Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients

ANALYTICAL

BIOCHEMISTRY

190,

193-197

(1990)

Isolation of Transcriptionally Active Nuclei from Striated Muscle Using Percoll Density Gradients Chang-Gyu Department

Received

Hahn

and Jonathan

of Physiology

October

Covault

and Neurobiology,

University

of Connecticut,

Storrs,

Connecticut

06269

10, 1989

A procedure is described utilizing Percoll density media for the separation of nuclei and myofibrils in homogenates of adult skeletal muscle. Using this method, transcriptionally active nuclei can be readily obtained in relatively high yield (-30%). In vitro RNA polymerase run-on-labeled RNAs isolated from these nuclei can be used in hybridization assays to study the transcriptional activities of specific genes. Percoll density gradient centrifugation should be useful for the isolation of nuclei from a variety of other tissues in which, like skeletal muscle, subcellular or tissue components cosediment with nuclei in conventional sucrose density centrifugation. Cl 1990 Academic Press. Inc.

Skeletal muscle has been used extensively as a model system for studies of cellular differentiation, tissue development, neuron-target cell-cell interactions, and adaptive changes to conditioning. Changes in the expression of specific genes stimulated by a variety of signals is thought to be a key event in each of these processes (l-6). Progress in understanding the factors involved in the regulation of such genes has been hampered by difficulties in the isolation of muscle nuclei imposed by the abundance of highly organized contractile elements in striated muscle. The isolation of nuclei is an important procedure for a variety of studies related to the regulation of gene expression, including nuclear run-on transcription, characterization of DNase I hypersensitive chromatin regions, and preparation of nuclear extracts for the identification of trans-acting regulatory factors. While several variations of sucrose density gradient techniques have been reported for use with skeletal muscle, our experience has been that these produce low yields of muscle nuclei that are variably contaminated from sample to sample by myofibrils. This problem appears to he a general one in this field as assays involving the isolation of nuclei from adult mus000%2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

Inc. reserved.

cle have generally been bypassed (9-11) or replaced by less definitive assays such as the quantification of premRNA intermediates rather than nascent transcripts as a measure of transcriptional activation (12). Here, we report a method to isolate nuclei using Percoll density media. By choosing conditions in which myofibrils float and nuclei sediment, we were able to obtain clean, transcriptionally active nuclei from adult skeletal muscle with a high yield. MATERIALS

AND

METHODS

Isolation of Nuclei White leghorn chickens (l-3 months old) were sacrificed by decapitation and tissues were quickly removed to ice-cold phosphate-buffered saline. Skeletal muscle was trimmed to remove bulk connective tissue and minced with scissors. Nuclei were prepared by a modification of the procedure described by Schibler et al. (13). One gram of trimmed muscle was homogenized in 25 ml of Buffer A (0.3 M sucrose, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM EGTA,’ 2 mM EDTA, 14 mM P-mercaptoethanol, 10 mg/ml BSA, 15 mM Hepes, pH 7.5) using a VirTishear (lo-mm shaft/micro fine generator, VirTis Co., Gardiner, NY) for 15 s each at settings 6,7, and 8. The homogenate was centrifuged in a Beckman JA-20 rotor for 5 min at 3000 rpm and the pellet was rehomogenized using the VirTishear in 20 ml of Buffer B (as Buffer A, but with 0.1 mM EGTA and 0.1 mM EDTA) for 15 s at a setting of 7. Triton X-100 was added to a final concentration of 0.5% (v/v) and the sample was hand homogenized using a Teflon-pestle Potter-Elvehjem tissue grinder. The resulting homogenate was filtered through lOO-pm-diameter nylon mesh

1 Abbreviations used: EGTA, ethylene ether)N,N’-tetraacetic acid, BSA, bovine thiothreitol; PMSF, phenylmethylsulfonyl acetic acid; SDS, sodium dodecyl sulfate.

glycol bin@-aminoethyl serum albumin; DTT, difluoride; TCA, trichloro-

193

194

HAHN

AND

to remove poorly disrupted tissue pieces. Alternatively, large tissue pieces were allowed to settle out at lg for 5 min. Percoll (Pharmacia, Piscataway, NJ) in Buffer B was added to the filtrate to a final concentration of 27% (v/v) and the mixture was centrifuged in a JA-20 rotor at 15,000 rpm (27,000g max) for 15 min. The nuclear layer near the bottom of the test tube was removed with a sialinized Pasteur pipette, diluted with 10 vol of Buffer B, layered on a l-ml pad of nuclei storage buffer (50% glycerol, 75 mM NaCl, 5 mM magnesium acetate, 0.85 mM DTT, 0.125 mM PMSF, 20 mM Tris-HCl, pH 7.9) in a sialinized 25-ml Corex test tube, and centrifuged at 1OOOg for 10 min. The nuclear pellet was resuspended in approximately 0.5 ml of the storage buffer pad and counted using a hemocytometer. Nuclei were repelleted in a 1.5-ml microcentrifuge tube and resuspended in a volume of storage buffer to give lo5 nuclei/pi. Nuclei were stored at -70°C until used. Although this procedure was optimized for skeletal muscle, it can be easily adapted to other samples. For nonfibrous tissues, the ratio of buffer to tissue can be halved and the tissue can be homogenized using a hand homogenizer rather than the VirTishear. There is generally no need to filter the resulting sample. For embryonic tissues and cultured cells, a single homogenization in Buffer B plus 0.2% Triton X-100 is typically sufficient. In some experiments the Ca/Mg-containing buffer (0.32 M sucrose, 3 mM CaCl, 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 10 mg/ml BSA, 10 mM TrisHCl, pH 8.0) of Marzluff and Huang (14) was used in place of the Ca/Mg-free Buffers A and B in the above procedure. When centrifugation through sucrose was compared with Percoll density gradient centrifugation, samples were layered on a lo-ml pad of 2 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl, pH 8.0, and centrifuged for 45 min at 12,000 rpm in a SW27 rotor. To measure DNA and protein levels, purified nuclei were diluted lo-fold in 1 M sodium phosphate, pH 6.8. DNA concentrations were assayed using a TKO-100 fluorimeter (Hoefer, San Francisco, CA) with linearized plasmid DNA as standard and protein concentrations calorimetrically using the Pierce BCA Reagent (Pierce Chemical, Rockford, IL) and BSA as standard. For microscopic examination, nuclei were stained with Hoecst 33258 (0.1 pg/ml) and were examined using either phase or uv optics.

In Vitro Transcription and Assay of Transcriptional Activity In vitro elongation of nascent transcripts was carried out in a 25-~1 reaction containing 300 mM (NH&SO,, 95 mM Tris-HCl, pH 7.9,50 mM NaCl, 6.6 mM MnCl,, 2 mM magnesium acetate, 1 mM DTT, 0.2 mM EDTA, 0.1 mM PMSF, 1 mM each of ATP, GTP, and CTP, 2.5 pM

COVAULT

a32P-UTP (400 Ci/mmol, Amersham), 10 mM creatine phosphate, 64 U/ml creatine phosphokinase, 4 U/ml NDP kinase, 200 U/ml recombinant placental RNase inhibitor, and 4 X 10’ nuclei/ml. the reaction was incubated at room temperature (23-26°C) for 20 min. Transcriptional activity was assayed by spotting 2-~1 aliquots of the reaction on Whatman GFC filters presoaked in 10% TCA, 1% sodium pyrophosphate. Filters were batch washed in 5% TCA and counted.

Filter Hybridization

of in Vitro-Labeled

RNA

CsCl density gradient centrifugation was used to isolate RNA from nuclear run-on transcription reactions (15). Reactions (250 ~1) were terminated by the addition of 12 vol of 7 M urea, 0.35 M NaCl, 2% sodium sarcosine, 10 mM Tris-HCl, pH 7.5, and the sample was sheared by repeated passage through successively finer needles (19 to 25 gauge). One gram of CsCl was added, and the RNA was pelleted through a 1.4-ml pad of 5.7 M CsCl, 100 mM EDTA, pH 7.0, by centrifugation in a SW60 rotor at 35,000 rpm for 24 h at 20°C. The pellet, dissolved in 10 mM EDTA and 0.2% SDS, was extracted with phenokchloroform (1:l) and ethanol precipitated. Hybridization filters were prepared by transferring 1 pg of linearized target plasmid DNAs (chicken glyceraldehyde-3-phosphate dehydrogenase cDNA, pGAD-28 (16), chicken fast myosin heavy chain gene fragment, pDCM3-6.0 (17), or control vector) to nitrocellulose filters (Millipore, Bedford, MA) using a blotting manifold (BRL, Gaithersburg, MD) and baking in vacua for 1 h at 80°C. Radiolabeled RNA was dissolved in hybridization buffer (0.2 M NaCl, 10 mM EDTA, pH 8,0.2% SDS, and 100 pg/ml yeast tRNA) at 10’ cpm/ml and a volume just sufficient to wet filters (-10 pl/cm2) was pipetted onto prehybridized filters. To prevent evaporation of buffer, filters were placed in sealed plastic bags containing mineral oil (18). After hybridization at 65“C overnight the filters were washed in 2X SSC (SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7) for 2 h with several changes of buffer and exposed to Kodak XAR-5 film at -70°C with intensifying screens. RESULTS

AND

DISCUSSION

The most widely used methods for the isolation of animal cell nuclei involve disruption of cellular membranes and sedimentation of nuclei through concentrated solutions of sucrose (13,14). For most tissues the nucleus is by far the largest structure in tissue homogenates and as such can be readily separated from other cellular components by centrifugation through dense sucrose. The presence of large numbers of contractile filaments in homogenates of skeletal muscle (Fig. 2A) severely limits the effectiveness of this approach. Protocols which produce acceptable yields of muscle nuclei (-lo-15%) involve the careful preparation of very

ISOLATION

OF

NUCLEI

USING

Density (g/ml) 1.04

20 -

60 -

1.06

1.08

1.10

Myofibrils

’

-

Nuclei I

80_

FIG. 1. Buoyant density separation of myofibrils and nuclei using Percoll. Color-coded density marker beads were used to measure the density gradient profile generated by a 15min centrifugation (bead buoyant densities were calibrated by weighing fractions from parallel samples centrifuged for 3 h to generate a much more extended gradient). The density distribution versus distance is plotted along with a diagram of the typical banding pattern of myofibrils (d - 1.05 g/ml) and nuclei(d - 1.10 g/ml).

shallow two-step sucrose gradients (e.g., 54.5% vs 56%) (19). To achieve optimal yields the concentrations of sucrose in each layer should be empirically adjusted depending on the specific muscle used (19). As an alternative to sedimentation through sucrose we tested the ability of isopycnic sedimentation in self-generating Percoll density gradients (20) to separate myofibrils and muscle nuclei. Such gradients had previously been used for the isolation of transcriptionally active wheat nuclei (21). Two buffer systems were compared in conjunction with Percoll density media, the CalMg-containing nuclear isolation buffers of Marzluff and Huang (14) and the Ca/Mg-free spermine and spermidine-containing buffers developed by Hewish and Burgoyne (22) and used more recently by Schibler’s group (13) for the isolation of transcriptionally active nuclei. As diagramed in Fig. 1, using the Hewish and Burgoyne buffer, distinct bands highly enriched for either myofibrils or myonuclei quickly form during a brief 15-min centrifugation. By choosing an initial sample density (-1.07 g/ml) midway between that reported for liver nuclei (23) and myofibrils (24), we were able to effect a rapid separation of nuclei and myofibrils before a significant Percoll density gradient formed (Fig. 1). In agreement with previous studies (23,24), the apparent density of the myofibril and nuclear layers was approximately 1.05 and 1.10 g/ml, respectively. The observed densities were much lower than would be found using other density techniques (e.g., sucrose) as the colloidal silica particles (-30 nm diameter) comprising Percoll are likely ex-

PERCOLL

DENSITY

MEDIA

195

eluded from the intermolecular spaces in myofibrils and nuclei. When the Ca/Mg-containing buffer was used, a nuclear layer similar to that shown in Fig. 1 was seen, but in contrast, the myofibril layer was less distinct. In addition to the major myofibril band near the top of the gradient, myofibrils were found throughout the gradient, including the nuclear layer. This heterogeneity may reflect a broader distribution of sarcomere lengths secondary to calcium-activated myofiber contraction during the initial homogenization in CalMg-containing buffer. Shortened sarcomere lengths, by reducing interfibril solvent volume, should cause an increase in the apparent density of myofibrils in Percoll as has previously been reported (24). Similar results were obtained if the same buffer but less Ca was used. Presumably the high levels of endogenous tissue Ca released upon homogenization in the absence of EGTA/EDTA can produce the same effect. Thus to obtain optimal purity of muscle nuclei in Percoll density gradients, Ca/ Mg-free spermine and spermidine containing homogenization solutions are preferred to those using CalMg to stabilize chromatin structure. Using the Ca/Mg-free Hewish and Burgoyne buffer, we routinely obtained a 30-40% final yield of purified

FIG. 2. Photomicrographs of muscle nuclei before and after Perco11 density purification. Homogenate of chicken leg muscle (A and B) and purified nuclei (C and D) stained with a fluorescent DNA binding dye and visualized with either phase contrast (A and C) or fluorescence (B and D) optics. A nuclei in the crude homogenate is indicated by the arrowhead in A. Purified nuclei contain both round and elongated nuclei, left and right hand arrowheads, respectively, in C. The bar in C is 25 pm.

196

HAHN

09 0.40

COVAULT

+ Musde

0.60 0.50

AND

1

--+--

Musde

-+-

Liver

TABLE

+ amanilin

Comparison of Yield and Transcriptional Activity of Nuclei Isolated Using Percoll versus SucroseDensity Media

Liver+ amanitin

5 3 030 3 5E 0.20

Tissue Liver Liver Muscle Muscle

Q 0.10

I

0.00

1

0

10

20

time (min) FIG. 3. RNA polymerase activity of isolated chicken muscle and liver nuclei. Purified nuclei elongate RNA transcripts for at least 20 min. in uitro. 32P-UMP incorporation in both liver and muscle nuclei is reduced -70% by 1 pg/ml cy-amanitin, a specific inhibitor of RNA polymerase II. The data shown are averages of quaduplicates from one experiment, similar results were obtained in two additional experiments.

muscle nuclei from adult chicken, rat, or mouse leg muscle (based on the number of nuclei present in the final homogenate prior to density gradient centrifugation). Typically -1-2 X lo7 nuclei are obtained per gram of wet muscle. Microscopically, purified nuclei were nearly free of myofibrillar contamination (Figs. 2C and 2D) and had a protein/DNA ratio of 2.4 f 0.2 (SE) which was similar to that found for liver nuclei (1.9 f 0.3) purified by the same procedure. Since myonuclei comprise only about 50-70% of the total nuclei in skeletal muscle, we were concerned that this procedure did not selectively isolate one class of nuclei. As can be seen in Figs. 2C and 2D, a variety of nuclear shapes were seen in the final preparation, including both round as well as long slender nuclei. The proportion of long slender nuclei (reminiscent of myonuclei in histologic sections) was approximately the same when samples were com-

^_I

GAPD

it 6.3

Vector My osin

FIG. 4. In uitro-labeled muscle nuclei RNAs hybridize to specific target DNAs. Nuclear run-on transcripts hybridized to cDNA probes for glyceraldehyde-3-phosphate dehydrogenase (GAPD) and mysoin heavy chain but not to vector DNA.

Density

media”

Percoll Sucrose Percoll Sucrose

Yield

(%) *

41 f 3 (n = 2) 41 31 f 6 (n = 3) 3 f 2 (n = 2)

Transcriptional activity (cpm/nuclei)’ 1.4 + 0.5 (n = 2) 1.2 + 0.1 (n = 2) 1.7 + 0.1 (n = 2) n.d.d

D Either 27% Percoll in the Ca/Mg-free Hewish and Burgoyne buffer or a sucrose step gradient (O-32/2 M) in the Ca/Mg-containing buffers of Marzluff and Huang as described under Materials and Methods. * Number of purified nuclei in storage buffer/number nuclei in tissue homogenate prior to centrifugation X 100. ’ Transcriptional activity in a standard 20-min assay. d Not determined.

pared before and after Percoll density gradient centrifugation. To demonstrate that the muscle nuclei isolated using this procedure maintained transcriptional activity, in vitro run-on transcription was carried out with these purified muscle nuclei. As shown in Fig. 3, muscle nuclei incorporate a similar amount of 32P-UTP as liver nuclei, both continued to elongate nascent transcripts for up to 20 min in vitro. In parallel with other systems (13,14), approximately 70% of this activity was inhibited by 1 pg/ml a-amanitin, a specific inhibitor of RNA polymerase II (25). In vitro-labeled RNA isolated from transcription reactions was of sufficient integrity to show a specific hybridization signal when incubated with filters containing relevant rDNA probes, including myosin heavy chain (Fig. 4). The ease with which nuclei can be isolated from muscle using Percoll suggests that it may be useful for the isolation of nuclei from a variety of fibrous tissues. In view of this we sought to demonstrate that the use of Percoll density media does not in itself have a deleterious effect on the yield or transcriptional activity of nuclei isolated from a standard, nonfibrous tissue such as liver. As detailed in Table 1, Percoll density gradient centrifugation produced an equivalent recovery of transcriptionally active nuclei from chicken liver as compared with the commonly used Marzluff and Huang (14) sucrose gradient ultracentrifugation procedure. Similar results were obtained for chicken brain and embryonic chick tissues. In summary, Percoll density gradient centrifugation provides a convenient method for the isolation of transcriptionally active nuclei applicable to a variety of tissues. Although not tested here, these nuclei should also be suitable for nuclease studies of chromatin structure and for the isolation of trans-acting factors. Using this

ISOLATION

OF

NUCLEI

USING

method it should now be possible to address more detailed questions about the regulation of specific genes in striated muscle.

PERCOLL

11. 12.

Tsay,

DENSITY

H. J., and Schmidt,

Shieh, B. H., Ballivet, 104,1337-1341.

13. Schibler,

This work was supported by NIH from the Alfred P. Sloan foundation

Grant NS25264 to J.C.

and a fellowship

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