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Genetic code expansion in stable cell lines enables encoded chromatin modification

Abstract

Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However, this approach has not been coupled to genome-wide measurements, because efficient incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-ɛ-acetyl-lysine in place of six lysine residues in histone H3 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.

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Figure 1: Stable PiggyBac integration carrying the unnatural amino acid incorporation machinery.
Figure 2: PiggyBac-mediated generation and differentiation of mouse ESC lines for unnatural amino acid mutagenesis.
Figure 3: RNA-seq analysis of E14 ESC lines incorporating unnatural amino acids (CpK or AcK).
Figure 4: Site-specific incorporation of acetyl-lysine into histone H3 in ESCs.
Figure 5: Genetically encoded, site-specific histone acetylation in chromatin regulates gene expression.

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Acknowledgements

This work was supported by the UK Medical Research Council (U105181009 and UPA0241008 to J.W.C.). S.J.E. was supported by a European Molecular Biology Organization Long-Term Fellowship (ALTF 1232-2011) and the Herchel Smith Fund. O.S.W. was funded through the PhD program in Chemical Biology and Molecular Medicine at the University of Cambridge. We are grateful to T. Elliott (Medical Research Council Laboratory of Molecular Biology (MRC-LMB), Cambridge, UK) for providing CpK, to R. Schneider (Institut Génétique Biologie Moléculaire Cellulaire, Illkirch, France) for providing anti-H3K64ac (ActiveMotif 39545), to the MRC-LMB FACS facility (M. Daly, F. Zhang and V. Romashova) for support, and to J. Sale for helpful comments on the manuscript.

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Authors and Affiliations

Authors

Contributions

S.J.E. and J.W.C. conceived the experimental strategy, analyzed the data and wrote the paper. S.J.E. performed most experiments and analyzed and interpreted the RNA-seq data. O.S.W. and R.J.E. performed qPCR experiments and analysis.

Corresponding author

Correspondence to Jason W Chin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Transient transfection leads to heterogeneous levels of an unnatural amino acid–containing protein, with many cells untransfected; this problem is solved by PiggyBac integration of the unnatural amino acid incorporation machinery.

(a) Western blot of transiently transfected HEK293 and mESC, with 4xPylT/PylS and 4xPylT/mCherry-TAG-EGFP. (b) Representative images of transient transfection experiments in HEK293 and mESC, 48 h after transient transfection with 4xPylT/PylS and 4xPylT/mCherry-TAG-EGFP, and addition of amino acid (0.5 mM CpK). Scale bars, 100 µm.

Supplementary Figure 2 PiggyBac-mediated generation of HEK293 cell lines for unnatural amino acid mutagenesis.

(a) Scheme for PiggyBac-mediated generation of stable cell lines bearing pyrrolysyl-tRNA synthetase/tRNA (PylS/PylT) pair for unnatural amino acid (UAA) incorporation. (b) Western blot of two HEK293 cell line stably expressing 4xPylT/PylS and 4xPylT /mCherry-TAG-EGFP cassettes, cultured 48 hours with or without addition of 0.5 mM CpK. α-FLAG detects N-terminally tagged PylRS. (c) Representative fluorescent microscopy images of a HEK293 cell line, cultured 48 hours with or without addition of 0.5 mM CpK.

Supplementary Figure 3 PiggyBac-mediated generation of HCT-116 human colorectal cancer and 3T3 mouse embryonic fibroblast cell lines for unnatural amino acid mutagenesis.

(a) FACS analysis of HCT-116 transient (top) and stable (bottom) amber suppression using the 4xPylT/PylS and 4xPylT /mCherry-TAG-EGFP cassettes targeting vectors shown in Figure 1a. A drug selected bulk population and a derived single clone is shown. Cells were grown for 48 hours with or without 0.5 mM CpK. (b) Representative image of experiment in a. (c) FACS analysis of MEF 3T3 transient (top) and stable (bottom) amber suppression using the targeting vectors shown in Figure 1a. A drug selected bulk population and a derived single clone is shown. Cells were grown for 48 hours with or without 0.5 mM CpK. (d) Representative image of experiment in c.

Supplementary Figure 4 PiggyBac-mediated generation and differentiation of mouse embryonic stem cell lines for unnatural amino acid mutagenesis.

FACS analysis of bulk drug selected ESC population bearing 4xPylT/PylS and 4xPylT/sfGFP150TAG and four clones derived from the bulk selection, grown in the absence (-UAA) or presence of 0.2 mM CpK or 2 mM BocK for 48 hours.

Supplementary Figure 5 PiggyBac-mediated generation of mouse embryonic stem cell lines for unnatural amino acid mutagenesis.

(a) FACS analysis of E14 mouse ESC generated with 4xPylT/PylS and 4xPylT/mCherry-TAG-EGFP targeting vectors shown in Figure 1a. A drug selected bulk population (top) and a derived single clone (bottom) is shown. Cells were grown for 48 hours without unnatural amino acid (UAA), with 2 mM BocK or 0.5 mM CpK. (b) Representative image of the bulk selected population from a. (c) Western blot of the bulk selected population, showing PylRS expression with α-FLAG and high read-through of the amber codon as judged by α-mCherry western blot. Actin is shown as a loading control. (d) Dose-dependent amber suppression (EGFP production) as measured by FACS in the clone shown in a. Cells were grown in the presence of indicated concentrations of CpK for 48 hours.

Supplementary Figure 6 RNA-seq analysis of E14 ESC and MEF cell lines incorporating CpK or AcK unnatural amino acids.

(a) Multidimensional scaling (MDS) plot summarizing all RNA-Seq datasets used in this study. The first two principle components are roughly corresponding to cell type specific differences (M1) and an effect of amber suppression in ESC (M2). Wild type E14 cell lines in the presence or absence of unnatural amino acid cluster closely with amber suppression cell lines in the absence of unnatural amino acid. (b) RNA-seq analysis of a second clone bearing 4xPylT/PylS and 4xPylT/sfGFP150TAG, a biological replicate of Figure 3a. Whole transcriptome FPKM values in the presence versus absence of 0.2 mM CpK for 48h are plotted. Common significantly (P<0.005) up- and downregulated genes are colored in red and blue, respectively. (c) Venn diagram showing the overlap of significantly (P<0.005) dysregulated genes among two independent clonal cell lines shown in Figure 3a and Supplementary Figure 6b. (d) Venn diagram showing the overlap of significantly (P<0.005) dysregulated genes among two independent MEF cell lines. (e) Venn diagram of significantly deregulated genes in ESC and MEF, further divided into up- and downregulated gene sets in ESC.

Supplementary Figure 7 Stem cell–specific gene expression changes in response to amber suppression.

(a) RNA-Seq analysis of a clone bearing 4xPylT/PylS and 4xPylT/sfGFP150TAG as shown in Figure 3. Whole transcriptome FPKM values in the presence versus absence of 0.2 mM CpK for 48h are plotted. All genes with ESC-specific expression are colored in green. ESC-specific genes were defined by an at least 10-fold higher expression in ESC over MEF cells. (b) Expression changes amongst a set of well-known core ESC genes, including master regulators Oct-4/Pou5f1, Sox2 and Nanog, upon addition of 0.2 mM CpK for 48 hours. (c) RNA-Seq comparison of E14 ESC and 3T3 MEF clones bearing 4xPylT/PylS and 4xPylT/sfGFP150TAG. Significantly up- and downregulated genes in ESC as defined in Figure 3a are colored in red and blue, respectively. Most of the genes in these gene sets are expressed highly in both ESC and MEF cells. (d) Gene ontology (GO) analysis of significantly up- or downregulated gene sets in ESC. Upregulated genes fall in a variety of ontologies, whereas downregulated genes are strongly enriched for proteins involved in metabolic processes.

Supplementary Figure 8 Scheme for creating cell lines to express acetylated histones.

(a) Illustration of two-step targeting strategy to derive closely matched polyclonal pools of cells expressing the panel of acetylated histones. (b) Panel of acetylated histones used in the study. Location of TAG amber codons with respect to tail (light orange) and folded core (dark orange) of histone protein are indicated. (c) Transient expression tagged full-length histone and N-terminal histone fragments in HEK293T cells followed by western blotting corroborates the notion that truncated histones are unstable and rapidly degraded.

Supplementary Figure 9 Dose-dependent amber suppression in response to AcK.

E14 ESC bearing 4xPylT/AcKS-TAGDendra2 were incubated for 24 hours with indicated AcK concentrations.

Supplementary Figure 10 Flow cytometry analysis using immunostaining to assess expression of HA-tagged histone transgenes shown in Figure 3b on a single-cell level.

Pairs of H3.2 and H3.3 cell lines are shown which were derived independently from the same parental E14 ESC 4xPylT/AcKS-TAGDendra2 clone. Expression of H3.2, H3.2(XX)TAG, H3.3, H3.3(XX)TAG bearing a C-terminal triple HA tag, as measured by fluorescent staining with anti-HA Alexa647 antibody and FACS analysis. All cells were grown for 24 hours in the presence of 10 mM AcK. The parental cell lines, which do not express any HA-tagged protein is used as a negative control for the staining procedure (gray).

Supplementary Figure 11 Genetically encoded, site-specific histone acetylation is detected on chromatin and soluble histones.

(a) Western blot analysis of chromatin extracts detecting genetically directed, site-specific acetylation, in comparison to endogenous levels of acetylation. Endogenous levels of H3K37ac and H3K56ac were detected on the same western blot. The dotted red line indicates where western blot was cut for imaging to spatially separate the upper and lower half during imaging, to reduce interference by the stronger endogenous signal. Loading was controlled using H3.3-specific antibody, and a membrane stain. (b) Western blot of histone H3.3(XX)TAG-HA, immunoprecipitated from an MNase-treated chromatin fraction or from a soluble extract. Histones were probed for H3K37 and H3K56 acetylation by western blot. HA tag was blotted as a loading control. H3K37 and H3K56 are only detected when templated by genetically encoded, site-specific incorporation of acetyllysine. (c) Additional western blot of histone H3.3(XX)TAG-HA, immunoprecipitated from an MNase treated chromatin fraction. Histones were probed for HA tag, H3K27 and H3K64 acetylation by western blot. (d) Histone H3.3(XX)TAG-HA mutant overexpression in HEK293T cells for detection of templated histone acetylation in direct comparison to endogenous histones. H3K9 H3K27 and H3K56 acetlation was probed by western blot of acid-extracted histones. HA-tag blot shows overexpressed histone mutant. H3 antibody (ab1791) recognizes epitope at C-terminus of H3 and thus does is not able to detect HA-tagged histone species.

Supplementary Figure 12 Expression analysis of E14 ESC lines bearing AcKS-Dendra2/4×PylT and H3.3K(XX)TAG/4×PylT.

(ac) Absolute RNA-Seq expression (FPKM) values of representative genes from Figure 5a are plotted. Controls are included as follows: Wild type E14 cells with and without AcK for 24 hours (E14); parental E14 cell line bearing AcKS-Dendra2/4xPylT (E14 AcKS); E14 cell line bearing 4xPylT/AcKS-TAGDendra2 and 4xPylT/H3.3 with and without AcK for 24 hours (H3.3). (d) RT-qPCR validation of XIST gene expression in three biological replicates. Compare to RNA-Seq in panel a. Error bars indicate standard deviation of three biological replicates. Significance is given as p value based on Student’s T test. (e,f) Amber suppression efficiency and XIST gene activation as a function of AcK addition. E14 ESC bearing 4xPylT/AcKS-TAGDendra2 and 4xPylT/H3.3K56TAG were incubated for 24 hours with indicated AcK concentrations. (e) Amber suppression as measured by the AcKS-TAG-Dendra2 reporter. (f) Expression of XIST gene as measured by RT-qPCR relative to the Parent cell line, as a function of AcK concentration. Error bars indicate standard deviation of three biological replicates. Significance is given as p value based on Student’s T test (* p<0.05).

Supplementary Figure 13 Site-specific acetylated histone H3.3 is incorporated similarly to wild-type histone H3.3.

(a) ChIP assay validating variant-specific incorporation of H3.3-HA3 and H3.3K56TAG-HA3, in the presence of 10 mM AcK for 24 h, at the XIST locus at primer sites tiling the XIST locus as indicated in Figure 5c. Error bars indicate 95% confidence interval of three technical replicates. (b) ChIP assay validating variant-specific incorporation of H3.3K37TAG-HA3, in the presence of 10 mM AcK for 24 h, at the XIST locus at primer sites tiling the XIST locus as indicated in Figure 5c. Error bars indicate 95% confidence interval of three technical replicates.

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Supplementary Figures 1–13 and Supplementary Table 1 (PDF 2083 kb)

Beating cardiomyocyte aggregates form in the presence of CpK

Beating cardiomyocyte aggregates differentiated from mouse ESC via embryoid body formation in the presence of 0.2 mM CpK. (MOV 2798 kb)

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Elsässer, S., Ernst, R., Walker, O. et al. Genetic code expansion in stable cell lines enables encoded chromatin modification. Nat Methods 13, 158–164 (2016). https://doi.org/10.1038/nmeth.3701

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