Plant Molecular Biology Reporter 21: 65-71, March 2003 9 2003 International Society for Plant Molecular Biology. Printed in Canada. Protocols
A Simple and Rapid Method for Nuclear Run-on Transcription Assays in Plants LING MENG* and PEGGY G. LEMAUX Department of Plant and Microbial Biology, University of California, Berkeley, 94720-3102 USA Abstract. Nuclear run-on transcription assays allow researchers to determine if a gene is regulated at the transcriptional level. Existing methods to perform this procedure involve the application of a large variety of biochemical reagents, numerous steps, and time-consuming procedures. Here we report a method to perform this assay on plant tissue that involves a minimal number of reagents and steps, is time-efficient, and produces clean definitive results within 24 h. Key words: method, nuclear, plants, run-on, transcription Introduction
Nuclear run-on transcription assays provide a snapshot of the levels of in vivo-initiated transcripts at the moment of nuclei isolation because initiation of new transcripts is negligible after isolation (Weber et al., 1977; Lohr and Ide, 1983). Moreover, because of the separation of isolated nuclei from the cytoplasm, nuclear RNA transcripts escape the posttranscriptional process that occurs in the cytoplasm. At present, the widely used nuclear run-on transcription assay is the only available method to determine if a gene is regulated at the transcriptional level. The run-on assay is generally performed by incubating intact isolated nuclei with saturating amounts of 3 unlabeled nucleotides and a limiting amount of a fourth labeled nucleotide. Nuclei are incubated for a period of time for transcription, which is followed by the isolation of labeled RNA transcripts and hybridization of RNA to DNA of interest blotted on a membrane. This assay is challenging because of difficulties in isolating and maintaining highly active nuclei and limited yields of labeled nuclear transcripts. Published protocols for the assays involve many steps to isolate and purify nuclei and the labeled RNA transcripts (e.g., density gradient centrifugation, DNase I treatment, proteinase K digestion, phenol-chloroform extraction, and column purification) (Chen and Pikaard, 1997; Gubler et al., 2002; Clark et al., 2002; Cox and Goldberg, 1988). Hybridization periods (Wang et al., 2002; Zhang et al., 2002) *Author for correspondence, e-mail:
[email protected]; fax: 510-642-7356; ph: 510-642-1347.
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and the time for the autoradiography necessary for visualizing the results (Korn et al., 2001) can be long, up to 4 and 7 d, respectively. Here we report a simple rapid method to perform nuclear run-on transcription assays on plant tissue. It involves a minimal number of biochemical reagents and time-efficient steps, leading to strong signals and clean results within 24 h using small amounts of plant tissue and radioisotope.
Materials and Methods Plant materials Plant tissue used was from T7-generation barley plants from a stably expressing transgenic subline, T 3 #30, which originated from line GP724B-4-9 (Wan and Lemaux, 1994) and from the partially silenced R 0 plants regenerated from a 3-month-old in vitro organogenic culture derived from T 7 immature embryos of subline T 3 #30 (Meng and Lemaux, unpublished data). GP724B-4-9 was produced by bombardment of cultivar, Golden Promise, with pAHC25 (Christensen and Quail, 1996), containing uidA and bar both driven by the maize ubiquitin-1 promoter plus the first untranslated exon and intron, referred as the ubi-1 promoter complex. Solutions 9 H buffer (modified Hamilton Buffer): 10 mM Tris-HC1 (pH 7.6), 1.14 M sucrose, 5 mM MgC12, 7 mM ~-mercaptoethnol (Hamiton et al., 1972) 9 Lysis buffer: H Buffer + 0.15% Triton X-100 9 Transcription buffer: 50 mM Tris-HC1 (pH 8), 5 mM MgC12, 5 mM KC1 9 Hybridization buffer: 0.25 M NaH2POa-Na2HPO 4 (pH 7.2), 7% SDS 9 Washing buffer # 1 : 2 0 mM NaH2PO4-Na2HPO 4 (pH 7.2), 5% SDS 9 Washing buffer # 2 : 2 0 mM NaH2POa-Na2HPO4 (pH 7.2), 1% SDS Isolating nuclei 9 Grind 1-5 g of leaves in liquid N 2 into a fine powder using a mortar and pestle, Resuspend powder in 8-40 mL of ice-cold H Buffer (-8 mL/g tissue). 9 Gently stir slurry into a low-viscosity liquid and filter through 2 layers of Miracloth pre-wet with H buffer on ice (Calbiochem, La Jolla, CA). 9 Centrifuge at 1000 g for 10 min at 4~ 9 Discard supernatant. Resuspend pellet in 5-25 mL of cold lysis buffer (-5 mLlg tissue). Note: pellet must be completely suspended in lysis buffer. Centrifuge at 1000 g for l0 min at 4~ Repeat once. 9 Resuspend resulting nuclei pellet in 1 mL transcription buffer and transfer to fresh 1.5-mL Eppendorf tube. Centrifuge at 1000 g for 2 min at 4~ Wash pellet in 1 mL transcription buffer once and suspend final pellet in 100-500 ~L transcription buffer (-100 p.L/g original tissue).
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Nuclear run-on transcription Transcription is initiated, using 92 gL nuclei in transcription buffer (containing ~1 X l 0 6 nuclei), by adding 5 gL 20X NTP mixture (containing 10 mM CTP, 10 mM GTP, 15 mM ATP; final working concentration: 0.5 mM CTP, 0.5 mM GTR and 0.75 mM ATP) and 3 gL [~_32p] UTP (10/.tCi/gL, 3000 Ci/mM) (ICN, Irvine, CA, USA). The transcription reaction is performed at 30~ for 40 min with gentle shaking. Isolating 3eP-labeled transcripts The following manipulations are performed at room temperature, except as noted. 9 Transcription is stopped by adding 1 mL TRIzoL reagent (GIBCO-BRL, Life Technologies, Rockviile, MD, USA). Invert the tube several times and centrifuge at 12,000 g for 1 min. 9 Transfer supernatant to a fresh 1.5-mL Eppendorf tube and add 200 gL of chloroform. Cap the tube securely and shake vigorously by hand for 15 s. Incubate for 2 min. Centrifuge at 12,000 g for 2 min. 9 Transfer supernatant (-600 I.tL) to an RNase-free 1.5-mL Eppendorf tube. Add 40 gg tRNA (Roche, Indianapolis, IN, USA) and 500 gL isopropanol. Incubate at -80~ for 20 min. 9 Centrifuge at 12,000 g for 10 min. Wash RNA pellet with 1 mL of 75% ethanol. Centrifuge at 12,000 g for 5 rain. 9 Discard supernatant. Air-dry RNA pellet and dissolve in 50 gL of 50% formamide in DEPC-treated water. Labeled transcripts are ready for RNA hybridization. RNA hybridization 9 Digest 1 or 2.5 ~tg DNA of pAHC 25 with restriction enzymes Bgl II and Pst I. The resulting restriction fragments, as well as 5 gg KDNA-Hind II1 (negative control) and 5 ~tg total DNA from barley var. Golden Promise (positive control), are separated in 0.8% agarose gel and blotted to Zeta-probe GT membrane (BIO-RAD, Hercules, CA, USA). DNA is fixed to the membrane by means of UV cross-linking (Auto crosslink) (STRATALINK, La Jolla, CA, USA). 9 Prehybridize membrane in 20 mL of hybridization buffer for 20 min at 60~ Denature labeled nuclear RNA transcripts in 50% formamide for 5 rain at 80~ chill on ice for 5 rain. Add denatured RNA probe to 3 mL of hybridization buffer and hybridize for 10 h at 60~ 9 Wash membrane at 60~ in washing buffer #1 for 20 rain, twice; in washing buffer #2 for 15 min, once. 9 Visualize bands by means of autoradiography on Kodak Biomax MS film with intensifying screens for approximately 3-6 h at -80~
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A pAHC25(9679bp) P(13) 8(954}
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Figure 1. Barley nuclear run-on transcription. (A) Map of pAHC25 and expected fragments from restriction enzyme digestion with Bgl II and Pst I. (B) Arrows on the right side indicate hybridization bands from the expected fragments at 2916 bp (containing entire nos 3' terminator and backbone of the plasmid), 2188 bp (entire coding region of uidA and nos 3' terminator), 1045 and 941 bp (entire ubi-I promoter complex, including core promoter and the first untranslated exon and intron, from uid A and bar, respectively), and 586 bp (entire coding region of bar, excluding 35 bp in 3'end). (i) RNA hybridization of the nuclear run-on transcripts from nontransgenic control, var. Golden Promise. (ii) RNA hybridization of the nuclear run-on transcripts from the partially expressing transgenic R0 plants regenerated from a 3-month-old in vitro organogenic culture of T 7 immature embryos of subline T 3 #30. (iii) RNA hybridization of the nuclear run-on transcripts from the stably expressing t r a n s g e n i c T 7 plants of subline T 3 #30. N: negative control, 5 I-tg LDNA restricted with Hind III. P: positive control, 5 gg barley genomic DNA from nontransgenic control, Golden Promise plants. 1 or 2 : 1 ~tg or 2.5 txg pAHC25 DNA restricted with Bgl II and Pst I. Molecular weight markers are indicated in kb on the left side.
Results W e e x a m i n e d t r a n s c r i p t i o n levels o f t h e t r a n s g e n e s b a r a n d u i d A ( g u s ) in s t a b l y e x p r e s s i n g T 7 b a r l e y p l a n t s a n d p a r t i a l l y e x p r e s s i n g R 0 p l a n t s (partial B a s t a r e s i s tance and undetectable GUS expression). Results demonstrate that transcription levels o f b o t h t r a n s g e n e s d r o p p e d d r a m a t i c a l l y in the p a r t i a l l y e x p r e s s i n g R 0 p l a n t s in c o m p a r i s o n to s t r o n g t r a n s c r i p t i o n o f b o t h t r a n s g e n e s in t h e s t a b l y
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expressing T 7 plant (Figure 1). This result suggests silencing of bar occurred at the transcriptional level (transcriptional gene silencing, TGS) in the partially silenced R 0 plants from tissue culture. The complete absence of GUS expression in the R 0 plants results from both transcriptional and posttranscriptional silencing. The results shown in Figure 1 also demonstrate that the transcription of the first untranslated exon and intron in the ubiquitin-I promoter complex is much stronger than that of the bar or uidA coding regions in the plants that partially express both transgenes. Transcription of the endogenous ubiquitin-1 gene contributed to the signal strength of this fragment because of its presence in nontransgenic Golden Promise. Rapid termination of transgene transcription might also result in a significant drop in hybridization signal in transgene coding regions. Imposing increasing stringency during washing by prolonging washing time (30 min X 2 in washing buffer #2) did not significantly change the intensity of the hybridization signals from the first untranslated exon and intron or the transgene coding regions (data not shown). This result suggests high homology between the first untranslated exon and intron of ubiquitin-1 of maize and barley. In addition to the barley plants partially expressing the bar and uidA transgenes, we also examined the transcription status of transgenes in plants with completely silenced transgenes. Results indicate that lack of transcription (transcriptional silencing) of the transgenes was solely responsible for the silencing (data not shown). Results of the nuclear run-on assay confirmed earlier results from Northern blot hybridization and RT-PCR assays. However, the nuclear run-on transcription assay provided a more direct, quantifiable, and accurate assessment of transcriptional activity. Discussion
Isolation of intact nuclei is the first step to successful completion of a nuclear run-on transcription assay. Animal nuclei can be released by directly lysing the cell with nonionic detergents, such as Nonidet P-40 or Triton X-100, which destroy cellular membranes but leave nuclear membranes intact. However, plant nuclei, because of the existence of plant cell walls, can be released only after disruption of the cell wall and cellular membrane. This is commonly achieved by grinding plant tissue in liquid N 2 (Cox and Goldberg, 1988) or chopping tissue with a razor blade (Thompson et al., 1997). Following the filtration of the homogenized tissue solution through Miracloth, which separates the subcellular particles from the tissue and cell fragments, the intact nucleus can be purified further by removing the chloroplast with nonionic detergents. Our results indicate that grinding plant tissue in liquid N 2 is an efficient means to fractionate plant cells and release intact nuclei. After cell disruption, keeping the homogenized tissue solution at a low temperature (0-4~ with a sufficient concentration of [3-mercaptoethanol is important to inhibit activities of endogenous cytoplasmic enzymes. Proper concentrations of the inorganic ion, Mg 2§ and sucrose are also essential to maintain morphological and functional integrity of isolated nuclei. Nuclear transcription in vitro from isolated nuclei is started in transcription buffer, a critical solution in this assay. First, transcription buffer has to provide the proper ionic strength to maintain morphological integrity of nuclei. The
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concentrations of K + and Mg 2+ both affect nuclear morphology. DNA leakage from the nucleus occurred when insufficient concentrations of K + and Mg 2+ were used. High ionic strength favored aggregation and disintegration of the nuclei (Strand et al., 1994). Second, the pH of the transcription buffer also affects transcription activities of nuclei. Published reports indicate that the highest transcription activity occurs at pH 8, and transcription elongation is inhibited by 50% when the pH is reduced from 7.9 to 6.7 (Breukelen et al., 2000). Third, data from transcription and RNA synthesis studies in bacteria and Ehrlich ascites cells suggest ATP and GTP concentrations may eventually determine rates of ribosomal RNA synthesis (Gaal et al., 1997; Grnmmt and Grummt, 1976). A decline in ATP concentration was observed during the first hour of anoxia (Rees et al., 1989). On the basis of these considerations, using saturating concentration of ATP and GTP, especially ATP, and aerating the transcription buffer are helpful in maintaining high levels of transcription in isolated nuclei. RNase inhibitors are commonly added to the transcription buffer in addition to other reagents, such as EDTA, glycerol, dithiothreitol, and spermidine. According to published results, glycerol is not necessary to maintain morphological integrity (Strand et al., 1994). Our results indicate that establishing the proper ionic strength (concentrations of K + and Mg 2+) in the transcription buffer is sufficient to maintain morphological and functional integrity of nuclei. If isolated nuclei are kept intact, it is not necessary to add RNase inhibitors, EDTA, or other reagents to inhibit RNAase or DNAse activities. Our use of a simple transcription buffer was sufficient to achieve high nuclear transcription levels. We used TRIzoL reagent to isolate and purify RNA transcripts simultaneously. After the TRIzoL reagent treatment, nuclear DNA enters the acid phenol-chloroform phase, and denatured proteins accumulate at the interface. Labeled RNA transcripts from the nuclear run-on transcription stay in the supernatant. The labeled RNA, following ethanol precipitation and washing, can be used directly for RNA hybridization because negligible amounts of unincorporated 32p-UTP co-precipitate during ethanol treatments. Compared to published methods involving multistep procedures, such as DNase I and proteinase digestion, phenol-chloroform extraction, and column purification, the method described here simplifies those procedures and increases the yield of labeled RNA because of minimal loss during purification. The method results in a simple, efficient, and reliable method for performing nuclear run-on transcription assays with plant tissue.
Acknowledgments The Torrey Mesa Research (TMRI) supported L. Meng, and the USDA Cooperative Extension Service through the University of California supported EG. Lemaux. The authors thank Dr Henrilk Albert for helpful suggestions on this paper.
References Breukelen FV, Maier R, and Hand S (2000) Depression of nuclear transcription and extension of mRNA half-life under anoxia in Artemiafranciscana embryos. J Exp Biol 203: 1123-1130.
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Chen ZJ and Pikaard CS (1997) Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance. Genes and Devel 11(16): 2124-2136. Christensen AH and Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 1-6. Clark RA, Li SL, Pearson DW, Leidal KG, Clark JR, Denning GM, Reddick R, Krause KH, and Valente AJ (2002) Regulation of calreticulin expression during induction of differentiation in human myeloid cells. Evidence for remodeling of the endoplasmic reticulum. J Biol Chem 277(35): 32369-32378. Cox KH and Goldberg RB (1988) Analysis of plant gene expression. In: Shaw CH (ed), Plant Molecular Biology: A Practical Approach, pp 1-35, IRL press, Oxford. Gaal T, Bartlett MS, Ross W, Turnbough CL, and Gourse RL (1997) Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria. Science 278: 2092-2097. Grummt I and Grummt F (1976) Control of nucleolar RNA synthesis by the intracellular pools of ATP and GTP. Cell 7: 447-453. Gubler F, Chandler PM, White RG, Llewellyn DJ, and Jacobsen JV (2002) Gibberellin signaling in barley aleurone cells. Control of SLNl and GAMYB expression. Plant Physiol 129: 191-200. Hamilton RH, Kunsch U, and Temperli A (1972) Simple rapid procedures for the isolation of tobacco leaf nuclei. Anal Biochem 49: 48-57. Korn T, K~ihlkamp T, Track C, Schatz 1, Baumgarten K, Gorboulev V, and Koepseli H (2000) The plasma membrane-associated protein RS1 decreases transcription of the transporter SGLTI in confluent LLC-PK1 cells. J Biol Chem 276: 45330-45340. Lohr D and Ide GI (1983) In vitro initiation and termination of ribosomal RNA transcription in isolated yeast nuclei. J Bio Chem 258: 4668-4671. Rees BB, Ropson I, and Hand SC (1989) Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest in Artemia embryos during anoxia. J Biol Chem 264: 15410-15417. Strand R, Boe R, and Flatmark T (1994) The choice of resuspension medium for isolated rat liver nuclei: effects on nuclear morphology and in vitro transcription. Mol Cell Biochem 139(2): 149-157. Thompson WF, Beven AF, and Shaw PJ (1997) Sites of rDNA transcription are widely dispersed through the nucleus in Pisum sativun and can comprise single genes. Plant J 12: 571-581. Wan Y and Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37-48. Wang H, Jiang X, Yang F, Chapman GB, Durante W, Sibinga NE, Schafer AI, and Cyclin A (2002) Transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition. Blood 99(3): 939-45. Weber J, Jelinek W, and Darnell JE (1977) The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: Mapping of nascent RNA molecules labeled in isolated nuclei. Cell 10: 6ll-616. Zhang MY, Wang X, Wang JT, Compagnone NA, Mellon SH, Olson JL, Tenenhouse HS, Miller WL, and Portale AA (2002) Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1 alpha-hydroxylase gene expression in the proximal renal tubule. Endocrinol 143(2): 587-595.
