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Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries

Abstract

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome, is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk mechanisms. We anticipate that the high throughput and sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

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Figure 1: Preparation of a DNL and its use in in vitro ChIP-seq experiments.
Figure 2: Profiling the substrate specificity of histone-mark readers.
Figure 3: Histone acetylation signaling is propagated through a positive feedback loop.
Figure 4: Systems-level synergistic modulation of histone PTMs.

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Acknowledgements

We thank the current and former members of the Muir laboratory for many valuable discussions; S.A. Blythe and S. Josefowicz for critically reading the manuscript; W. Wang and D. Storton (High Throughput Sequencing and MicroArray Facility, Princeton University) for Ion Torrent Sequencing; D.H. Perlman (Princeton Proteomics & Mass Spectrometry Core) for the mass spectrometry data; the Roeder lab (Rockefeller University) for providing the Flag-p300 plasmid; the Allis lab (Rockefeller University) for the BPTF plasmid; the Kraus lab (UT Southwestern) for the Flag-ΔBD-p300 plasmid; the Kang lab (Princeton University) for the MDA-MB and NMuMG cells; and the Cristea lab (Princeton University) for the U2OS and HFF cells. This research was supported by the US National Institutes of Health (grants R37-GM086868 and R01 GM107047). U.T.T.N. was funded by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft. Funding by the Swiss National Science foundation (postdoctoral fellowships to M.M.M. and B.F.) is gratefully acknowledged.

Author information

Authors and Affiliations

Authors

Contributions

U.T.T.N., M.M.M. and T.W.M. conceived of the research. U.T.T.N. and T.W.M. designed the research with substantial contribution by B.F. U.T.T.N. and L.B. prepared reagents and performed experiments. M.M.M., B.F., Y.D. and B.H.-L. provided reagents with the help of V.F. and G.P.D. U.T.T.N., M.M.M. and T.W.M. wrote the manuscript.

Corresponding author

Correspondence to Tom W Muir.

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Competing interests

U.T.T.N., M.M.M. and T.W.M. are co-inventors of a patent application (PCT/US2013/044537) on DNA barcoding of nucleosomes for profiling chromatin effectors.

Integrated supplementary information

Supplementary Figure 1 Characterization of semisynthetic histones by RP-HPLC/ESI-MS.

C18 analytical RP-HPLC chromatograms (insets, gradient: 30-70 % HPLC buffer B, except for mono- and diacetylated H3, which were analyzed over a 45-70 % HPLC buffer B gradient) and corresponding ESI-MS spectra of indicated modified human histone variants prepared by protein semi-synthesis (see Materials and Methods for further information). The H3 sequence was based on the human H3.1 variant with C96A and C110A mutations. The ubiquitylated H2A and H2B sequences contained a G76A mutation. All semi-synthetic H4 proteins contained an additional acetyl mark at their respective N-terminus. ub: ubiquitin; ac: acetyl; me: methyl; Da: Dalton.

Supplementary Figure 2 Modular ligation strategy for streamlined production of barcoded nucleosomal 601 DNA.

(a) DNA ligation using non-palindromic overhangs for the preparation of barcoded nucleosomal 601 DNA (‘BC-601’). Complementary single-stranded DNA spanning nucleotides (nt) 10-30 of the forward Ion Torrent Adaptor A (‘Adaptor A10-30’) and the respective 6 bp nucleosome (NUC) barcodes (‘BC-NUC’) were annealed to afford ‘BC’. Note that an AA overhang was introduced at the 3’-end of the BC bottom strand to prevent blunt end self-ligation. Additionally, a 5’ overhang of 4 bases was incorporated into the bottom strand for the subsequent ligation reaction, in which 1.1 eq. of the BC was combined with 1.0 eq. of a BsaI-DraIII-digested nucleosomal 601 sequence (‘601’). The mixture was in situ phosphorylated using T4 DNA PNK and ligated with T4 DNA ligase. The respective sequences are shown at the bottom. (b) Analysis of starting material (601 and BC, left) and reaction products (right) by native gel electrophoresis followed by ethidium bromide (EtBr) DNA staining revealed quantitative ligation. For the identity of the NUC barcodes, see Supplementary Table 1. H: A/T/C.

Supplementary Figure 3 Streamlined preparation of barcoded NUCs for DNL-1.

(a) Schematic of NUC assembly process. Step 1 - Octamer formation: Equivalent amounts of wt recombinant and/or modified semi-synthetic histones H2A, H2B, H3 and H4 (scale: 1 nmole/15 μg per histone per variant) are denatured, combined and refolded by dialysis into TE buffer (10 mM Tris, pH 7.8, 0.1 mM EDTA, 1 mM DTT) containing 2 M NaCl. Step 2 - NUC formation: Without further purification, these histone complexes are combined with a limiting amount of barcoded nucleosomal DNA (‘BC-601’, 0.6 eq. compared to histone octamer) and biotinylated buffer DNA based on the MMTV DNA1 (0.4 eq.) and dialyzed from TE containing 1.4 M KCl into TE containing 10 mM KCl. Step 3 - NUC workup: Removal of buffer DNA and any bound histones is achieved by streptavidin affinity pulldown (SAP). (b,c) The integrity and purity of NUCs was checked by native gel electrophoresis and EtBr DNA staining. In (c), an unmodified NUC is shown before SAP (lane 1) and after SAP (lane 2). A single band migrating on the native gel at the height of a 500-600 bp DNA marker was detected for most NUCs, demonstrating a homogenous NUC preparation. Note that the different migration behavior of the various NUCs was a result of pre-installed histone PTMs. Most notably, lysine ubiquitylation and H3 hyperacetylation induced a shift in retention in gel migration. A faint band associated with free DNA was detected, representing an artifact resulting from analysis by gel electrophoresis. (d) Denaturing polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Brilliant Blue (CBB) staining demonstrated that the SAP-purified NUC species contain equal amounts of each histone, exemplified by variants 1, 4, 7, 8, 12, and 14 (lanes 1-6), or the entire library (lane 7; note that the intensity of the ubiquitylated histone bands are much weaker since only a fraction of DNL-1 nucleosomes contained H2AK119ub or H2BK120ub). For NUC IDs, see Supplementary Table 1. Different migration behavior of the H4 variants were a result of the degree of acetylation.

Supplementary Figure 4 Schematic overview of the in vitro ChIP-seq experiment workflow and downstream data processing.

Step I: The DNA-barcoded NUC library is subjected to a resin-bound histone mark reader, a soluble histone mark writer or nuclear extract in the presence of the appropriate cofactors, which is followed by antibody pulldown, if needed (e.g. against the deposited mark, when a histone mark writer is used). Alternatively, for scrambling experiments illustrated in this cartoon (Supplementary Fig. 5b-e), the library is directly incubated with the histone PTM-targeting antibody. After thorough washing steps, the DNA is eluted from the histone-DNA complex by proteinase K treatment and purified using the Qiagen DNA purification kit. The concentration of the DNA is determined using Invitrogen’s Qubit DNA quantification kit. Shown are the barcoded DNA sequence mixtures (‘BC-601’) corresponding to the isolated substrates of two distinct experiments (exp1 and exp2) as well as the library input (IN; for normalization during subsequent data analysis). For illustration purposes, only four NUC library members (NUC 1-4) are shown. Step II: DNA experiment multiplexing. The experimental origin of the DNA mixtures is encoded by individual PCR reactions with uniquely barcoded reverse primers. The pulldown DNA mixtures from the experiments are diluted to a concentration of 2 pg/μL, and the dilution factor is employed later for data analysis (see step VI). 9 pg of diluted DNA pulldown mixture and 1 pg of DNA standard (see Materials and Methods) are subjected to 15 cycles of PCR using the common forward Ion Torrent Adaptor A primer (FW Adaptor A, black) and an appropriate reverse primer (‘RV-MP’; containing a common 601 annealing site, ‘60133-62’, a unique multiplexing experiment barcode, ‘BC-EXP’, as well as Adaptor P1 for Ion Torrent sequencing). Step III: The multiplexed DNA molecules from all experiments are pooled and subjected to emulsion PCR and Ion Torrent DNA sequencing; the Ion Torrent Suite software removes the adaptors subsequently. Step IV: Sorting of the DNA sequencing reads according to the list of experiment (multiplex) barcodes located at the 3’-end of the sequences (see Supplementary Table 2). Step V: Sorting of these experiment-specific DNA sequencing reads according to the list of NUC barcodes located at the 5’ end of the sequences (see Supplementary Table 1 and 3 for DNL-1 and DNL-2, respectively). Step VI: Normalization to IN. The sorted raw DNA reads for each variant and each experiment are first multiplied by the dilution factor and subsequently normalized against the sequenced IN to correct for (potential) differences in the initial amounts of library members in the library mixture. If no further normalization is performed, the final input-normalized data is averaged and displayed as ‘(mean ± SD) PD (% IN)’, resulting from 3 independent experiments (Supplementary Figs. 6c, 7c) or, in case of n=2, the data is plotted separately without averaging (Supplementary Figs. 9c, 13d, Supplementary Data 2a). Step VII: Internal normalization. In most cases, input-normalized values for each experiment are internally normalized to one specific variant within that experiment (the identity of the normalization variant is indicated in the respective figure legend with an asterisk; this variant was set as 1, see also Supplementary Data 1). The internally normalized values are then averaged from 3 independent experiments and displayed as ‘(mean ± SD) Normalized reads (a.u.)’. In case of n=2, the data is plotted separately without averaging.

Supplementary Figure 5 In vitro ChIP-seq scrambling experiments with site-specific histone PTM antibodies confirm the integrity of the library.

(a) The linear range of the downstream DNA processing was confirmed by subjecting 10 pg of the standard DNA mixture containing 4 BC-601 sequences equipped with a unique barcode each (combined at a ratio of 1:10:100:1000 eq for standards A, B, C, D; see Materials and Methods) to the identical multiplex PCR and Ion Torrent next generation sequencing conditions as for the library in vitro ChIP-Seq exeriments described later. Plotting of the obtained raw DNA reads (log-log display), averaged from 3 independent PCR/sequencing experiments, showed both an excellent reproducibility of the data (power law curve fit, R2 value = 0.9992) and the preservation of the DNA ratios in the sequencing range typically employed in the current study. (b-d) Analysis of DNL-1 stability towards histone exchange. 12 fmoles of total NUCs were combined with an α-H3K9ac (b), α-H4K8ac (c) or α-H3K4me3 (d) antibody. (e) The experiment was identical as in (d), except this NUC library had been stored for > 6 months at 4 °C. All downstream processing was performed as described for Supplementary Fig. 4. Internal normalization was performed to the following variants (set as 1, red asterisks): H3Kac5 (b), H4Kac5 (c), or H3K4me3 (d,e). Values are shown as (mean ± SD, n=3) for (c,d) or plotted separately for (n=2, b,e) employing the same grid as in Fig. 1b.

Supplementary Figure 6 Chromatin recruitment experiments employing recombinant BPTF.

(a) Schematic representation of Glutathione-S-transferase (GST)-tagged BPTF constructs employed in this study. (b) N-terminally GST-tagged human BPTF constructs were expressed recombinantly in E.coli and purified by glutathione affinity, ion exchange and size exclusion chromatography. Denaturing PAGE followed by CBB staining demonstrated > 95 % purity of the protein constructs. (c) Affinity pulldown of DNL-1 using resin-bound GST-BPTF-BD. Binding conditions and DNA processing were as shown in Fig. 2a, except that no internal normalization was performed. The input (IN)-normalized reads were averaged from 3 independent pulldown experiments and are expressed as (mean ± SD) employing the same grid as in Fig. 1b. (d) The degree of binding of the individual NUCs (unmodified; H3K4me3-modified; H3K4me3-H4K16ac modified; H3K4me3-H4Kac5 modified) to resin-bound GST-BPTF-PHD-BD was detected by native gel electrophoresis and DNA EtBr staining. Inset: Quantification of band intensities normalized to the H3K4me3-containing NUC (black asterisk, set as 1). Errors = SD (n=3).

Supplementary Figure 7 Chromatin recruitment experiments employing recombinant p300.

(a) Schematic representation of recombinant GST-tagged p300 constructs employed in this study. (b) N-terminally GST-tagged human p300 constructs were expressed recombinantly in E.coli and purified by glutathione affinity, ion exchange and size exclusion chromatography. Denaturing PAGE followed by CBB staining demonstrated > 95 % purity of the protein constructs. (c) Affinity pulldown of DNL-1 using resin-bound GST-p300-PHD. Experimental binding conditions and data processing were identical as in Fig. 2b. The input (IN)-normalized reads were averaged from 3 independent pulldown (PD) experiments and are expressed as (mean ± SD) employing the same grid as in Fig. 1b.

Supplementary Figure 8 Chromatin recruitment experiments employing recombinant histone mark readers.

(a) Top: Schematic representation of the recombinant Flag-tagged Brd4 construct, spanning BD1 and BD2. Bottom: Flag-Brd4-BD1-BD2 was expressed in Sf9 cells and purified by anti-Flag immunoprecipitation. The purity of the protein was assessed by denaturing PAGE followed by CBB staining. (b) DNL-1 incubation of resin-bound Flag-Brd4-BD1-BD2 identical to Fig. 2c (replicate 2). (c) Combinations of histone modifications (‘mod’) selected for the second version of the library (‘DNL-2’). Unmodified (‘–mod’) or mono-/di-/hyperacetylated H3 proteins (horizontal axis) were combined with otherwise unmodified histones (‘–mod’) or mono-/hyperacetylated H4 (vertical axis). (d) Analysis of the combined DNL-2 by native gel electrophoresis and ethidium bromide (EtBr) DNA staining. (e) Incubation of resin-bound GST-tagged p300-BD-PHD with DNL-2. All subsequent steps were performed as in Fig. 2b. Internal normalization: H4Kac5 variant, set as 1 (red asterisk). Experiment was performed in duplicate.

Supplementary Figure 9 Chromatin recruitment experiments employing recombinant Brd4 constructs and DNL-2.

(a) Resin-bound Brd4-BD1-BD2 was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c. Internal normalization: H4Kac5 variant, set as 1 (red asterisks). The experiment was performed in duplicate, and the resulting data is plotted employing the same grid as in Supplementary Fig. 8c. (b) Left: Schematic representation of His6-Brd4-BD1-Flag construct and PAGE analysis of the protein, which was expressed recombinantly in E.coli and purified via Nickel-NTA and size exclusion chromatography. Right: Resin-bound Brd4-BD1 was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c. Internal normalization: H4Kac5 variant, set as 1 (red asterisks). The experiment was performed in duplicate. (c) Left: Schematic representation of His6-Brd4-BD2-Flag, expressed and purified identically as described in (b). Right: His6-Brd4-BD2-Flag was incubated with DNL-2. All subsequent steps were performed as in Fig. 2c, except that no internal normalization was performed. The input (IN)-normalized reads are shown from 2 independent pulldown (PD) experiments. (d) Analysis of recombinant full-length human p300 constructs. N-terminally Flag-tagged full-length human p300 constructs were expressed recombinantly in Sf9 cells, purified on M2 anti-Flag affinity matrix and analyzed by denaturing PAGE followed by CBB staining (lane 1: Y1089A/F1090S; lane 2: ΔBD; lane 3: wt).

Supplementary Figure 10 Confirmation of p300 positive feedback loop using antibody-independent experiments.

(a) 4.8 pmoles of the individual NUCs (unmodified; H4K12ac-modified; H4Kac5-modified; or H3Kac5-modified) were incubated with 120 fmoles of wt p300 in the presence of excess 3H-acetyl-CoA at 30 °C. The reactions were quenched after 0, 5, 15 and 45 min with 4x SDS sample buffer and analyzed by denaturing PAGE followed by CBB staining (top) or fluorography (bottom). Note that hyperacetylated H3 and H4 migrate faster than the respective unmodified versions, with hyperacetylated H3 comigrating with unmodified H2B. For analysis of the reaction products by native gel electrophoresis, see Fig. 3c. (b) The acetyltransferase assay was performed essentially the same as described for (a), using cold acetyl-CoA. The peptides were trypsinized, propionylated and subjected to ultra-high performance liquid chromatography-coupled high-resolution mass spectrometry. The resulting LC-MS/MS data were searched against a database consisting of human histones H2A, H2B, H3, and H4, and filtered to the 90 % peptide and 95 % protein probability levels. Ascore derived PTM site localization probabilities were calculated for each PTM, and fragmentation spectral assignments were subject to manual inspection and validation using the original tandem mass spectra acquired in profile mode. (c) Chromatin acetylation experiment employing ΔBD p300 mutant as shown in Fig. 3b (replicate 2).

Supplementary Figure 11 p300 positive feedback loop results from tighter nucleosome binding and is mediated by the BD.

(a,b) The HAT assay and downstream DNA and data processing was performed using DNL-1 identically as described in Fig. 3b with an α-H3K18ac specific antibody in the absence (blue) or presence (gray) of 20 mM Nω-Acetylhistamine (NAH, a) or in the absence (blue, b) or presence of 20 μM Bio-H4Kac4 (green, b) or 100 µM Bio-H4Kac4 (dark gray, b). For (b), the biotinylated H4 peptide was removed prior to α-H3K18ac affinity pulldown using streptavidin-coated magnetic beads. Internal normalization: H3Kac5 variant, set as 1 (red asterisks). Values are shown as (mean ± SD, n=3) or plotted independently (n=2) employing the same grid as in Fig. 1b.

Supplementary Figure 12 Nuclear extract library acetylation experiments.

(a-e) Library acetylation experiments, all further downstream processing and data visualization were performed identically as in Fig. 4b employing nuclear extract derived from U2OS (replicate 2, a), 293T (b), HFF (c) MDA-MB (d), and NMuMG (e) cells. Internal normalization: H3Kac5 variant, set as 1 (red asterisks). Different shades of blue were used only for illustration purposes.

Supplementary Figure 13 Nuclear extract acetylation and methylation experiments.

(a) Comparison of de novo library acetylation results using recombinant wt p300 (black) or U2OS nuclear extract (red). Shown are only nucleosomes containing unmodified H3. The normalized reads were extracted from the sequencing data shown in Fig. 3b and 4a (replicate 2 of each experiment), respectively. For illustration purposes, the reads of the unmodified variant were set as 1. (b,c) HAT validation assays were performed with individual NUCs using acetyl-CoA (b) or 3H-acetyl-CoA (c) as a substrate in the absence of SAM and ATP. The extent of site-specific H3 acetylation was investigated by Western blot using a H3K14ac-specific antibody (b, top). For loading control, the blot was stripped and reblotted with a H3-specific antibody (b, bottom). Alternatively, 3H-acetyl-CoA was used in the reaction to monitor bulk histone acetylation by denaturing PAGE followed by CBB staining (c, top) and fluorography (c, bottom). (d) Anti-H3K4me1 specificity assessed using DNL-1. The experiment was performed as in Fig. 4c, except no nuclear extract and cofactors were added in the reaction and no further internal normalization was performed (n=2). (e) Histone methyltransferase assays were performed using SAM as a substrate in the absence acetyl-CoA and ATP. The extent of site-specific H3 methylation was investigated by Western blot using a H3K4me1-specific antibody (e, top). For loading control, the blot was stripped and reblotted with a H3-specific antibody (e, bottom).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Tables 1–5 (PDF 4021 kb)

Supplementary Data 1

Collated input-normalized DNA reads for all library ChIP-seq experiments (XLSX 373 kb)

Supplementary Data 2

Library NUC acetylation profiles of recombinant full-length p300 detected by in vitro ChIP-seq experiments with site-specific Kac antibodies. (PDF 2188 kb)

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Nguyen, U., Bittova, L., Müller, M. et al. Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nat Methods 11, 834–840 (2014). https://doi.org/10.1038/nmeth.3022

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