Utilizing intein trans-splicing for in vivo generation of site-specifically modified proteins

Abstract

Many cellular processes as well as their associated pathologies are regulated by protein post-translational modifications (PTMs). Understanding the precise roles of these adducts hinges on the development of methods to robustly and site-specifically manipulate proteins in their physiological environments. Recently, ultrafast intein protein trans-splicing (PTS) was harnessed to incorporate site-specific modifications on cellular chromatin in live cells. In this chapter, we present the protocols for the generation of synthetic modifications on native chromatin as well as highlight the capabilities of this methodology.

1. Introduction

Post-translational modifications (PTMs) of a protein can determine its activity, localization, interactions, and half-life (Mann & Jensen, 2003). The dynamic nature of PTM regulation drives maintenance of cellular homeostasis with broad impacts on cellular physiology. Among the processes regulated by PTMs, chromatin structure and function are of major interest because they facilitate all DNA-templated processes and ultimately, cell fate (Jenuwein & Allis, 2001). Understanding the fundamental mechanism of the biochemical processes dictating the function of these modifications requires their precise manipulation in vivo. Current approaches primarily rely on employing robust genetic methodologies, which provide systems-wide information. Through this, the existence of distinct chromatin states has been revealed, linking specific histone modifications and transcriptional gene states (Bonasio, Tu, & Reinberg, 2010; ENCODE Project Consortium, 2004; Soshnev, Josefowicz, & Allis, 2018). However, much of the delineated information is correlative and often lacks mechanistic information. Alternatively, reductionist methods that allow in vitro and site-specific manipulation of proteins offer the advantages of synthetic specificity and unambiguous results but may not capture key aspects of the physiological system (David & Muir, 2017; Muller & Muir, 2015). An ideal approach would therefore emulate attributes from both paradigms by utilizing synthetic chemistry to generate exquisite control of a PTM placement while simultaneously addressing the system in a native in vivo setting.

One such complementary synthetic biology approach utilizes ultrafast split inteins to incorporate site-specific modifications in cellular proteins and has the advantages of temporal control, flexibility of the PTM and the capacity to incorporate multiple marks simultaneously (David, Vila-Perello, Verma, & Muir, 2015). Inteins are a family of protein domains that are inserted in host proteins and catalyze their own excision as a post-translational modification of several essential proteins in lower organisms (Vila-Perello & Muir, 2010). Split inteins are a sub-family where the contiguous intein is split into two domains, each expressed as part of a separate polypeptide, that rapidly associate and catalyze splicing in trans (see Fig. 1A) (Shah & Muir, 2011). These split inteins can be ultrafast, completing the entire reaction in seconds to minutes, making them ideal for in vivo manipulation of proteins (Shah & Muir, 2014). In the cell, a truncated form of the native protein of interest is expressed fused to one intein partner while a probe consisting of the other intein partner and the remaining portion of the protein with the desired modification is simultaneously chemically synthesized. The synthesized portion with the probe is designed to have a disulfide linkage to a cell penetrating peptide. This apo-probe can be delivered to the cell, at which point the cell-penetrating peptide-probe disulfide is reduced by the reductive cytosolic environment. The resulting probe rapidly associates with the chromatin-bound intein and facilitates its own excision thus mediating the ligation of the modified and expressed histone fragments (see Fig. 1B).

Fig. 1.

Canonical mechanism of intein splicing and in vivo PTS. (A) Binding of Int^N and Int^C domains leads to rapid splicing via an intramolecular N-S acyl shift, followed by transthioesterification, succinimide formation and finally the release of the products by an S-N acyl shift. (B) Scheme of intein splicing showing (1) cellular uptake of the apo-probe via endocytosis, (2) release of peptide into cytoplasm by endosomal lysis, (3) disulfide bond reduction by endogenous glutathione removes CPP, (4) reaction of delivered Int^C-QSY-TMR-HA with native chromatin containing incorporated H2B-Int^N resulting in fluorophore-histone labeling and release of quencher.

Beyond their fast kinetics there are several additional advantages of using inteins: (1) Inteins are exogenous to mammalian cells and thus do not have any off-target effects or toxicity. (2) The attachment of the intein to a protein target does not affect the activity of intein splicing, making this a flexible and global manipulation. (3) The choice of cargo is very flexible with the only limiting factor on size being the efficiency of delivery. (4) The traceless nature of the intein chemistry results in a final labeled protein without a fused intein or other logistical motif (David & Muir, 2017).

It is important to note that the robustness of the reaction relies on the expression of the intein-fused construct and its incorporation into chromatin. Furthermore, truncating the expressed protein might perturb its function, or incorporation in the case of histones. Third, the probe itself is generated by solid phase peptide synthesis, which limits the size of the probe to about 50–60 amino acids in length, of which about 30 is the intein portion, thus limiting the modified peptide portion to about 30 amino acids (Behrendt, White, & Offer, 2016). However, a unique advantage inherent to the intein system stems from the convergent nature of the strategy; each module of the process may be optimized and modified separately. Several labs have been working to overcome these hurdles in order to make the system general and ubiquitous. This approach, while originally developed as an epigenetic tool, could potentially be applied to a variety of cellular proteins of interest, provided both the protein and the site are accessible. In this chapter, we discuss the design, synthesis, expression, transfection, and delivery of intein-pairs to introduce modified histones into chromatin to analyze the effects of specific marks on chromatin dynamics and epigenetics.

2. In vivo synthesis of H2B-TMR-HA using split inteins

Here, we describe a step-by-step protocol using as an example the traceless incorporation of a fluorescent probe, tetramethylrhodamine (TMR), and an HA tag onto H2B in live 293 cells. In this case, the truncated histone H2B is fused to an N-intein (Int^N) and a C-terminal Flag tag and cloned into a mammalian construct. The final H2B-Int^N-Flag construct is transfected into cells where the truncated H2B is incorporated into chromatin. In parallel, a peptide probe consisting of Int^C, the C-intein compliment to the N-intein used, as well as the TMR fluorophore, a quencher QSY to quench signal of unspliced reactant, and an HA tag to track the reaction progress, is synthesized via solid-phase peptide synthesis (see Figs. 2 and 3) (see Note 1 on split inteins). It is then conjugated to a designated HA2-TAT CPP (cell penetrating peptide) through a directed disulfide bond and the final Int^C-QSY-TMR-HA~CPP apo-probe is delivered onto the cells. Once the apo-probe enters the cytoplasm, the disulfide bond is reduced by endogenous glutathione, releasing the Int^C-QSY-TMR-HA cargo. This reduced probe reacts with its intein counterpart in the nucleus forming H2B-TMR-HA on cellular chromatin releasing both the Int^C-QSY and Int^N-Flag as a heterodimer/complex (see Fig. 1B). Upon successful splicing, the fluorophore is incorporated into chromatin while the quencher remains attached to the intein side-product. This confers the advantage of a dramatic decrease in background signal in selective imaging of semisynthetic chromatin in living cells (see Fig. 4B).

Fig. 2.

Synthetic synthesis scheme for the Int^C-QSY-TMR-HA~CPP apo-probe (see Section 2.2 for details). (a) Standard Fmoc-based SPPS protocols (b) 3% hydrazine in DCM (v/v) (c) TMR-SE, DIEA in DMF (d) 95% TFA, 2.5% TIPS, and 2.5% water; RP-HPLC purification (e) QSY9-maleimide, pH 7.0 (f) TCEP in water; RP-HPLC (g) Standard Fmoc-based SPPS protocols; RP-HPLC (h) Int^C-QSY-TMR-HA and HA2-TAT CPP.

Fig. 3.

Representative hypothetical purification chromatograms for (A) Int^C-cargo peptide, (B) HA2-TAT cell penetrating peptide (CPP), (C) Disulfide-conjugated CPP~cargo peptide.

Fig. 4.

Protein splicing analysis. (A) Western blot analysis from in nucleo experiments showing time-course of protein trans-splicing. Lanes 1–4: no product formation in non-transfected sample; lanes 5–8: rapid splicing product formation concurrent with depletion of cargo peptide; lanes 9–12: no formation of splicing product in cells transfected with catalytically inactive Int^N (C1A). (B) Live-cell imaging of live cells with delivered Int^C-QSY-TMR-HA. Top: fluorescent (left) and bright-field (right) images of non-transfected cells after cargo delivery. Bottom: representative fluorescent images of transfected cells after cargo delivery.

2.1. Engineering the H2B-Int^N-flag construct

A few key considerations must be made when designing the splicing site for PTS. First, residues that can be replaced with the N-terminal cysteine residue from the delivered cargo should be identified. Second, the splicing reaction may be affected by neighboring N- or C-extein residues. To attenuate this issue, a site that reflects the optimal extein sequence should be selected. If no such site exists, an extra linker should be added to increase the reaction efficiency (David et al., 2015).

2.1.1. Equipment

1. 200 μL thin-walled PCR tubes

2. 1.7mL Eppendorf tubes

3. 15mL conical tubes

4. Thermal cycler

2.1.2. Buffers and reagents

1. 100mg/mL ampicillin solution

2. Bacterial strains for DNA preparation: E. coli DH5α (New England Biolabs)

3. Gibson Assembly Cloning Kit (New England Biolabs)

4. Luria Broth-agar

5. Luria Broth (LB) powder

6. Phusion High-Fidelity DNA Polymerase (Thermo Fisher)

7. Plasmids: pCMV-H2B, pET3a-Int^N (see Table 1)

8. QIAprep Spin Miniprep Kit (Qiagen)

9. QIAquick PCR Purification Kit (Qiagen)

10. Synthetic DNA oligo primers (see Table 2)

Table 1.

Parent vectors for Gibson Assembly.

Gibson cloning component	Vector backbone	MCS insert
Vector, H2B truncation	pCMV	H2B
Insert, IntN Fusion	pET3a	IntN

Table 2.

Gibson Assembly primer sequences.

Primer name	Sequence (5′ to 3′)
pCMV_H2B Forward	TAATACGACTCACTATAGGGAG
pCMV H2B Reverse	CTTGCTGCTGGT GTACTTG
IntN Insert Forward	CCAAGTACACCAGCAGCAAGTGCCTGAGCTATGATACCG
IntN Insert Reverse	CCCTATAGTGAGTCGTATTATTCCGGCAGGCCTTTCAC

2.1.3. Procedure

1. PCR amplify gene of protein of interest using Gibson Assembly primers (see Note 2) in thermal cycler.

2. Combine PCR purified product from step 1 as well as vector plasmid containing the N-intein with NEB Gibson Assembly Kit master mix according to the manufacturer’s instructions.

3. Transform the resulting vector into E. coli DH5α cells, select a colony to be grown in LB broth and isolate the plasmid.

2.2. Synthesis of Int^C-QSY-TMR-HA~CPP apo-probe

All peptides are synthesized by Fmoc SPPS on resin with Rink amide linkers to generate C-terminal amides upon cleavage resulting in more stable peptide C-termini (see Note 3 for automated peptide synthesis and Note 4 for stopping points). The Int^C-HA parent peptide has a Lys³⁹ and Cys³⁶ orthogonally protected by ivDde and StBu groups, respectively. After the final Boc-Ile-OH has been added, the Lys³⁹ side chain is deprotected using 3% hydrazine allowing the fluorophore, TMR, to be attached by coupling its N-hydroxysuccinimidyl ester derivative (TMR-SE) to the free ε-amine of Lys³⁹. The peptide is cleaved off resin using a TFA:TIPS:H₂O cleavage cocktail and then purified by reverse phase HPLC (RP-HPLC). Thereafter, the peptide can be dissolved in maleimide reaction buffer and the free thiol group of Cys²² is free to react with QSY9-maleimide. The resulting Int^C-QSY-TMR-HA product is then purified by RP-HPLC (see Fig. 3).

The HA2-TAT composed of the TAT penetrating peptide fused to an HA2 endosome escape motif, is similarly synthesized on resin by Fmoc SPPS, cleaved and then purified by RP-HPLC (Guidotti, Brambilla, & Rossi, 2017; Suhorutsenko et al., 2011). The HA2-TAT is thereafter combined with the requisite Int^C-cargo peptide (in this case, Int^C-QSY-TMR-HA) and the purified product isolated by RP-HPLC (see Figs. 2 and 3).

2.2.1. Equipment

1. 20mL gravity flow reaction columns

2. LC-ESI-MS system with Agilent C18 column (5 μm, 4 × 150 mm)

3. Lyophilizer

4. pH probe

5. RP-HPLC system with Agilent C18 preparative column (15–20 μm, 20 × 250mm) or Waters semi-preparative column (12μm, 10mm × 250mm)

6. Vacuum manifold (see Note 5)

2.2.2. Buffers and reagents

1. 3% hydrazine solution (v/v) in DCM

2. 2-(7-Aza-1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)

3. 20% piperidine solution (v/v) in DMF

4. Acetic anhydride

5. Acetonitrile for HPLC (≥99.9%)

6. ChemMatrix resin with Rink amide linker

7. Conjugation buffer: 6M guanidine HCl (GnHCl), 100 mM phosphate buffer, pH 6.0, Dichloromethane (DCM)

8. Diethyl ether

9. Diisopropylethylamine (DIPEA)

10. Dimethylformamide (DMF)

11. HPLC buffer A (0.1% TFA in water)

12. HPLC buffer B (90% acetonitrile in water with 0.1% TFA)

13. Hydroxybenzotriazole (HOBt)

14. l-Cysteine

15. Maleimide reaction buffer: 6M Guanidine HCl (GnHCl), 1 × PBS, 1 mM EDTA, pH 7.0

16. N,N′-Diisopropylcarbodiimide (DIC)

17. N-terminally Boc-protected amino acids: Boc-Ile-OH.

18. O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)

19. QSY9-maleimide

20. Standard Fmoc-SPPS amino acids, with Fmoc protecting groups on the α-amine and the following side-chain protecting groups: Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Glu(OtBu), Gln(Trt), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc) and Tyr(tBu), as well as Lys(ivDde) and Cys(StBu).

21. Tetramethylrhodamine N-hydroxysuccinimidyl ester (TMR-SE)

22. Trifluoroacetic acid (TFA) for HPLC (≥99.9%)

23. Triisopropylsilane (TIPS)

24. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)

2.2.3. Procedure: Synthesis of Int^C-QSY-TMR-HA

1. Weigh out 845mg (5 eq.) of HOBt and 1.858 g (4.9 eq.) of HBTU and dissolve in DMF to 10mL (sufficient for 10 couplings at 0.1 mmol scale).

2. Weigh out 182mg (0.1mmol) of Rink amide Chem Matrix resin (0.54mmol/g loading capacity) into a 20mL gravity flow reaction column.

3. Swell resin (see Note 6 about swelling and drying resin) in about 3mL of DMF for 20min with agitation (see Note 7 about agitation) before draining over a vacuum line.

4. Add 3–4mL of 20% piperidine in DMF solution and allow the mixture to agitate for 30s (see Note 8 about aspartimide formation). Drain over a vacuum line and wash the resin using DMF.

5. Repeat step 4 for 20min instead of 3min.

6. Weigh out 0.5mmol of the first amino acid (Fmoc-l-Ala-OH in this case, 5 eq.) and dissolve it in 1mL of the stock made in step 1.

7. Add 87 μL (0.5mmol, 5eq.) of DIEA to 6 and pour this mixture into the column. Agitate the mixture for 45min (see Note 9). Drain and wash the column thoroughly with DMF.

8. Add a mixture of 47.3 μL (0.5mmol, 5eq.) of acetic anhydride and 174.2μL (1mmol, 10eq.) of DIEA in 1mL of DMF to the column and agitate for 20min. Drain and wash the column thoroughly with DMF.

9. Repeat step 8 once.

10. Repeat steps 4–7 until desired length of peptide is reached (in our case until Boc-Ile-OH added, see Table 3 for peptide sequence).

11. Treat the resin with 5mL of 3% hydrazine in DCM for 2min 6–8 times while washing with DCM and DMF in-between.

12. Weigh out 264mg (0.5mmol, 5eq.) of TMR-SE and dissolve it in about 4mL of DMF, add 174.2 μL (1mmol, 10eq.) of DIEA then pour this solution into the column and agitate for 12h. Drain and wash the resin thoroughly in DMF.

13. Add 3–5mL of 95% TFA, 2.5% TIPS and 2.5% water to the resin and agitate the mixture for 2h. Then drain the filtrate into a 50mL conical tube and evaporate the solution by blowing nitrogen gas onto the liquid.

14. Add 10mL of cold diethyl ether to the residue in 13, spin this sample down at 5000 rcf for 5min and 4 °C. Dump the supernatant and repeat twice before letting the resulting pellet air dry for 20–30 min.

15. Dissolve the pellet in 5–10mL of a 50:50 mixture of LCMS buffer A: LCMS buffer B.

16. Load 100 μL of this solution onto an LC-ESI-MS system and determine gradient required for HPLC purification.

17. Freeze the peptide solution in liquid nitrogen and remove liquid via lyophilizer.

18. Redissolve peptide in combination of LCMS buffer A:LCMS buffer B and purify peptide by semi-preparative or preparative RP-HPLC. Evaluate fractions by LCMS, combine pure fractions and load 100 μL of pure fraction onto LCMS for quantification (see Note 10 on quantification) before freezing and lyophilizing peptide.

19. Redissolve peptide in maleimide reaction buffer (4mM solution). Dissolve QSY9-maleimide in this same volume (but 8mM, 2eq.) and combine the two solutions with agitation at room temperature. Verify pH is at 7.0 using pH probe and adjust accordingly using 1N NaOH (aq.) and 1 N HCl (aq.). Monitor the reaction every hour by LCMS until reaction completion.

20. Quench the reaction by adding L-cysteine (50 eq.) and incubate at room temperature with agitation. Monitor the disappearance of free QSY9-maleimide every 30min by LCMS.

21. Remove the StBu group by adding a TCEP in water stock to a final concentration of 90mM. Agitate the mixture at room temperature and monitor the reaction every hour by LCMS.

22. Isolate the product by semi-preparative or preparative HPLC as in step 18 (see Fig. 3A).

Table 3.

Amino acid sequence of transfected protein and deliver peptide.

Component	Amino acid sequence
H2B-IntN Fusion	PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVLKQV
	HPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVR
	LLLPGELKHAVSEGTKAITKYTSSKGGKFAEYCLSYDTEVLTVEYGFVPI
	GEIVDKGIECSVFSIDSNGIVYTQPIAQWHHRGKQEVFEYCLEDGSIIKAT
	KDHKFMTQDGKMLPIDEIFEQELDLLQVKGLPE
IntC-Cargo Peptide	IKIATRKYLGKQNVYDIGVERC(QSY)HNFALKNGFIASNCFNK(TMR)YPY DVPDYA
HA2-TAT CPP	C(Npys)GLFEAIAEFIENGWEGLIEGWYGGRKKRRQRRR

2.2.4. Procedure: Synthesis of HA2-TAT (CPP)

Use the SPPS protocol from Section 2.2.3 (namely, steps 1–10 for synthesis and 14–18 for purification) with minor alterations to prepare the CPP. The CPP’s sequence, which may be found in Table 3, contains a high abundance of arginine residues which may affect the efficiency of coupling and so should be double coupled (see Note 11 on double coupling) (Behrendt et al., 2016). The cleavage of the peptide from resin requires a longer reaction time (3h instead of 2h). The peptide is also very hydrophobic and needs to be dissolved in 6M GnHCl for purification on RP-HPLC (see Fig. 3B).

2.2.5. Procedure: Conjugation of Int^C-QSY-TMR-HA to HA2-TAT

1. Resuspend HA2-TAT and Int^C-QSY-TMR-HA in degassed conjugation buffer to give final concentrations of 2.25 and 1.5mM, respectively, and agitate the mixture at room temperature for 45 min.

2. Isolate the product by RP-HPLC as in step 18 of Section 2.2.3 (see Fig. 3C).

2.3. Transfection of H2B-Int^N-flag

We utilize a lipofectamine-mediated transfection protocol to introduce the histone-Int^N fusion into HEK 293T cells (see Note 12). The transfection protocol outlined below is for a single 10cm plate of ~1 × 10⁷ cells and unless otherwise stated, all steps should be conducted under sterile conditions. A control transfection without any DNA should also be performed (see Note 13 about controls).

2.3.1. Equipment

1. 1.7mL Eppendorf tubes

2. Falcon Standard Tissue 10cm Culture Dishes

3. Fisherbrand Sterile Polystyrene Disposable Serological Pipets

4. Sterile tissue culture hood

5. Tissue culture CO₂ incubator

2.3.2. Buffers and reagents

1. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 500units/mL penicillin/streptomycin antibiotic (full media)

2. HEK 293T cells (ATCC)

3. Lipofectamine 2000

4. Opti-MEM Reduced Serum Media (Gibco)

2.3.3. Procedure

1. To 2–5 μg of DNA for the H2B-Int^N-Flag construct, add 400 μL of Opti-MEM in a 1.7mL Eppendorf tube.

2. In a separate 1.7mL Eppendorf tube, add 15 μL of Lipofectamine 2000 and 400 μL of Opti-MEM.

3. Carefully add the solution in step 2 to the solution in step 1 and allow the mixture to incubate at room temperature for 30 min.

4. Drain and replace the media on a plate containing HEK 293T cells at 70% confuency with 5 mL of full media. Then carefully add the mixture from 3 and incubate the plate at 37 °C in a tissue culture incubator for 6h.

5. Drain and replace the media in the plate with 10mL of full media and incubate the plate at 37°C in a tissue culture incubator for 18–24 h.

2.4. Peptide delivery

2.4.1. Buffers and reagents

1. Dimethyl sulfoxide (DMSO)

2. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 500 units/mL penicillin/streptomycin antibiotic (full media)

3. Opti-MEM Reduced Serum Media (Gibco)

2.4.2. Procedure

1. Dissolve Int^C-QSY-TMR-HA~CPP apo-probe in DMSO to make 1mM solution. This stock can be frozen and reused.

2. Make delivery stock solutions of 1 using Opti-MEM to a volume of 2mL and concentration of between 1 and 10 μM.

3. Wash the H2B-Int^N expressing cells from Section 2.3 with Opti-MEM.

4. Add the solution from step 2 to the cells and incubate for 1h at 37°C.

5. Supplement the cells with equal volume of full media and incubate at 37°C for 1–4h.

2.5. Harvesting cells and western blot analysis

Following the delivery of the fluorophore cargo, the cells may be harvested and lysed. Then, the nuclear fraction may be isolated and success of the splicing reaction verified by western blot. In this case, the starting materials, desired and side-products may be tracked using antibodies specific for Flag, and HA epitopes (see Fig. 4A).

2.5.1. Equipment

1. 1.7mL Eppendorf tubes

2. Gel/membrane transfer apparatus including transfer sandwich, box, power source, etc.

3. Gel running apparatus including plates, gaskets, running box, power source, etc.

4. Dounce pestle homogenizer

5. Odyssey CLx Imaging System

6. PVDF membrane

7. Rod sonicator with microtip

8. Tabletop centrifuge

2.5.2. Buffers and reagents

1. 3-(N-morpholino) propanesulfonic acid (MOPS)

2. 4 × Protein loading sample buffer (SB): 200mM Tris-HCl, 280mM sodium dodecyl sulfate, 6mM bromophenol blue, 40% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol

3. 12% polyacrylamide Bis-Tris gel

4. 0.8 M iodoacetamide solution

5. Antibodies: rabbit-αHA, mouse-αH2B, α rabbit 800 and α mouse 680 (see Note 14 about secondary antibodies).

6. Bovine Serum Albumin (BSA)

7. Hypotonic buffer: 10mM Tris, 15mM NaCl, 1.5mM Mg(Cl)₂, protease inhibitors, pH 7.6

8. PBST: phosphate buffered saline with 1% Tween

9. SDS RIPA lysis buffer: 25mM Tris pH 8.0, 150mM NaCl, 1% sodium dodecyl sulfate, 1% NP-40, 1% deoxycholate

10. Transfer buffer: 25mM Tris base, 192mM glycine, 20% (v/v) Methanol

2.5.3. Procedure

1. Wash cells with cold 1 × PBS then add 1mL of cold hypotonic buffer and scrape cells into 1.7mL Eppendorf tube.

2. Spin down cells at 400g for 5min at 4°C in table-top centrifuge and discard the supernatant.

3. Resuspend the pellet in 1mL hypotonic buffer supplemented with 1% Triton X-100 and lyse with 10 strokes of a Dounce pestle homogenizer.

4. Add 0.8 M iodoacetamide to final concentration of 80 mM to quench the splicing reaction (see Note 15).

5. Spin down at 10,000g for 10min at 4°C in table-top centrifuge to separate nucleoplasm. Discard the sup.

6. Resuspend the chromatin in 0.1mL SDS-RIPA lysis buffer and sonicate for 5s at 35% amplitude. The procedure can be paused here and the solutions frozen at −20 or −80 °C.

7. Add 5μL of SB to 15 μL of sample, boil for 10min and load onto 12% polyacrylamide Bis-Tris gel.

8. Run gel in MOPS running buffer for 1h at 150V.

9. Transfer protein onto PVDF membrane using transfer buffer at constant voltage, 85V for 75min at 4 °C (see Note 16 about imaging blot).

10. Block the membrane for 1h at room temperature or overnight at 4 °C in PBST solution supplemented with 5% BSA.

11. Blot the membrane with rabbit-αHA and mouse-αH2B in PBST (1:1000) for 1h at room temperature or overnight at 4 °C.

12. Blot the membrane with α rabbit 800 and α mouse 680 (1:15,000) in PBST with 5% BSA for 45min at room temperature.

13. Image the blot using an Odyssey blot imager.

2.6. Microscopy

Cargo peptide delivery and successful incorporation of the TMR fluorophore into cellular chromatin may be visualized and tracked via live-cell imaging using confocal microscopy (see Fig. 4B).

2.6.1. Equipment

1. 1.7mL Eppendorf tubes

2. Fisherbrand Sterile Polystyrene Disposable Serological Pipets

3. Leica SP5 microscope (Leica Microsystems GmbH, Germany)

4. Sterile tissue culture hood

5. Thermo Fisher glass bottom 35mm dish

6. Tissue culture CO₂ incubator

2.6.2. Buffers and reagents

1. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 500units/mL penicillin/streptomycin antibiotic (full media)

2. HEK 293T cells (ATCC)

3. Leibovitz L-15 Media (Thermo Fisher Scientific)

4. Lipofectamine 2000

5. Opti-MEM Reduced Serum Media (Gibco)

2.6.3. Procedure: Live confocal microscopy

1. Plate approximately 2 × 10⁵ HEK 293T cells on a glass bottom 35mm dish.

2. Appropriately scale-down and follow transfection procedure from Section 2.3 and peptide delivery procedure from Section 2.4.

3. Wash cells with Opti-MEM medium.

4. Follow manufacturer’s protocols to perform confocal microscopy at 63 × magnification using a Leica SP5 microscope, and take several cross-sectional images.

2.6.4. Procedure: Time-lapse confocal microscopy

1. Plate approximately 2 × 10⁵ HEK 293T cells on a glass bottom 35mm dish.

2. Appropriately scale-down and follow transfection procedure from Section 2.3.

3. Wash cells with Opti-MEM medium.

4. Prior to peptide delivery, place the plate of cells on microscope stage and cover to maintain 5% CO₂ and 37 °C conditions.

5. Appropriately scale-down and follow peptide delivery procedure from Section 2.4.

6. Follow manufacturer’s protocols to perform confocal microscopy at 63 × magnification using a Leica SP5 microscope, and take several cross-sectional images.

7. Take time-lapse images of the fluorescence signal using several crosssections per condition per cell. Record the cells at 5min intervals for the first hour and 15min intervals for an additional 2h.

4. Summary and outlook.

Here, we outlined the recent development of ultrafast-trans splicing intein technology toward the site-specific incorporation of modifications on histones in live cells (David et al., 2015). The immediate applications include the investigation of individual modifications as well as their roles in regulating epigenetic states, transcription, and ultimately, cellular physiology. A detailed protocol of the incorporation of a fluorophore modification on Histone H2B in live cells has been included as an example. While this method was originally developed to assist in deconvoluting the histone code, its versatility renders it a powerful tool toward understanding the roles of other PTMs. Additionally, orthogonal intein pairs could be utilized to introduce multiple modifications toward the understanding of protein interplay in an in vivo context (Carvajal-Vallejos et al., 2012; Shah et al., 2012). Finally, in the long-term, similar methodologies could be developed in a whole organism in a site-specific manner in order to elucidate the role of these modifications system-wide. Understanding the mechanisms behind these chemical switches in cellular physiology will lead to the decoupling of millions of modifications in live systems.