Unmasking a hidden code during transcription: A practical strategy to reveal Thr4 phosphorylation in RNA polymerase II

Abstract

RNA Polymerase II (Pol II) is core to cell survival as the responsible enzyme for transcription of mRNA as well as various non-coding RNA species. The post-translational modification (PTM) of the C-terminal domain (CTD) of its largest subunit RPB1, principally through phosphorylation, enables docking of relevant proteins to govern the transcription cycle and RNA processing. The detection of phosphorylated Thr4 of the CTD heptad motif is exacerbated by epitope masking due to phosphorylations on nearby residues, thereby decreasing antibody recognition of the target epitope. Here, we introduced ChIP-phos-seq, wherein we conducted chromatin immunoprecipitation with phosphatase treatment followed by sequencing. This method addressed the overlooked phosphorylated Thr4 by removing masking phosphomarks with the targeted, specific phosphatase, Ssu72. In order to perform ChIP-phos-seq, Ssu72 was purified and characterized for specificity and activity through mass spectrometry and kinetics assays. The phosphatase treatment was optimized and used as a tool to target the masked phosphorylated Thr4 epitopes recognized in an antibody pulldown. Sequencing the DNA bound to Pol II then allows the CTD Thr4 phosphorylation to be profiled and analyzed for its presence on different gene types, lengths, and ontologies. With a clearer view of phosphorylated Thr4, a better understanding of how Thr4 phosphorylation contributes to transcription regulation and transcript processing is achievable.

1. Introduction

In eukaryotic cells, RNA Polymerase II (Pol II) are responsible for transcribing all protein-coding mRNAs and numerous non-coding RNAs (Eick & Geyer, 2013). A key structural feature of Pol II to handle such a huge workload is its C-terminal domain (CTD) of its largest subunit, composed of tandem repeats of a conserved heptapeptide sequence, Tyr₁-Ser₂-Pro₃-Thr₄-Ser₅-Pro₆-Ser₇ (Corden, 1990; Eick & Geyer, 2013). This modular architecture, present in various repeats (26 repeats in yeast and 52 in humans), acts as a dynamic regulatory platform, integrating transcription with RNA processing through patterns of post-translational modifications (PTMs) (Buratowski, 2003; Eick & Geyer, 2013; Venkat Ramani et al., 2021).

The dominant PTMs of Pol II are site-specific phosphorylations, which recruit or release regulatory factors at defined stages of the transcription cycle (Kim et al., 2021; LeBlanc et al., 2021). The phosphorylation of Ser5 and Ser2 have been studied in detail, revealing distinct roles in initiation and elongation/termination, respectively (Ho & Shuman, 1999; West & Corden, 1995; Zhou et al., 2012). However, other modifiable residues within the heptad repeat—namely Tyr1, Ser7, and Thr4—remain less understood (Chapman et al., 2007; Hintermair et al., 2012; Hsin et al., 2014). In particular, the loss of phosphorylation at Thr4 (pThr4) in human cells is fatal and this modification has been implicated through genetic studies in transcriptional elongation, proper termination, and mitotic progression (Hintermair et al., 2012, 2016). Despite these compelling associations, molecular mechanistic understanding of pThr4’s function has remained frustratingly elusive, largely due to technical barriers in detecting it (Corden, 2013; Hintermair et al., 2012; Kempen et al., 2023; LeBlanc et al., 2021; Venkat Ramani et al., 2021). Antibodies generated against the pThr4 epitope are typically specific in vitro, but the pThr4 specific antibodies only map to weak signal within gene body with an increase 500 bp after transcription termination in protein-coding genes (Hintermair et al., 2012; Schlackow et al., 2017).

The reason for lack of detection of pThr4 Pol II might lie in the densely phosphorylated environment of the Pol II CTD in vivo, where nearby modifications—especially at Ser2 and Ser5—can physically hinder antibody access to pThr4 (Hintermair et al., 2012; Schüller et al., 2016). This “epitope masking” effect leads to an underrepresentation of pThr4 signals, particularly during early and mid-transcription when Ser2 and Ser5 are heavily phosphorylated, and has created misleading impressions about its timing and function (Hintermair et al., 2012; Moreno et al., 2024; Schüller et al., 2016). To overcome this problem, we introduce a simple but powerful approach: using CTD-specific phosphatases to selectively remove masking phosphorylations (Moreno et al., 2024). By treating crosslinked chromatin samples with a well-characterized phosphatase prior to immunoprecipitation, we can eliminate obstructive Ser5 phosphorylation while preserving Thr4 phosphorylation (Moreno et al., 2024). This unmasking strategy restores antibody access to the pThr4 epitope, allowing for a more faithful detection of its genomic distribution through ChIP-seq. This method is fast, technically straightforward, and compatible with existing ChIP workflows. Importantly, it preserves the native chromatin context and does not require genetic manipulation or specialized reagents. Researchers studying the CTD—or any multisite modified protein—can readily apply this approach to resolve masked epitopes and avoid the false negatives of their ChIP data when using appropriate phosphatase treatment.

The impact of this method lies in its potential to clarify the true biological roles of Thr4 phosphorylation (Hintermair et al., 2012; Kempen et al., 2023; Moreno et al., 2024). Previous models based on masked detection suggested that pThr4 appears only late in transcription. However, this view is at odds with genetic evidence linking Thr4 to transcription elongation and pre-mRNA processing, both of which occur earlier (Hintermair et al., 2012; Hsin et al., 2011). By lifting the veil of epitope masking, researchers are now equipped to re-examine the timing, function, and physiological importance of this elusive CTD modification. Given its conservation and essentiality in higher eukaryotes, Thr4 phosphorylation likely plays a fundamental role in gene regulation. Its precise dynamics and molecular partners have remained obscure not because they are absent, but because standard tools failed to detect them. The unmasking method we describe enable the community to revisit this overlooked modification and explore its contribution to transcriptional elongation, RNA maturation, and cell cycle control.

2. Approach

2.1. Ssu72 can dephosphorylate Ser5 CTD but not at Thr4

Previously, we have shown that Ssu72 can dephosphorylate Ser5 without reducing the phosphorylation marks on Thr4 when CTD peptide is doubly phosphorylated on these sites (Irani et al., 2019; Luo et al., 2013). Since Thr4-Ser5 double phosphorylation is reported to be quite abundant in human cells (Schüller et al., 2016), we first confirmed the Ssu72 specificity using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Fig. 1A, B and C). CTD phosphopeptides at ~100 μM concentration were incubated with 5 μM Ssu72 complexed with Symplekin for 30 min at 28 °C. After incubation, the samples were prepared by desalting in preparation to be subjected to MALDI-TOF mass spectrometry to improve signal. Similar experiments were conducted for phosphopeptides of CTD with different phosphorylation sites. Consistently, when we treated CTD phosphopeptide with Ser5 phosphorylation, the phosphate group was effectively removed by Ssu72 while no such effect occurred with the same treatment on CTD peptide with Thr4 phosphorylated (Figs. 1A and B). Treatment of Ssu72 on a CTD peptide with doubly phosphorylated Thr4 and Ser5 led to the removal of one phosphate on Ser5 while pThr4 remained intact (Fig. 1C). The reason for the high specificity is due to the high specificity binding of Ssu72 to phosphorylated Ser5 due to the recognition of cis-Pro6 by a hydrophobic pocket (Fig. 1D).

Fig. 1. Ssu72/Symplekin dephosphorylates Ser5 specifically.

Mass spectrometry comparison of treated and untreated CTD peptides. pSer5 (A), pThr4 (B), and pThr4pSer5 (C) peptides. Intensity was normalized to percent, with the lowest value equivalent to zero and the highest intensity set to 100 % using GraphPad Prism (version 10.5.0). (D) Drosophila Ssu72 complexed with Symplekin bound to pSer5 CTD peptide (PDB: 6NPW, (Irani et al., 2019)). Hydrogen bonding between CTD peptide and Ssu72 is shown. Proteins were shown graphically with analyses performed using UCSF ChimeraX (version 1.9) (Meng et al., 2023).

The detailed protocol for testing the phosphatase activity using MALDI is shown below:

2.2. Before you begin

• Ssu72 enzyme (at least 5 μM, may include Symplekin as a complex)

• Ssu72 reaction buffer: 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0, 200 mM NaCl

• Ssu72 protein buffer: 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM β-mercaptoethanol (BME)

2.3. Key resources table

Reagent or resource	Source	Identifier
Chemicals, Peptides, and Recombinant Peptides
18-mer biotinylated CTD peptide (10 mg) (phosphorylated on Tyr1, Ser2, Thr4, Ser5, Ser7, and Thr4Ser5)	Biomatik	Custom Peptide Synthesis
Trifluoroacetic acid (99% extra pure)	Thermo Scientific Chemicals	139721000 CAS: 76-05-1
Acetonitrile (Certified ACS)	Fisher Scientific	A21-4 CAS: 75-05-8
2,5 Dihydroxybenzoic acid (DHB) MALDI Matrix, Single Use, 3 × 8 microtube strip, 4 mg/microtube	Thermo Scientific	90033
Software and Algorithms
flexAnalysis	Bruker Daltonik GmbH
GraphPad Prism Software	Dotmatics	GraphPad Prism 10.5.0

2.4. Materials and equipment

• Pierce C18 tips, 10 μL (Catalog: 87782, Thermo Scientific)

• 18-mer singly phosphorylated phosphopeptides (diluted to 10 mg/mL in water, Biomatix)

• ThermoMixer C (Catalog: 5382000023, Eppendorf), with 1.5 mL SmartBlock (Catalog: 5360000038, Eppendorf) and ThermoTop (Catalog: 5308000003, Eppendorf)

• Bruker autoflex maX MALDI-TOF (Bruker Scientific) and MTP 384 target plate polished steel BC (Catalog: 8280781, Bruker Scientific), MTP target frame III (Catalog: 8074115, Bruker Scientific)

2.5. Specificity of Ssu72 with MALDI-TOF

Timing: 4 h.

1. In a 1.5 mL microcentrifuge tube combine 108 μM phosphopeptide with 5 μM Ssu72 in Ssu72 reaction buffer to a final reaction volume of 20 μL. The assay should be setup for singly phosphorylated CTD peptides: pTyr1, pSer2, pThr4, pSer5, and pSer7, as well as double phosphorylation peptide pThr4pSer5.

2. Incubate reaction in thermomixer for 30 min at 28 °C, 650 rpm.

   Pause Point: The samples may be frozen at −20 °C at this juncture for storage until the available of mass spectrometry measurement.

3. Adjust sample to 0.1 % TFA.

4. Desalt samples with Pierce C18 tips according to manufacturer’s protocol. The wetting solution was adjusted to 100 % acetonitrile (ACN), the rinse solution was modified to remove ACN. Elute samples in 0.1 % TFA/50 % ACN solution mixed with DHB.

5. Spot a calibrated mix of proteins with known molecular weights on a MALDI-TOF plate and blow dry. Spot the desalted samples on a plate (2 μL) and blow dry, without heating. (Note: Calibration of the MALDI-TOF should use the same matrix, DHB, as used in the samples.)

6. Insert plate into the Bruker autoflex maX MALDI-TOF, controlled by flexControl software.

7. Auto-calibrate Bruker autoflex maX MALDI-TOF using the calibration mix.

8. Hit samples with the beam with a minimal percentage laser power to get good signal, with a beam walk.

9. Compare data first in flexAnalysis, followed by analysis in GraphPad Prism.

2.6. Safety considerations and standards

Trifluoroacetic acid is toxic and corrosive, an irritant to the respiratory system, and causes damage to eyes and skin. Acetonitrile is flammable, an irritant to the skin, respiratory system, and eyes, and toxic, potentially causing damage to multiple organs. BME is a hazardous and volatile chemical with a strong, unpleasant odor. These chemicals should be used in a chemical fume hood and the tips and tubes contaminated with these chemicals should be disposed of properly. Likewise, DHB, which serves as a matrix for the sample, is an irritant to the eye, skin, and respiratory system, and should be disposed of properly as it is unsafe for marine life and the environment.

2.7. Troubleshooting and experimental optimization

Excessive exposure of peptide to large quantities of phosphatase may increase the likelihood of Ssu72 engaging in more promiscuous activity toward different peptides. Alternatively, a batch of Ssu72 with low activity may be insufficient to dephosphorylate all the pSer5 peptide in 30 min. If there are issues, the peptide incubation time and protein amount may be adjusted. Conducting a preliminary test with a constant peptide concentration and varied Ssu72 concentrations for pThr4, pSer5, and pThr4pSer5 can determine an ideal ratio prior to incubation with pTyr1, pSer2, and pSer7 CTD peptides.

2.8. Experimental results and expected outcomes

Ssu72 effectively dephosphorylated pSer5 CTD peptide; the molecular weight decreased from 2313 to 2233 Daltons (Da), consistent with the loss of a phosphate group (Fig. 1A). Conversely, pThr4 peptide remained the same molecular weight upon Ssu72 treatment, indicating the lack of dephosphorylation (Fig. 1B). This pattern was consistent in the double phosphorylated peptide pThr4pSer5, in which a single dephosphorylation occurred, indicating that specificity could be retained in cases of multiple phosphorylation (Fig. 1C). Ssu72 exhibited little activity against other CTD phosphopeptides: pTyr1, pSer2, pSer7.

3. Activity of Ssu72 from various organisms

To identify the optimal ortholog of Ssu72 for removing Ser5 phosphorylation on the CTD, we tested the phosphatase activity of Ssu72 from yeast (residues 1–206), Drosophila (residue1–195), and human (residue 1–194). Enzymatic activity was then assessed using the non-specific substrate para-nitrophenylphosphate (pNPP).

The detailed protocol for pNPP assay is shown below:

3.1. Before you begin

• Ssu72 enzyme at least 10 μM, selection of orthologs, or with Symplekin (purification described later)

• 5× reaction buffer: 100 mM MES pH 6.0, 200 mM NaCl

• Ssu72 storage buffer: 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM BME

3.2. Key resources table

Reagent or resource	Source	Identifier
Chemicals, Peptides, and Recombinant Peptides
4-Nitrophenyl phosphate disodium salt hexahydrate	Sigma-Aldrich	71768-5g CAS: 333338-18-4
4-Nitrophenol	MP Biomedicals	102465 CAS: 100-02-7
Software and Algorithms
Microsoft Excel	Microsoft
GraphPad Prism Software	Dotmatics	GraphPad Prism 10.5.0

3.3. Materials and equipment

• Tecan plate reader Infinite M200 (Tecan Life Sciences)

• 96 well plate, non-treated surface, no lid, non-sterile, pack of 5 (Catalog: 269620, Thermo Scientific)

• P20 multichannel pipette

3.4. Generate absorbance standard curve

Timing: 1 h.

1. Using a multichannel pipette, add 5x reaction buffer, Ssu72 buffer, and pNP into a clear, flat-bottomed 96-well plate, to a final concentration of 1x reaction buffer, 10 % Ssu72 buffer, and pNP ranging from 0 to 2 mM, in triplicate. Suggested final concentrations are 0, 125, 250, 375, 500, 1000, 1500, and 2000 μM pNP.

2. Measure three times on Tecan infinite M200 plate reader and average the readings for each concentration.

3. Plot the absorbance (y-axis) versus the concentration (x-axis) in Excel.

   Note: The resultant plot should show a linear relationship between absorbance and concentration.

   Divide the absorbance by the molar concentration to find the value of the molar extinction coefficient multiplied by the path length. Average the value. This value will be used to convert the absorbance reading into the quantity of products formed.

   Critical: The molar extinction coefficient of pNP can vary depending on the reaction buffer, so it is essential that the standard curve is generated in the same buffer used in the phosphatase assay to accurately determine the amount of product formed.

3.5. Set up pNPP assay for Ssu72 phosphatase

Timing: 1.5–2 h

1. Using a multichannel pipette, add 5x reaction buffer, 10 μM Ssu72 (for each phosphatase condition desired, including different orthologs or complex status), and pNPP into a clear, flat-bottomed 96-well plate in triplicate, to a final concentration of 1x reaction buffer, 1 μM Ssu72, and pNPP ranging from 0 to 150 mM. Suggested final concentrations are 0, 15, 30, 45, 60, 90, 120, and 150 mM pNPP.

2. In parallel, replicate conditions in triplicate, substituting volume of Ssu72 with Ssu72 buffer, as a control for any increase in absorbance not related to enzymatic activity. Mix by pipetting up and down and collect absorbance readings on Tecan plate reader.

   Note: Steps 13 and 14 should be performed to proceed quickly to measuring absorbance on Tecan plate reader.

3. Collect absorbance readings every 45 s for 30 min, at 28 °C, shaking for 3 s prior to each measurement.

4. Find the slope of the absorbance reading increase over the change in time (in seconds) for the first 900 s

5. Using the constants calculated through Beer-Lamberts law with the absorbance standard curve, convert absorbance/second to μM/s product (pNP) formed.

6. Average the rate of increase for each concentration for the blank. Subtract rate of increase of each concentration of pNPP to control for increases in absorbance that are unrelated to phosphatase activity.

7. Plot Michaelis-Menten curve with three replicates at each concentration for each condition to find V_max and K_m. Divide V_max by the concentration of Ssu72 (1 μM) to find k_cat (Fig. 2).

   Note: Both pNPP and its product pNP are light sensitive and should be covered with foil when possible.

Fig. 2. Kinetics of orthologous Ssu72 with the pNPP assay.

Drosophila, yeast, and human Ssu72 were monitored for absorbance increases, indicative of pNP product formation (μM/s), when assayed with 0–150 mM pNPP. A nonlinear regression curve fit was performed GraphPad Prism (version 10.5.0).

3.6. Advantages and limitations

The pNPP assay can be used as a quality control for each batch of phosphatase before sample treatment. This assay is easy to do and can yield comparative turnover rates between phosphatases which can be applied to whether a Ssu72 ortholog or complex is a viable candidate for ChIP-phos-seq as quality control.

3.7. Troubleshooting and optimization

The pNPP assay can be adjusted in several ways to suit the experimental needs. To accurately determine k_cat, the Michaelis-Menten curve must reach a plateau to define V_max. If the range of pNPP concentrations used is insufficient to capture this plateau, higher concentrations should be added. Conversely, if greater resolution at low substrate concentrations is needed, additional lower pNPP concentrations can be included. In cases where pNP formation is too rapid to collect data over a full 15-minute period, the concentration of Ssu72 can be reduced to slow the reaction and enable more accurate measurement.

3.8. Experimental results and expected outcomes

The pNPP assay should yield a fully realized Michaelis-Menten curve with the rate (1/s) (y-axis) versus the pNPP substrate concentration (μM) (x-axis) (Fig. 2). Drosophila and human Ssu72 displayed comparable catalytic rates (Drosophila Ssu72 with a k_cat of 1.42 ± 0.08 s⁻¹ and human Ssu72 a k_cat of 1.10 ± 0.03 s⁻¹), whereas the yeast enzyme showed a markedly lower k_cat of 0.008 ± 0.005 s⁻¹ (Fig. 2). Accordingly, the yeast ortholog was excluded from subsequent ChIP-seq experiments.

4. Purification of Ssu72/Symplekin

We and others have previously shown that Ssu72 forms a stable complex with the scaffold protein Symplekin, which enhances its stability and catalytic activity (Luo et al., 2013; Xiang et al., 2010). The pNPP assay results demonstrated comparable catalytic activity between Drosophila Ssu72/Symplekin (k_cat = 1.88 ± 0.07 s⁻¹) and human Ssu72/Symplekin (k_cat = 1.69 ± 0.03 s⁻¹).

The detailed purification protocol for the Drosophila Ssu72/Symplekin complex (Fig. 3A) is provided below, human complex method is identical:

Fig. 3. Purification and reconstruction of Ssu72/symplekin complex.

Depiction of Ssu72/Symplekin complex purification. The process to purify the Ssu72/Symplekin is shown (A). An elution profile of the Ssu72/Symplekin complex using size exclusion chromatography (B). The Ssu72/Symplekin complex is indicated with an arrow. Created in BioRender. Stephenson, R. (2025) https://BioRender.com/95673y5.

4.1. Before you begin

• His-tagged Drosophila Ssu72 (1–194) and Drosophila Symplekin (19–351) plasmids in a pET28a backbone should be transformed into BL21 (DE3) and streaked onto a kanamycin positive Luria Broth (LB) plate

• Lysis Buffer: 500 mM NaCl, 10 % glycerol, 50 mM Tris-HCl pH 8.0, 10 mM imidazole, 0.1 % Triton X-100, 10 mM BME

• Wash Buffer: 500 mM NaCl, 50 mM Tris-HCl pH 8.0, 10 mM imidazole, 10 mM BME

• Elution Buffer: 250 mM imidazole, 500 mM NaCl, 100 mM Tris-HCl pH 8.5, 10 mM BME

• Dialysis/Gel Filtration Buffer: 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM BME

  Note: Buffers can be made in advance; however, the BME should be added the day of use.

4.2. Key resources table

Reagent or resource	Source	Identifier
Bacterial and Virus Strains
E. coli		BL21(DE3)

4.3. Materials and equipment

• 0.5 M Isopropyl β-D-1-thiogalactopyranoside (IPTG)

• 50 mg/mL kanamycin

• LB

• SnakeSkin Dialysis Tubing 3500 MWCO (molecular weight cut-off) (Catalog: 68035, Thermo Scientific)

• Nanodrop 1000 Spectrophotometer (Thermo Scientific)

• His-Pur Ni-NTA Resin (Catalog: 88221, Thermo Scientific)

• 250 mL flasks

• 2800 mL flasks

• Beakers (250 mL, 400 mL)

• Beaker (plastic, for sonication)

• Sonicator (Misonix Ultrasonic Processor S-4000)

• Avanti J-E Centrifuge

• Sorvall Lynx 6000 Centrifuge and 1 L centrifuge bottles

• Stir plate

• Centrifuge 5425 R (Eppendorf)

• G24 Environmental Incubator Shaker (New Brunswick Scientific)

• Excella E25 Incubator Shaker Series (New Brunswick Scientific)

• Column PD-10, Empty (Catalog: 17-0435-01, GE Healthcare)

• HiLoad 16/600 Superdex 75 pg column (Catalog: 28989333, Cytiva Life Sciences) connected to a NGC Chromatography System (BioRad)

• Spectrophotometer (Ultrospec 10 cell density meter, Amersham Biosciences) and cuvettes (semi-micro, 1.5 mL, Catalog: 14955127, Fisher Scientific)

• Vivaspin 6 (Catalog: VS0602) and 20 (Catalog: VS2002, Sartorius), centrifugal concentrators, membrane: 10,000 MWCO PES

• 5 mL BD Luer-Lok syringe (Catalog: 309646, BD)

• −80 °C freezer

4.4. Phosphatase Ssu72 purification

Timing: 4 days

1. Add 50 uL of 50 mg/mL kanamycin to two flasks of 50 mL LB and inoculate with a colony of E. coli BL21 (DE3) transformed with Symplekin and Ssu72, respectively. Incubate overnight at 37 °C with shaking.

2. Add 1 mL of 50 mg/mL kanamycin to as many 1 L LB flasks as desired for Symplekin and Ssu72 (more is preferable, 3 of each is reasonable). Inoculate with 10 mL of 50 mL overnight cultures.

3. Incubate 1 L LB flasks at 37 °C with shaking at 200 rpm.

4. Induce 1 L cultures with 1 mL 0.5 M IPTG when OD reaches 0.6–0.9, monitored with a spectrophotometer and 1.5 mL cuvettes, blanked with sterile LB.

   Note: This will likely take approximately two to four hours. The cultures should be monitored throughout this time period so that the window is not missed.

5. Reduce shaker temperature to 16 °C with shaking and incubate 1 L flasks overnight, 16–20 h.

6. Spin down the culture at 5000 g, 20 min at 4 °C.

7. Dispose of supernatant and scrape resultant pellets into a tube.

   Pause Point: The pellet can be scraped into a 50 mL conical tube and stored at −20 °C for a week.

8. Move the bacterial pellet into lysis buffer. Add 50−100 mL of lysis buffer per 1 L flask of culture and stir on a stir plate at 4 °C until homogeneous.

9. While stirring on ice, break open cells with a sonicator set to 60 % or 90 amp, with 6 cycles of sonication with 1 s on, 5 s off for 3 min, and 5 intermediate periods of 3 min.

10. Spin down cell lysate for 45 min, 27,200 g at 4 °C. During centrifugation, equilibrate regenerated nickel beads by pouring lysis buffer through PD-10 column. Quantity of nickel beads should reflect quantity of protein.

11. After centrifugation, separate supernatant from pellet, saving supernatant. Dilute small amount of pellet in water and dilute small aliquot of supernatant (1 μL of supernatant: 3 μL water) for quality control sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

12. Incubate supernatant with nickel beads, stirring slowly for 15 min to allow protein binding to the beads. Ensure beads are not clumping.

13. Pour lysate and bead mixture into a disposable column. Take a small aliquot of the flow-through to save for sodium dodecyl sulfate (SDS) gel.

14. Wash column and beads with 60 mL wash buffer. Take a small aliquot of the wash buffer after it runs through the column to save for SDS gel.

15. Elute with 20 mL of elution buffer, saving eluate. Save a small aliquot of eluate as well.

16. Concentrate proteins with prewet Vivaspin 20 and measure concentration on Nanodrop.

17. Combine Symplekin and Ssu72 in a 1:1.2 ratio in a 3500 MWCO dialysis bag.

    Note: Extinction coefficient information for the proteins can be derived from ProtParam on Expasy.

18. Dialyze Ssu72/Symplekin overnight in dialysis buffer while stirring, at 4 °C.

19. Wash HiLoad 16/600 Superdex 75 pg column with gel filtration buffer.

20. Slowly inject protein into NGC Chromatography system with syringe and run through size exclusion chromatography HiLoad 16/600 Superdex 75 pg column, equilibrated with Ssu72 buffer. Collect fractions consistent with a final product of approximately 60 kDa.

    Note: For the purification of Ssu72/Symplekin complex, the final product should be approximately 60 kDa. An SDS-PAGE can serve as quality control to ensure both proteins are present in the collected fractions after size exclusion chromatography, which should be at approximately 37 kDa and 23 kDa for Symplekin and Ssu72, respectively.

21. Concentrate protein and flash freeze in liquid nitrogen in aliquots. Record concentration of phosphatase.

5. Ssu72 optimization in cell lysate for unmasking pThr4

After selecting and characterizing the appropriate Ssu72 ortholog, we optimized its use for phosphatase treatment of cell lysate in preparation for ChIP-seq analysis. Drosophila and human Ssu72/Symplekin complex have similar results and below our protocol was conducted using Drosophila complex but human one should be similar. Both treatment time and enzyme dosage were adjusted to achieve maximal removal of Ser5 phosphorylation while enhancing the detection of pThr4. HEK293 cell lysates were incubated with Ssu72/Symplekin at 28 °C and analyzed by Western blot. pThr4 demasking was detectable as early as 15 min post-treatment (Fig. 4), and by 30 min, Ser5 phosphorylation was largely removed while the pThr4 signal started to wane.

Fig. 4. Ssu72/Symplekin treatment unmasks HEK293 pThr4.

Time course of HEK293 cell lysate treatment with Ssu72. HEK293 cell lysate was treated with Ssu72 or its protein buffer for different treatment time lengths, ranging from 15 to 60 min, then blotted for pThr4 (A) and pSer5 (B) with a loading control of alpha tubulin. (A) and (B) feature the same samples loaded onto different Western blots.

To optimize dosage, lysates were treated with varying concentrations of Ssu72/Symplekin. A two- to three-fold increase in detectable Thr4 phosphorylation was typically observed upon demasking. A final concentration of 80 μM Ssu72/Symplekin was sufficient to achieve effective demasking, although the exact concentration required varied depending on the activity of the enzyme batch.

6. ChIP-seq analysis with Ssu72/Symplekin phosphatase treatment

Once the treatment time and dosage were optimized, we integrated this phosphatase treatment step into the ChIP-seq workflow. To avoid inactivation of the enzymatic activity, the phosphatase was added after sonication —once lysates were extracted but before incubation with the anti-pThr4 antibody.

The detailed protocol for ChIP-phos-seq treatment to detect pThr4 genome location is shown below (Fig. 5).

Fig. 5. ChIP-phos-seq protocol.

Depiction of HEK293 undergoing ChIP-phos-seq protocol with Ssu72/Symplekin treatment and antibody pulldown. Created in BioRender. Stephenson, R. (2025). https://BioRender.com/079u4w8.

6.1. Before you begin

• HEK293 cells (human cells, under passage 20–25) should be grown to confluency (approximately 20 million cells for a pairwise set of samples) on BioLite 150 mm Dish, Cell Culture-treated Surface (Catalog: 130183, Thermo Scientific) in Dulbecco’s Modified Eagle’s Medium – high glucose (Catalog: D6429-mL, Sigma-Aldrich) with 1 % Penicillin-Streptomycin solution (Catalog: SV30010, Cytiva) and 10 % FBS Opti-Gold, Performance Enhanced (F0900–050, GenDEPOT) at 37 °C, 5 % CO₂ (Isotemp CO₂ Incubator, Fisher Scientific). Confluency and cell health can be monitored on a Leica DMIL microscope and with a Luna II Automated Cell Counter (see manufacturer’s protocol).

• Lysis Buffer 1: 50 mM 4-(2-hydroxethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH pH 7.5, 140 mM NaCl, 1 mM ethylenediamine tetra-acetic acid (EDTA), 10 % glycerol, 0.5 % NP-40, 0.25 % Triton X-100, 1x Halt Protease and Phosphatase Inhibitors (Catalog: 78442, Thermo Scientific)

• Lysis Buffer 2: 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM ethyleneglycol- bis(β-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA), 1x Halt Protease and Phosphatase Inhibitors.

• Lysis Buffer 3: 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 % sodium deoxycholate, 0.5 % N-lauroylsarcosine.

• Ssu72 Buffer: 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM BME

• Low Salt Wash Buffer: 50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 % sodium deoxycholate, 1 % Triton X-100.

• High Salt Wash Buffer: 50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1 % sodium deoxycholate, 1 % Triton X-100.

• Lithium Chloride Wash Buffer: 10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5 % NP-40, 0.5 % sodium deoxycholate.

• Tris-EDTA Buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

• Reverse Crosslinking Buffer: 0.1 M Sodium bicarbonate, 1 % SDS.

6.2. Key resources table

Reagent or resource	Source	Identifier
Antibodies
RNA Pol II CTD phospho Thr4 antibody (mAb) 6D7	Active Motif	61362
Rat IgG Isotype Control	Invitrogen	10700
Chemicals, Peptides, and Recombinant Peptides
Calbiochem OmniPur Bovine serum albumin (BSA), Fraction V (Heat Shock Isolation)	Sigma-Aldrich EMD Millipore Corp.	2960-500GM CAS: 9048-46-8
Dulbecco’s Phosphate Buffered Saline (Modified, without calcium chloride and magnesium chloride)	Sigma -Aldrich	D8537-mL
16% Formaldehyde Solution (w/v), Methanol free	Thermo Scientific	28908
RNase A, pancreatic High Purity Grade	VWR Life Science	0675-250MG CAS:9001-99-4
Proteinase K	Ambion	2542
Phenol/Chloroform/Isoamyl alcohol (25:24:1), stabilized, saturated with 100 mM Tris-EDTA to pH 8.0, for molecular biology, DNAse, RNAse, and Protease free, pH 7.8–8.2	Thermo Scientific	327111000 CAS: 136112-00-0
Glycogen, molecular biology grade	Thermo Scientific	R0561
Critical Commercial Assays
Qubit dsDNA HS Assay kit	Invitrogen	Q32851
Deposited DNA
HEK293 Ssu72 ChlP-seq Data	Gene Expression Omnibus	Accession Code: GSE262826
Experimental Models: Cell Lines
HEK293	ATCC	CRL-1573
J774A.1	ATCC	TIB-67
Software and Algorithms
Rstudio	Posit Software	RStudio 2025.05.0 + 496
UseGalaxy		Galaxy version 25.0.2.dev0

6.3. Materials and equipment

• 1 M glycine

• Ethanol

• Vortex

• User Enzyme (Catalog: M5505L, New England Biolabs)

• PCR grade nuclease-free water

• Luna II Automated Cell Counter (Catalog: L40001 Logos Biosystems) and slides (Catalog: L12001, Logos Biosystems)

• Roto-Mini Plus Rotator - R2024 (Catalog: 470313–912, Ward’s Science)

• Qubit 2.0 Fluorometer (Catalog: Q32866, Invitrogen)

• Invitrogen Protein G Dynabeads (Catalog: 10004D, Invitrogen)

• Cell Lifter (Catalog: 3008, Corning Incorporated)

• Qsonica Q800R3 Sonicator, DNA & Chromatin Shearing System, with 0.5 mL natural polypropylene, frosted cap PCR tubes (Catalog: PCR-05F, Globe Scientific)

• Magnetic strip stand (for both PCR tubes and 1.5 mL centrifuge tubes)

• Tris-HCl pH 7.5 and 8.0

• 5 M NaCl

• AMPure XP beads (Catalog: A63881, Beckman Coulter)

• ThermoMixer C (Catalog: 5382000023), with 1.5 mL SmartBlock (Catalog: 5360000038) and ThermoTop (Catalog: 5308000003, Eppendorf)

• Thermocycler

• NEBNext Ultra II DNA Library Prep Kit for Illumina (Catalog: E7645S, New England Biolabs)

• NEBNext Multiplex Oligos for Illumina Index Primers (Different 12-index sets include Catalog: E7335S, E7710S, and E7730S, New England Biolabs)

• Nuclease-free PCR tubes

• Qubit assay tubes (Catalog: Q32856, Invitrogen)

• Centrifuge 5425 R (Eppendorf)

• 1.5 mL microcentrifuge tubes

• −80 °C freezer

  Optional

• Safe-Lock Tubes 1.5 mL, natural (Catalog: 022363204, Eppendorf)

6.4. Cross-link cells, sonication and IP

Timing: 5–6 days

• 1Grow as many 150 mm dishes of 20 million HEK293 cells as required for an experiment, plus an extra dish to be counted on a cell counter.
• 2When the cells are ready, prepare the Protein G beads. Aliquot 50 μL of vortexed Protein G Dynabeads into as many tubes as desired and place on magnet strip to remove solution.

  Note: Remember that each Ssu72-treated sample will have a pairwise control when setting up the beads. An extra sample can be used as an IgG control.

• 3Resuspend Protein G beads in 1 mL of 5 mg/mL bovine serum albumin dissolved in Dulbecco’s Phosphate buffered saline (BSA/DPBS) and rotate for 5 min at 4 °C. Using a magnetic strip stand, remove the supernatant. Wash these beads in this manner for a total of three washes.
• 4Bind 10 μg pThr4 antibody to protein G beads for at least 4 h in 350 μL 5 mg/mL BSA/DPBS at 4 °C. Beads can bind overnight. An equivalent quantity of 10 μg anti-rat IgG antibody can be used as the control.
• 5HEK293 plates should come to room temperature, by leaving at room temperature for 5 min. Remove media and add room temperature 1 % formaldehyde in Dulbecco’s Phosphate Buffered Saline (DPBS) to plates. Incubate 8 min. Do not crosslink for too long. In parallel, treat J774A.1 mouse cells to the same procedure for an internal control.

  Note: Pipette slowly and to the side of the plate to avoid loss of cells throughout HEK293 treatment. This can minimize the sample yield and create inconsistencies.

• 6Add 1 M glycine to plate with formaldehyde to bring it to a final concentration of 0.125 M glycine and incubate for 5 min.
• 7Wash cells with 15 mL ice cold DPBS two times. Keep cells cold and avoid extended periods of HEK293 cell exposure to DPBS as cells may cease to adhere to plate, leading to cell loss.
• 8Using DPBS, gently scrape cells from dish with a cell lifter and pipette into collection tube. Keep cells on ice.
• 9Spin cells down at 2000 g for 3 min at 4 °C and remove supernatant.

  Pause Point: Cell pellet can be frozen at −20 °C for up to a week, or for longer at −80 °C. If pellet is frozen, the sonication protocol may require additional optimization.

• 10Resuspend pellet in lysis buffer 1, then rotate for 10 min on a rotator at 4 °C.
• 11Spin for 5 min 2000 g, at 4 °C and remove supernatant.
• 12Resuspend pellet in lysis buffer 2, then rotate for 5 min on a rotator at 4 °C.
• 13Spin for 5 min 2000 g, 4 °C and remove supernatant.
• 14Add 300 μL of lysis buffer 3 and resuspend cell pellet.
• 15Sonicate cell pellets in lysis buffer 3 to shear DNA to a 200−500 base pair length for 15 min on-time, with a pulse of 30 s on, 30 s off, and an amplitude of 70 %. Water in which the samples are sonicated should be kept at 4 °C.

  Note: Different sonication machines may vary and optimization of sonication time may be necessary. DNA sizes can be assessed following DNA extraction on a 2 % agarose gel.

• 16During sonication, remove Protein G Dynabeads from rotator and place on magnetic strip to remove DPBS/BSA solution. Antibodies should have bound to beads at this juncture.
• 17Resuspend Protein G beads in 1 mL of 5 mg/mL BSA/DPBS, rotating for 5 min at 4 °C. Remove supernatant on magnetic strip, leaving beads. Repeat for three washes and resuspend in 100 μL of 5 mg/mL BSA/DPBS. Step 17 may overlap with following steps but must be completed before step 24.
• 18Following sonication, samples should be split in half, with one sample set aside for treatment with wild-type active Ssu72. The other half will be treated with either mutant Ssu72 or Ssu72 buffer. Exclude mouse control cells from phosphatase treatment.
• 19Phosphatase is added in a 3:1 ratio with cell lysate, to a final concentration of 125 μM Ssu72/Symplekin (for example 150 μL cell lysate, 50 μL phosphatase or buffer) and incubated at 28 °C for 30 min at 650 rpm on a ThermoMixer.

  Note: A quality control check may be performed at this juncture if desired, by saving 15 μL of sample following treatment for western blotting to determine if demasking occurred.

• 20Add 30 μL of the J774A.1 mouse lysate to the HEK293 treated samples.
• 21Add 10 % Triton X-100, diluted in lysis buffer 3, to samples to a final concentration of 1 % Triton X-100.
• 22Spin for 10 min, 4 °C, at 21,130 g. Save the supernatant as ChIP IP samples.

  Note: Pellets are typically not very big.

• 23Remove 10 % of the sample to save as an input. Keep the input at 4 °C.
• 24Add 1x Halt protease and phosphatase inhibitors (final concentration), 1 % Triton X-100 (final concentration), 100 μL protein G beads (from step 17), and lysis buffer 3 to sample to a total volume of 1300 μL and rotate overnight at 4 °C.

Reverse crosslink beads

• 25Place beads on magnetic strip and remove supernatant.
• 26In 1 mL washes, wash beads with low salt buffer twice, removing supernatant on magnetic strip between each wash. For each wash, the beads should be resuspended through inversion and tapping prior to a 5 min rotation on the rotator at 4 °C.

  Note: Add the wash buffer while the beads are off the magnetic strip as it will facilitate resuspension. Keep beads on ice when not at 4 °C.

• 27Wash the beads as in step 26, once with high salt buffer.
• 28Wash beads as in step 26, once with lithium chloride buffer.
• 29Wash beads as in step 26, twice with Tris-EDTA buffer. Remove supernatant.
• 30Resuspend beads in 200 μL reverse crosslinking buffer. Add reverse crosslinking buffer to inputs, to a total volume of 200 μL.

  Note: Do not put reverse crosslinking buffer on ice. The beads can be at room temperature for step 30.

  Reverse crosslink beads for a minimum of 16 h at 65 °C, 750 rpm on a ThermoMixer

  Note: Samples may build up pressure at this step. To avoid evaporation caused by caps popping open, use locking-lid tubes or seal the tubes with parafilm.

DNA extraction

• 31Vortex to resuspend beads and place on a magnetic strip. Save the supernatant.
• 32Add 200 μL Tris-EDTA buffer to all samples, including inputs, which should be treated the same as samples.
• 33Add 8 μL 10 mg/mL RNase A, pancreatic dissolved in 10 mM Tris-HCl pH 7.5, 15 mM NaCl to the samples and inputs. RNase A should be kept at −20 °C and made within a month.
• 34Incubate for 30 min to 1 h at 37 °C. 1 h is preferable to degrade RNA.
• 35Add 4 μL 20 mg/mL Proteinase K dissolved in 50 % Tris-EDTA buffer, 50 % glycerol to the samples and inputs. Proteinase K should be stored at −20 °C.
• 36Incubate for 1–2 h at 55 °C. 2 h is preferable to degrade proteins.
• 37In a chemical fume hood, add 400 μL Phenol:Chloroform:Isoamyl Alcohol and vortex for 30 s. Remain in chemical fume hood for duration of usage of Phenol:Chloroform:Isoamyl Alcohol.
• 38Spin samples for 5 min at 21,130 g.
• 39Save the aqueous top layer in a separate tube. The bottom layer contains phenol chloroform and should be disposed of properly.
• 40Add 16 μL 5 M NaCl and vortex 10 s.
• 41Add 1 μL 20 mg/mL glycogen and vortex 10 s.
• 42Add 800 μL 100 % ethanol and mix well, leave samples and inputs at −80 °C for at least 30 min to overnight.
• 43Spin samples at 4 °C at 21,130 g for 30 min. Find visible precipitate and remove supernatant.

  Note: Precipitate may be very small; however, in the case that the precipitate cannot be seen, another 1 μL 20 mg/mL glycogen can be added and followed by incubation at −80 °C overnight again.

• 44Being careful not to dislodge pellet, wash with 500 μL of 70 % ethanol.
• 45Spin for 5 min at 4 °C at 21,130 g.
• 46Air dry pellet, then add 20–30 μL PCR grade nuclease-free water, preheated to 65 °C and resuspend the pellet by pipetting up and down.

  Note: Adding less water will decrease sample yield, but increase the DNA concentration.

• 47On ThermoMixer, heat samples to 65 °C for less than a minute. DNA samples can be stored at −80 °C.
• 48Using manufacturer’s protocol, determine DNA concentration using Qubit dsDNA HS Assay kit. 1–3 μL of each sample should be sufficient for testing.

  Note: An optional quantitative polymerase chain reaction (qPCR) experiment may be performed after DNA extraction as a quality control to confirm enrichment of IP samples when compared to inputs.

  Pause Point: The DNA samples are stable at −80 °C.

Library preparation

• 49In a nuclease-free PCR tube, dilute 2 ng of DNA in sterile 10 mM Tris-HCl pH 8.0–50 μL.
• 50Add 7 μL of NEBNext Ultra II End Prep Reaction Buffer and 3 μL NEBNext Ultra II End Prep Enzyme Mix to DNA and pipette to mix.
• 51On a thermocycler with a lid heated to at least 75 °C, incubate samples for 30 min at 20 °C and 30 min at 65 °C, holding at 4 °C.
• 52Dilute NEBNext Adaptor for Illumina 25-fold in sterile-filtered 10 mM Tris-HCl pH 8.0, then add 2.5 μL to the DNA mixture. Add 30 μL NEBNext Ultra II Ligation Master Mix, and 1 μL NEBNext Ligation Enhancer as well, pipetting to mix thoroughly.

  Note: The NEBNext Ultra II Ligation Master Mix should be premixed prior to its addition to the DNA mixture.

• 53On a thermocycler with an unheated lid, incubate samples for 15 min at 20 °C and holding at 4 °C. Allow AMPure XP beads to equilibrate to room temperature. Beads must have been at room temperature for at least 30 min for step 57.
• 54Add 3 μL USER enzyme and pipette to mix.
• 55On a thermocycler with a lid heated to at least 47 °C, incubate samples for 15 min at 37 °C and holding at 4 °C.
• 56After AMPure XP beads have equilibrated at room temperature for 30 min, vortex, then add 87 μL beads to the samples. Pipette up and down a minimum of 10 times to mix. Do not lose any beads from pipetting.
• 57Incubate at room temperature for at least 5 min before placing on magnetic strip stand, discarding the supernatant when clear.
• 58Wash beads twice with 200 μL 80 % fresh ethanol, allowing to incubate for 10 s for each wash.

  Note: Do not disturb the beads during this step. Do not allow beads to over dry, which can be visualized as the beads becoming a flat brown color and cracked.

• 59Elute DNA by resuspending beads with 17 μL 10 mM Tris-HCl pH 8.0, mixing well or vortexing. Incubate at least 2 min at room temperature.
• 60Save supernatant and add 15 μL of the sample combine with 25 μL NEBNext Ultra II Q5 Master Mix, 1 μL unique Index primer for each sample, 1 μL universal PCR primer, and 8 μL PCR grade nuclease-free water, pipetting up and down to mix.

  Note: Keep a record of which sample corresponds to each index primer.

• 61On a thermocycler, set the initial denaturation to 98 °C for 30 s, with 12 cycles of 98 °C for 10 s and 65 °C for 75 s, a final extension of 65 °C for 5 min, before holding at 4 °C.
• 62Combine samples with 27.5 μL AMPure beads to samples, pipetting to mix thoroughly before incubating for 10 min at room temperature.
• 63Place samples on a magnetic stand and remove and save supernatant. Note the quantity of supernatant.
• 64Add an equal amount of AMPure beads to the supernatant, mixing thoroughly and incubating at room temperature for 10 min.
• 65Place samples on magnetic strip stand and discard the supernatant when clear.
• 66Wash beads twice with 200 μL 80 % fresh ethanol, allowing to incubate for 10 s for each wash.
• 67Elute DNA by resuspending beads with 20 μL PCR grade nuclease-free water, mixing well or vortexing. Incubate at least 2 min at room temperature before placing on magnetic strip stand. Wait until supernatant is clear.
• 68Save supernatant and add 30 μL PCR grade nuclease-free water to a total volume of 50 μL.
• 69Add an equal amount of AMPure beads to the supernatant (50 μL), mixing thoroughly and incubating at room temperature for 10 min.
• 70Place samples on magnetic strip stand and discard the supernatant when clear.
• 71Wash beads twice with 200 μL 80 % fresh ethanol, allowing to incubate for 10 s for each wash.
• 72Elute DNA by resuspending beads with 20 μL PCR grade nuclease-free water, mixing well or vortexing. Incubate at least 2 min at room temperature before placing on magnetic strip stand and waiting until supernatant is clear.
• 73Save supernatant. Do not remove any beads. The supernatant is now prepared for sequencing. A second round of dsDNA HS Qubit Assay can be performed at this step to ascertain that DNA is present in sufficient concentrations.

Safety considerations and standards

Formaldehyde is flammable and acutely toxic if through exposure to the skin, orally, to organs, and through inhalation. Dispose of formaldehyde and broken glass waste appropriately, donning appropriate PPE. SDS is flammable, corrosive, and an irritant, and potentially damaging to eyes. Phenol:Chloroform:Isoamyl is toxic through inhalation and oral and skin exposure, damaging to the eyes, skin, reproductivity, and organs, and is potentially a mutagen and carcinogen. Only handle inside a chemical hood with appropriate PPE and dispose of all contaminated materials carefully and appropriately.

7. CTD Thr4 phosphorylation profile in human cells

Upon receipt of ChIP-seq results, sequencing results must be analyzed. Begin with quality assessment using FastQC, adapters sequences and low-quality reads (Phred score < 20) are trimmed using fastp. Paired-ends reads are aligned to the human genome (hg38) with Bowtie2. The resulting files are converted to bam files, then sorted, indexed, and filtered using samtools. Peak calling is performed with macs2. Bigwig files and matrix files are generated to visualize the genomic profiles and compare Ssu72-treated samples with controls by deepTools (Fig. 6). Peaks called from Ssu72 samples with their corresponding controls can be further analyzed for differential binding using the DiffBind pipeline. Additional analyses can include gene type classification, gene ontology annotation (molecular function, biological process, and cellular component), and gene length distribution.

Fig. 6. Ssu72 pThr4 ChIP-phos-seq genomic profile in HEK293.

Genomic profile of pThr4 ChIP-seq processed data normalized to inputs and plotted for protein-coding genes, ranging from 2 kilobases (kb) prior to the transcription start site (TSS) to 2 kb after the transcription end site (TES). The controls, Ssu72 samples, and inputs were reprocessed from gene expression omnibus (GEO) accession code GSE262826 using Ssu72 treatment (Moreno et al., 2024).

8. Limitation of phosphatase treatment method to unmask the flanking phosphorylation

While the application of Ssu72 phosphatase enables detection of Pol II CTD Thr4 phosphorylation in the genomic context, several improvements could enhance the efficiency and practicality of this method. We selected Ssu72 for its exceptional selectivity, as it exhibits no detectable activity against pThr4. However, Ssu72 exhibits relatively low catalytic activity, necessitating the use of large amounts of enzyme. To improve both activity and stability, we employed the Ssu72/Symplekin complex rather than the Ssu72 phosphatase domain alone (Luo et al., 2013; Xiang et al., 2010; Zhang et al., 2011). However, the method would benefit from a more active phosphatase that requires lower protein input while maintaining the same high specificity. Future efforts—either through screening of novel phosphatases or protein engineering to enhance Ssu72’s catalytic efficiency without compromising specificity—could significantly improve this approach.

Moreover, although Ssu72 efficiently removes Ser5 phosphorylation within a short incubation, its activity toward Ser2 is limited (Irani et al., 2019). Given that Ser2/Thr4 diphosphorylated heptads are reportedly rare (Schüller et al., 2016) and that kinases typically do not install Ser2 and Thr4 modifications on adjacent residues within the same heptad (Hintermair et al., 2012), the limited Ser2 activity of Ssu72 is unlikely to significantly distort the observed pThr4 profile. Nevertheless, identifying or engineering a phosphatase capable of broadly removing interfering modifications while sparing the target phosphorylation site would further refine this method.

9. Conclusion

For researchers studying CTD modifications or working with multiply phosphorylated proteins, we strongly recommend incorporating this phosphatase-based unmasking strategy. It’s a simple addition to your existing ChIP workflow that can dramatically improve detection of otherwise hidden epitopes like pThr4. This approach not only resolves longstanding inconsistencies in CTD ChIP profiles for pThr4 but also opens the door to exploring important regulatory modifications that may have been overlooked due to technical limitations of epitope masking. With this additional step, researchers may uncover new insights into transcriptional regulation and protein signaling. Furthermore, phosphatases with distinct substrate specificities could be exploited to detect other phosphorylation sites masked by similar epitope interference.