supplementary File 2 for complete datasets from peptide (n = 287) and nucleosome (n = 77) target discovery screens.

Figure 2.

Lysine-methyl readers show refined PTM specificity in nucleosome versus histone peptide assays. (A) GST-L3MBTL1 MBT query titration to H4_[11–27]K20 methyl peptide targets (maximal S/B at EC₈₀^rel = 3 nM) identifies the reported preference for Kme1 and Kme2. (B) GST-L3MBTL1 MBT (3 nM) discovery screen against 287-member peptide panel identifies a preference for Kme1 and Kme2 independent of histone residue (data subset shown). (C) [Inset] GST-L3MBTL1 MBT titration to nucleosome targets bearing distinct methyl states at H4K20 identifies no binding preference. [Main] GST-L3MBTL1 MBT (3 nM) discovery screen shows background binding to nucleosomes independent of lysine-methyl status. (D) [Inset] GST-RAG2 PHD titration to H3_[1–20]K4 methyl peptides (EC₇₀^rel = 1.8 nM). [Main] GST-RAG2 PHD (1.8 nM) peptide discovery screen shows selective binding to methylations at H3K4 (me1-2-3) over all other histone methyl states (data subset shown). (E) [Inset] GST-RAG2 PHD titration to nucleosomes with H3K4 methyl states (EC₈₀^rel = 11.9 nM). [Main] GST-RAG2 PHD (11.9 nM) nucleosome discovery screen reveals a refined preference for ([H3K4me3]₂). For (B–E), see Supplementary File 2 for complete discovery screen datasets. (F) Native top-down mass spectrometry (nTDMS) distinguishes a 1:1 mix of unmodified and [H3K4me3] nucleosomes (both 1 μM). (G) GST-RAG2 PHD (5 μM) selectively associates with ([H3K4me3]₂) over unmodified nucleosomes. Reader was incubated with a 1:1 nucleosome mix (both 1 μM) and analyzed by nTDMS. Mass-to-charge (m/z) peaks correspond to dimerized GST-RAG2 PHD bound to ([H3K4me3]₂) nucleosomes (further characterized in Supplementary Fig. S7).

Figure 3.

Nucleosome context is required for an accurate determination of multivalent reader engagement. Titration of GST-HP1β (CBX1) CD (A) and GST-ATRX ADD (B) to indicated nucleosomes identifies differential impact of the ([H3K9me3S10ph]₂) combinatorial relative to ([H3K9me3]₂). (C) salDNA optimization for 6His-GLYR1 PWWP. Nonspecific nucleosome binding is reduced by the free DNA, revealing a preference for ([H3K36me3]₂). Dashed line represents optimal salDNA concentration (120 ng/ml) used for (D and E). (D) Salt optimization for 6His-GLYR1 PWWP. Query was titrated at noted NaCl concentrations against ([H3K36me3]₂) and unmodified nucleosomes, where increasing salt reduced binding to latter (EC₈₀^rel = 170 nM). (E) GLYR1 PWWP (170 nM) binding in a nucleosome discovery screen with optimized buffer conditions (120 ng/ml salDNA, 175 mM NaCl) confirms a preference for ([H3K36me3]₂). (F) GST-CBX7 CD-ATL titration to histone peptides identifies equivalent binding to H3_[1–20]K9me3 and H3_[15–34]K27me3. (G, H) CBX7 CD-ATL titration to nucleosomes reveals a preference for ([H3K27me3]₂), but only in presence of salDNA competitor (123 ng/ml: compare G and H) (EC₅₀^rel = 7.1 nM). (I) CBX7 CD-ATL (7.1 nM) binding in a nucleosome discovery screen with optimized buffer conditions (120 ng/ml salDNA), confirms a preference for ([H3K27me3]₂). See Supplementary Figs S9 and S10 for Captify-Alpha 2D optimizations of a range of lysine-methyl readers; Supplementary Figs S12 and S13 for further dissection of the CBX7 CD-ATL tandem; and Supplementary File 2 for complete datasets from all discovery screens.

Figure 4.

Captify-Luminex enables interrogation of chromatin reader binding to multiplexed nucleosome targets. (A) Biotinylated nucleosome targets (the K-MetStat panel: me0-1-2-3 at H3K4, H3K9, H3K27, H3K36, and H4K20) are individually conjugated to distinct Luminex avidin-coated MagPlex bead regions, pooled, and probed in multiplex with GST-tagged reader domains. Interactions are detected using anti-GST and anti-IgG*PE secondary, with bead/PE signals measured on a FLEXMAP-3D system. (B) GST-HP1β CD, GST-TAF3 PHD, and GST-CBX7 CD-ATL binding across the K-MetStat panel. For each query concentration (nM; labeled columns), heatmap depicts signal as percentage of max MFI. (C) Signal range for each query in panel (B). Figure 4A created in BioRender https://biorender.com/178t3sy.

Figure 5.

Readers can deliver antibody-like PTM profiling in CUT&RUN genomic mapping. (A) DNA-barcoded nucleosome spike-in (K-MetStat panel) recovery from Reader-CUT&RUN reactions. Columns group data by antibody/GST-tagged reader; rows depict recovery of each PTM in K-MetStat panel normalized to predicted target (100%; orange in heatmap), except for anti-IgG and anti-GST (% of total spike-in reads). (B) Pearson correlation plot compares CUT&RUN read overlap from antibodies versus GST-readers (1 = perfect correlation). (C–F) Venn overlap of called peaks between PTM-specific antibodies and GST-readers (≥50% peak overlap classed as positive). Table shows average peak width and % overlapping peaks in each pairwise comparison (target 1, GST-reader; target 2, PTM-specific antibody). Representative peak comparisons: H3K4me3 and GST-TAF3 PHD (G); H3K9me3, H3K27me3, and GST-HP1β CD (H); H3K9me3, H3K27me3, and GST-CBX7 CD-ATL (I). Each IGV browser window (genomic region noted) is group-scaled to * track. IgG and αGST are background controls.