In situ chromatin interactomics using a chemical bait and trap approach

Abstract

Elucidating the physiological binding partners of histone post-translational modifications (hPTMs) is key to understanding fundamental epigenetic regulatory pathways. Determining such interactomes will enable the study of how perturbations of these interactions affect disease. Here we use a synthetic biology approach to set a series of hPTM-controlled photo-affinity traps in native chromatin. Using quantitative proteomics, the local interactomes of these chemically customized chromatin landscapes are determined. We show that the approach captures transiently interacting factors such as methyltransferases and demethylases, as well as previously reported and novel hPTM reader proteins. We also apply this in situ proteomics approach to a recently disclosed cancer-associated histone mutation, H3K4M, revealing a number of perturbed interactions with the mutated tail. Collectively our studies demonstrate that modifying and interrogating native chromatin with chemical precision is a powerful tool for exploring epigenetic regulation and dysregulation at the molecular level.

Figure 1.. Schematic for the in-situ chromatin interactomics approach.

Protein-trans splicing between a chromatinized C-intein and an N-intein construct bearing hPTM(s) and a diazirine crosslinker introduces photoaffinity traps onto native chromatin. Upon UV-irradiation the chromatin-relevant hPTM interactome is determined.

Figure 2.. In-situ installation of hPTMs with ultra-fast split inteins.

a. Protein-trans splicing between delivered material 1 and expressed construct 2 results in the semi-synthetic histone 3 bearing H3K9me3 with an adjacent photocrosslinker (H3T11pLeu). b. Expanded view of splice product 3 installing H3K9me3 and H3T11pLeu into native chromatin (Bt = biotin). c. Incorporation of 2 into chromatin is ~15%, when compared to endogenous H3. n = 2, with representative data shown. d. Time course for the trans-splicing reaction showing build-up of splice product 3. n = 2, with representative data shown. e. LCMS/MS analysis of the splice product band 3 confirms a unique peptide sequence containing the splice junction. f. An H3K9me3-dependent crosslink to HP1α is observed on addition of recombinant HP1α to nuclei bearing H3K9me3 or the wild-type tail. Right: western blot analysis displaying an H3K9me3-dependent histone-HP1α crosslink with anti-HP1α and streptavidin-800 detection. anti-HP1α and histone H4 input controls are shown below. n = 2, with representative data shown. Uncropped western blots are provided in Supplementary Fig. 26.

Figure 3.. Determining the in-situ interactome of H3K9me3.

a. Schematic for the SILAC-based quantitative proteomics workflow to determine the interactome of H3K9me3, compared to the wild-type H3 tail. b. Installation of wild type (Me0) and H3K9me3-bearing tails (Me3) to endogenous chromatin in heavy (H) and light (L) labeled nuclei. Splice product (green) is observed by western blot, with excess delivered material (≈ 32 kDa) removed after the reaction. Histone H4 levels served as a loading control. c. Immunoblot analysis of UV-treated samples displaying crosslinked bands from H3K9me0- and H3K9me3-containing chromatin. Histone H4 levels served as a loading control. d. Volcano plot displaying proteins enriched in H3K9me3 (orange) and H3K9me0 samples (green); FDR < 0.05, >1.5-fold change, with tryptic peptides observed in both biological replicates. FDR values were calculated using the Benjamini-Hochberg procedure, as described in the Methods. For panels b-d experiments were performed as “forward” and “reversed” biological replicates (n = 2). Uncropped western blots are provided in Supplementary Fig. 27.

Figure 4.. Determining the interactome of H3K4 as a function of hPTMs.

a. hPTMs and mutations are incorporated at H3K4 and H3K27, along with photoleucine at T6. b. Enrichment of the splice product shows installed methylation and acetylation PTMs are present throughout the in-situ crosslinking workflow. Immunoblots were probed with indicated antibodies. n = 2 independent experiments, representative data shown. Uncropped western blots are provided in Supplementary Fig. 28. c. Volcano plot displaying interactors enriched by H3K4me3 (blue) and H3K4me0 (green). Cutoffs as for Fig. 2d. FDR values were calculated using the Benjamini-Hochberg procedure, as described in the Methods. Proteins with Log₁₀ FDR <25 were set to 25 for ease of visualization. d. Volcano plot displaying interactors enriched by H3K4me1 (red) and H3K4me0 (green). Cutoffs as for Fig. 2d. e. Volcano plot displaying interactors enriched by H3K4norleucine (purple) and H3K4me0 (green). Cutoffs as for Fig. 2d. For panels c-e experiments were performed as “forward” and “reversed” biological replicates (n = 2). f. Fluorescence anisotropy binding experiments for H3K4me0 (green), H3K4me3 (blue), and H3K4norleucine (purple) peptides binding to PHF14 PHD finger 1. Errors are reported as SEM (n = 3 independent experiments). g. Fluorescence anisotropy experiments for H3K4me0 (green), H3K4me3 (blue), and H3K4norleucine (purple) peptides binding to JADE1 PHD finger 1. Errors are reported as SEM (n = 3 independent experiments).