A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Summary

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

Introduction

Skeletal muscle plays important roles in physiology and behavior. The multi-nucleated muscle fiber consists of myofibrils where actin and myosin form functional units called sarcomeres to generate contractile force. Skeletal muscle is also the largest metabolic organ in the body, accounting for >80% postprandial glucose intake and regulating insulin response and metabolic homeostasis^1,2. Muscle physiology and metabolism are closely regulated by the circadian clock, an intrinsic biological timer^3,4,5,6. For example, the skeletal muscle-specific deletion of Bmal1, one of the core circadian clock components, resulted in insulin resistance and diminished glucose uptake in skeletal muscle, and the animals were found to develop type 2 diabetes⁷. In addition, skeletal muscle is also increasingly being appreciated as an endocrine organ⁸, secreting myokines to regulate systemic metabolism and physiology. Mechanistic studies are required to fully understand these regulatory functions in skeletal muscle.

ChIP is a powerful approach to delineate promoter recruitment of DNA binding proteins. ChIP was initially developed to identify nucleosome organization on chromatin DNA^9,10. A variety of methods have since been reported to cross-link proteins and chromatin DNA using formaldehyde, dimethyl sulfate or ultraviolet irradiation (UV)^11,12. Formaldehyde cross-linking is the most commonly used, preserving chromatin structure and DNA-protein interactions^9,13,14. Cross-linked chromatin is shredded by sonication and immunoprecipitated with antibody against the particular DNA binding protein of interest^15,16. In recent years, ChIP-sequencing (ChIP-seq), a method combining ChIP with NGS, has been developed to interrogate genome-wide transcription factor binding¹⁷, and in some cases to monitor dynamic changes over a time course^18,19,20. For example, circadian ChIP-seq studies have revealed a highly orchestrated sequence of genomic binding of circadian clock components and histone markers, which drives temporally precise gene expression throughout the ~ 24 h circadian cycle¹⁸.

Most available ChIP protocols are designed for soft tissues (e.g. liver, brain, etc.), and very few have been published for hard tissues including skeletal muscle. It is technically challenging to homogenize fiber-rich skeletal muscle and isolate high-quality nuclei²¹, especially for ChIP experiments which require cross-linking. In a recent muscle ChIP study²², satellite cells were separated from myofibers, and nuclei were prepared from both cell types through a prolonged procedure involving tissue digestion. The entire process took approximately three hours to complete before formaldehyde cross-linking was performed on isolated nuclei. While this procedure avoided cross-linking muscle fiber, which makes muscle tissue even more refractory to efficient homogenization, and was able to produce high-quality nuclei, the significant time-lag from tissue collection to nuclei cross-linking incurs the risk of altered DNA-protein interaction. In contrast, most studies performed cross-linking immediately after experimental treatment or tissue collection in order to preserve the real-time DNA-protein binding¹². A second drawback of nuclei isolation before cross-linking is that it precludes time-sensitive applications such as circadian sample collection which typically occurs at 3 - 4 h intervals. Without cross-linking the nuclei, the isolation needs to proceed immediately after dissection, whereas cross-linked samples can be processed together after the entire time course is completed, thus ensuring greater experimental consistency.

Other protocols for nuclei isolation from uncrosslinked skeletal muscle have also been reported. Two studies described the use of gradient ultracentrifugation to separate nuclei from remaining myofibrils and cell debris^23,24. While sucrose or colloidal gradient ultracentrifugation is effective with uncrosslinked muscle tissues, our experiments revealed that after crosslinking, gradient ultracentrifugation failed to separate nuclei from cell debris on the gradient.

We therefore developed a procedure to isolate high-quality nuclei using cross-linked mouse skeletal muscle tissues. Rather than gradient ultracentrifugation, we devised a serial filtration method to effectively separate nuclei from debris. Following ultrasonication, the chromatin samples were successfully applied for ChIP studies which showed a circadian pattern of BMAL1 protein binding to target promoters. Our method can be broadly applicable to various mechanistic studies of muscle tissues.

Protocol

Animal care was performed under Institutional Animal Care and Use Committee (IACUC) guidelines, and the procedures were conducted according to an animal protocol approved by the University of Texas Health Science Center at Houston.

1. Nuclei Isolation from Cross-linked Skeletal Muscle

1. Weigh and mince hind limb skeletal muscle, isolated from approximately 20-week old C57BL/6 male mice, in ice-cold phosphate buffered saline (PBS) as described in detail previously²².
   NOTE: We usually obtain 1.0 - 1.5 g of skeletal muscle from one mouse.
   1. Place minced skeletal muscle tissue in a 50-mL conical tube containing 10 mL PBS on ice.
2. Centrifuge at 300 x g at 4 °C for 5 min.
3. Carefully remove PBS supernatant by aspiration.
4. Estimate pellet volume in each 50-mL tube by using reference tubes with 1, 2, 3 and 4 mL of water.
5. Add 7 volumes of ice-cold 1% formaldehyde in PBS and homogenize the samples on ice using a probe tissue homogenizer (see Table of Materials).
   NOTE: Optional: The homogenization procedure can be conducted in a cold room.
6. Cross-link the sample by incubating at room temperature for 10 min.
7. Quench the cross-linking reaction by the addition of 1 M glycine to a final concentration of 0.125 M and incubate for 5 min at room temperature.
8. Centrifuge the samples at 3,000 x g for 5 min at 4 °C. Remove the supernatant via aspiration.
9. Rinse with 10 mL of ice-cold base buffer (10 mM HEPES-KOH at pH 7.3, 10 mM EDTA, 10 mM KCl, 5 mM MgCl₂, 0.5 mM DTT) containing freshly added protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/mL leupeptin). Centrifuge at 3,000 g for 5 min at 4 °C and carefully remove the supernatant by aspiration.
10. Resuspend the pellet in 6 mL of lysis buffer (10 mM HEPES-KOH at pH 7.3, 10 mM EDTA, 10 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 1% Triton X-100) containing freshly added protease inhibitors (0.2 mM PMSF, and 10 µg/mL leupeptin).
11. Transfer the sample to a prechilled 15 mL Dounce homogenizer and incubate for 10 min on ice.Dounce homogenize each sample on ice with 15 slow strokes using loose pestle followed by 15 strokes with tight pestle to release the nuclei.
12. Filter the homogenate (6 mL) through cell strainer (pore size: 100 µm) and rinse with 4 mL of lysis buffer. Centrifuge the samples at 1,000 x g for 10 min at 4 °C. Resuspend into 5 mL of ice-cold base buffer and dounce 10 times with tight pestle to release nuclei.
    1. Remove an aliquot of 20 µL for DNA concentration measurement with a spectrophotometer (OD260/OD280) and microscopic observation with trypan blue if necessary (see below) (Figure 1A).
13. Filter the suspension through a cell strainer (Pore size: 70 µm), rinse the tube and filter again with 2 mL base buffer as above.
14. Repeat the filtration as in step 1.13 with cell strainers of gradually reduced pore size (40 µm, 30 µm, 20 µm and 10 µm).
    NOTE: In total, strainers of 6 pore sizes were used in steps 1.12-1.14.
15. Centrifuge at 1,000 g at 4 °C for 10 min. Remove supernatant and resuspend the pellet in 500 µL base buffer.
16. Measure DNA concentration as above.
    NOTE: Approximately 200 µg of nucleic DNA was obtained from combining both hind limbs from one animal. Optionally, save 20 µL for microscopic observation with trypan blue staining (stock concentration 0.4%, 1:5 dilution); nuclei will stain blue (Figure 1B).
17. Centrifuge at 1,000 g at 4 °C for 10 min, and discard the supernatant. Store pellet at -80 °C.

2. Sonication

1. Thaw samples in 500 µL of SDS lysis buffer and resuspend with gentle pipetting.
2. Transfer the nuclei DNA suspension to a glass vial on ice.
3. Run the sonication with focused bursts of ultrasonic acoustic energy from a dish-shaped transducer. See Table of Materials for equipment details. Use the following setting: cycles per burst: 200; intensity: 5; duty cycle: 20; temperature 4 - 6 °C; 30 s on/off.
   NOTE: An example for evaluating sonication efficacy is shown in Figure 2.
4. Transfer the sonicated chromatin to a 1.5 mL tube. Centrifuge at 12,000 x g for 15 min and transfer the supernatant to a new tube. Transfer 50 µL of sonicated samples (~ 7 - 10 µg/muscle sample) into a new 1.5 mL tube for reverse crosslinking overnight at 65 °C. Freeze the remaining samples at -80 °C.

3. Evaluation of Sonication and Quantitation

1. Add 1.0 µL of RNase A (500 U/mL RNase A: 20,000 U/mL RNase T1) to the saved 50 µL sonicated samples and incubate for 30 min at 37 °C.
2. Add 8 µL of protease K (10 mg/mL) and incubate 30 min at 55 °C.
3. Harvest the DNA by using a DNA extraction kit.
   1. Add 5 volumes of binding buffer, apply it to spin column, and centrifuge at 4,000 x g, 10 min.
   2. Apply the pass-through onto the same column and centrifuge again.
   3. Wash with washing buffer two times at 13,000 x g, 1 min.
   4. Centrifuge again at 13,000 x g, 1 min to dry the column.
   5. Add 50 µL of H₂O and centrifuge at 13,000 x g, 1 min to elute DNA.
   6. Apply the pass-through onto the same column and centrifuge again.
4. Quantify the amount of DNA with a spectrophotometer (OD260/OD280). Run samples on 0.8% gels to evaluate the size and amount (1 µg) of the sonicated products (Figure 2).
   NOTE: The average chromatin yield is approximately ~ 20%.

4. ChIP

1. Thaw the previously saved chromatin at -80 °C and aliquot the chromatin to ~ 100 - 120 µg per tube. Dilute 1:10 with radioimmunoprecipitation assay (RIPA) buffer (20mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1mM NaF, 1mM Na3VO4, 1mM PMSF, 1x Protease inhibitor cocktail (PIC), 0.1% Na-deoxycholate (w/v), 1% Triton X-100).
   1. If there are multiple samples in each group, combine equal amounts of chromatin DNA to the final amount of ~ 100 - 120 µg/sample. Save 10% as input.
2. Add 40 µL (50% v/v slurry in RIPA buffer) of BSA-pre-blocked (~ 2 h, 4 °C) Protein A/G agarose (non-Chicken antibody) or IgY beads (Chicken antibody) and incubate on a rotator for 3 h at 4 °C.
3. Centrifuge at 1,000 x g, 10 min and carefully transfer the supernatant to new tubes.
4. Add antibodies at 1 µg antibody per ~ 25 - 100 µg chromatin DNA.
5. Rotate gently at 4 °C overnight at approximately 15 rpm.
6. Add 10 µL of BSA-pre-blocked (~2 h, 4 °C) Protein A/G agarose (non-Chicken antibody) or IgY beads (Chicken antibody) mix and rotate in the cold room for 2 h.
7. Wash beads as follows. Wash with 1 mL of RIPA buffer for 3 min twice, wash with 1 mL of high salt buffer (20mM Tris-HCl (pH 7.4), 500mM NaCl, 2mM EDTA, 1mM PMSF, 1% Triton X-100) for 3 min twice, wash with 1 mL of LiCl buffer (20mM Tris-HCl (pH 7.4), 250mM LiCl, 2mM EDTA, 1mM PMSF, 1% Na-deoxycholate, 0.5% NP40) for 3 min twice.
   1. Wash with 1 mL of Tris-EDTA (TE) buffer (10mM Tris-HCl (pH 7.4), 1mM EDTA) for 3 min once. Then elute DNA with elution buffer (20 mM Tris-HCl, 5 mM EDTA and 0.5% SDS).
8. Centrifuge at 1,000 g, 1 min and carefully remove the supernatant.
9. Resuspend the bead with 50 µL of elution buffer.
10. Incubate for 10 min at 65 °C.
11. Centrifuge at 12,000 g for 5 min.
12. Transfer the supernatant to a new tube, add another 50 µL and centrifuge once again at 12,000 g for 5 min. The final elution volume will be 100 µL.
13. Reverse crosslinking and DNA elution.
    1. Incubate the eluted chromatin overnight at 65 °C.
    2. Add 1.0 µL of RNase A and incubate for 30 min at 37 °C.
    3. Add 8 µL of protease K (10 mg/mL) and incubate 30 min at 55 °C.
    4. Harvest the DNA by using a PCR cleanup kit with elution volume at 50 µL according to the manufacturer's protocol.
14. Analyze the profiles by Real-time qPCR as previously described^25,26.
    NOTE: The primer sequences are listed in Figure 3 Legend. Data are presented as mean ± SEM (Figure 3).

Results

Here we performed formaldehyde cross-linking immediately after tissue collection to preserve real-time DNA-protein interaction. However, we found that sucrose or colloidal gradient, commonly used for nuclei isolation^23,24, was not effective in separating nuclei from myofibrils (data not shown). The reason may be that cross-linking conferred similar gravity for nuclei and myofibrils. Therefore, we developed a serial filtration proc...

Discussion

Here we describe a robust method where cross-linked skeletal muscle tissues were used to isolate high-quality nuclei. Sequential filtration was carried out to effectively separate nuclei from debris, and ultrasonic acoustic energy from dish-shaped transducer sheared the chromatin for ChIP analysis. The results showed circadian time-specific binding of BMAL1 to target promoters.

ChIP can be employed to capture real-time protein occupancy on genomic DNA when cross-linking takes place. To take ad...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Formaldehyde solution	Sigma Aldrich	F8775
Glycine	Fisher Scientific	BP381-5
Trypan Blue solution (0.4%)	Fisher Scientific	15250061
RNase A	Sigma Aldrich	10109142001
Protease K	Sigma Aldrich	3115887001
Chicken Anti-BMAL1 antibody			Generated in chicken (Cocalico Biologicals) against antigen aa 318 - 579, and IgY was affinity purified using the same antigen.
Chicken IgY Precipitating Resin	GenScript	L00405
Equipment
KINEMATICA Polytron PT2100 Benchtop Homogenizer	Fisher Scientific	08-451-178
15 mL Dounce tissue grinder	Whearton	357544
Falcon Cell Strainers 100 µm	Fisher Scientific	08-771-19
Falcon Cell Strainers 70 µm	Fisher Scientific	08-771-1
Falcon Cell Strainers 40 µm	Fisher Scientific	08-771-2
pluriStrainer 30 µm	PluriSelect	43-50030-03
pluriStrainer 20 µm	PluriSelect	43-50020-03
pluriStrainer 10 µm	PluriSelect	43-50010-03
Covaris S2 Focused-ultrasonicator	Covaris	Model S2
Labquake	Thermo Scientific	C415110
CCD Microscope Camera	Leica Microsystems	DFC3000 G
Reagent Kit
DNA extraction kit	Thermo Scientific	K0691
Buffers
All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich