Cell, Vol. 13, 213-214,
February
1978,
Copyright
8 1976 by MIT
Correspondence
Transcription of Chromatin in Isolated Nuclei by Exogenous RNA Polymerases In the March 1977 issue of Cell (70,405414), Sklar and Roeder report their studies on the “Transcription of Specific Genes in Isolated Nuclei by Exogenous RNA Polymerases.” The endogenous RNA polymerases in nuclei obtained from mouse plasmacytoma 460 cells actively synthesize RNA, including the 5s and 4.5s RNAs. Sklar and Roeder show convincingly that addition of exogenous purified RNA polymerases Ill, and III, from the same cell type stimulates the synthesis of these low molecular weight RNAs about 3-6 fold. The implication of their report, and indeed the interpretation of the title of their paper, is that the added RNA polymerases penetrate into the nuclei and initiate transcription of the particular RNAs species. While this might be correct for their particular nuclei, our experience in a different system suggests the alternative hypothesis that exogenous polymerases transcribe chromatin fragments released from nuclei. We have recently transcribed chick embryonic erythroblast and myoblast nuclei with E. coli RNA polymerase (Table 1). We believe that the large stimulation of transcription of low molecular weight RNAs which we observe is due largely or entirely to the action of the added enzyme on chromatin fragments which have leaked out of the nuclei during the incubation period. A different enzyme and different nuclei and probably different experimental conditions are involved in our experiments. We wonder, however, whether Sklar and Roeder have considered the possibility that their stimulation of low molecular weight RNA synthesis by exogenous RNA polymerases might not also be due to the action of these enzymes on chromatin fragments leaking out of the nuclei. We compared the total incorporation into RNA by the endogenous polymerases with and without added E. coli RNA polymerase under a variety of salt conditions (Table 1). The salt used to adjust the ionic strength of the incubation mixture was a mixture of potassium chloride and sodium chloride in a ratio of 3:l. The ionic composition of nuclei has been reported to contain approximately this ratio of the two monovalent ions (Siebert and Larrgendorf, Naturwissenschaften 57, 119-124, 1970). Erythroblast nuclei at low salt concentrations gave a high rate of incorporation with E. coli RNA polymerase which, as expected, was insensitive to cy-amanitin. Transcription by the enzyme at high salt was much lower and was mostly accounted for by the endogenous high salt transcription. Preincubation of the erythroblast nuclei in 0.1 M salt at 37°C for 15 minutes in the usual incubation mixture containing the usual amount of unlabeled UTP, but no radioactive tracer, produced a supernatant fraction which was highly active when E. coli RNA
polymerase was added and incubation was continued for another 30 minutes with labeled UTP. The nuclei themselves were inactive when incubated with the polymerase. We conclude that E. coli polymerase in this system acts on something, presumably chromatin fragments, released from the nuclei. When myoblast-fibroblast nuclei were incubated with E. coli RNA polymerase at 0.1 M salt, a high rate of synthesis was obtained. The product consisted almost entirely ‘of material of about 5S, which end-group analysis proved to be approximately 100 nucleotides long. Preincubation of the myoblast-fibroblast nuclei under the usual incubation conditions in 0.1 M salt for 15 minutes at 37°C gave rise to a supernatant fraction which was active in transcription with added E. coli RNA polymerase during 30 minutes (Table 1). In comparison, the nuclei were much less active, although not as inactive as the corresponding erythroblast Table 1. Endogenous coli Polymerase
Transcription
of Nuclei
Stimulated
by E.
+ Polymerase (pmoles LIMP Per Pi! DNA?
(pmoles UMP per pg DNA”)
5.0 (4.4)C 2.7 (1 .I) NTe
0.2 (O.l)C 1.5 (0.1) 1 .I (0.2)
13.6 0
NT NT
Untreated 0.1 M (K. Na)CI 0.7 M (K, Na)CI 0.2 M (NH&SO,
18.1 NT NT
0.2 2.0 (0.1) 2.0
Preincubated,” Supernatant Nuclei
10.1 8.2
NT NT
Erythroblast 5 Day Chick
Nuclei (from Embryos)
Untreated 0.1 M (K, Na)Clb 0.7 M (K, Na)CI 0.2 M (NH&SO, Preincubated,dO.l Supernatant Nuclei
M (K, Na)CI
MyoblastlFibroblast (from 11 Day Chick Embryos)
0.1 M (K, Na)CI
a Units are pmoles of 3H-UMP per pg “chromatin DNA’ incorporated in 30 min at 3PC (Pomerai, Chesterton and Butterworth, Eur. J. Biochem. 48, 461-471, 1974). E. coli RNA polymerase is at 32 units per ml. Erythroblast nuclei were prepared in 0.25 M sucrose 50 mM Tris, 25 mM KCI, 5 mM MgCh (pH 7.0), to which 0.2% Triton X-100 and IO-’ M PMSF were added. Nuclei were washed in the same buffer without Triton and resuspended in the same buffer without Triton or PMSF. Mixed myoblast/ fibroblast cells were obtained by trypsinization of thigh tissue from 11 day chick embryos. Nuclei were obtained by the above procedure. b (K. Na)CI is a 3:l mixture of KCI and NaCl. ’ Numbers in parentheses are incorporation in the presence of 0.4 pglml of a-amanitin. d Preincubated in transcription mixture without “H-UTP tracer, but with cold UTP. for 15 min at 3PC. * Not tested.
cell 214
nuclei. Myoblast-fibroblast nuclei prelabeled with 14C-thymidine lost almost 1% of their TCA-precipitable radioactivity into the supernatant in the usual incubation mix without incubation. An additional 1.8% of total radioactive precipitable DNA was released during preincubation at 37°C for 15 minutes. In our system at least, the pronounced stimulation of synthesis of low molecular weight RNA by the addition of E. coli RNA polymerase to nuclei might be largely or entirely accounted for by action of the enzyme on released chromatin, particularly in low salt. The possibility of obtaining mostly external transcription with exogenously added polymerases is a real one and must be considered in experiments involving these enzymes. Helen K. Hagopian and Vernon M. Ingram Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139
In a previous report (Sklar and Roeder, Cell 70, 405-414, 1977), we demonstrated greatly increased rates of synthesis of 5s RNA and 4.5s RNAs when isolated mouse plasmacytoma nuclear templates were incubated with purified class III RNA polymerases. In the accompanying letter, Hagopian and Ingram report that an exogenous bacterial RNA polymerase stimulates total RNA synthesis when incubated with isolated erythroblast or myoblast nuclei. They further speculate that the increased level of RNA synthesis results from the transcription of chromatin released from nuclei during the incubation. As a result of these findings, they suggest that our earlier results could reflect transcription of released chromatin by exogenous RNA polymerase III rather than the transcription of intranuclear genes as was implied in the title of our manuscript. Although the point raised by Hagopian and Ingram is indeed a valid and important one, there are several reasons why their results and conclusions might not be directly comparable to our own. First, they examined the synthesis of undefined RNAs, whereas we analyzed the transcription of specific genes known to be active in vivo. Second, the nature of the presumed template for the E. coli RNA polymerase was not established in the erythroblast-myoblast studies. It is relevant to note that the bacterial RNA polymerase may exhibit aberrant reactions in vitro, including the use of RNAs as primers (Zazloff and Felsenfeld, Biochemistry, in press). In our studies, the synthesis of the low molecular weight RNAs has been shown to be sensitive to DNAase and to require all four ribonu-
cleoside triphosphates (unpublished observations). Third, when incubated with isolated nuclei, E. coli RNA polymerase stimulates RNA synthesis much more extensively than do eucaryotic RNA polymerases. Moreover, the E. coli enzyme transcripts are very heterogeneous in size (Jaehning and Roeder, J. Biol. Chem. 252, 8752-8781,1977) and appear to result from random transcription events. Although the question raised has not been addressed directly by us, it seems most probable that the templates for 5s and 4.5s synthesis by class III RNA polymerases are intranuclear or nucleus-associated. This hypothesis is based in part on analogous studies of the transcription of specific genes (those encoding cellular 5s and 4.5s and adenovirus 5.5s RNAs) by purified class III RNA polymerases in another nuclear system (Jaehning and Roeder, J. Biol. Chem. 252, 8752-8781, 1977). In this system, control nuclei and nuclei preincubated under synthesis conditions and recovered by centrifugation were equally active as templates for specific gene transcription by the exogenous class III RNA polymerases (Jaehning and Roeder, unpublished observations). It is, of course, highly probable that some transcription of released DNA or chromatin fragments by the class III polymerases occurs in these nuclear systems. If the fragments result from a random degradation of intranuclear chromatin, however, then a random array of transcripts would be expected. Such transcripts need not necessarily complicate the analysis of discrete sized transcripts of specific genes. Finally, it should be mentioned that the major objective of our isolated nuclei transcription studies has been to develop reconstructed systems which exhibit faithful gene transcription by exogenous eucaryotic RNA polymerases. Whether or not the actual templates are intranuclear or extranuclear (soluble) in no way detracts from the significance of these studies or the general applicability of this approach. In fact, since the next major goal of these studies is to simplify the nuclear template systems (first to total chromatin and then to chromatin subfractions), it would be extremely fortunate if the active templates in these systems were in a soluble extranuclear form. R. G. Roeder, J. A. Jaehning and V. E. F. Sklar Division of Biology and Biomedical Sciences Department of Biological Chemistry Washington University St. Louis, Missouri 63110
February
1978,
Copyright
8 1976 by MIT
Correspondence
Transcription of Chromatin in Isolated Nuclei by Exogenous RNA Polymerases In the March 1977 issue of Cell (70,405414), Sklar and Roeder report their studies on the “Transcription of Specific Genes in Isolated Nuclei by Exogenous RNA Polymerases.” The endogenous RNA polymerases in nuclei obtained from mouse plasmacytoma 460 cells actively synthesize RNA, including the 5s and 4.5s RNAs. Sklar and Roeder show convincingly that addition of exogenous purified RNA polymerases Ill, and III, from the same cell type stimulates the synthesis of these low molecular weight RNAs about 3-6 fold. The implication of their report, and indeed the interpretation of the title of their paper, is that the added RNA polymerases penetrate into the nuclei and initiate transcription of the particular RNAs species. While this might be correct for their particular nuclei, our experience in a different system suggests the alternative hypothesis that exogenous polymerases transcribe chromatin fragments released from nuclei. We have recently transcribed chick embryonic erythroblast and myoblast nuclei with E. coli RNA polymerase (Table 1). We believe that the large stimulation of transcription of low molecular weight RNAs which we observe is due largely or entirely to the action of the added enzyme on chromatin fragments which have leaked out of the nuclei during the incubation period. A different enzyme and different nuclei and probably different experimental conditions are involved in our experiments. We wonder, however, whether Sklar and Roeder have considered the possibility that their stimulation of low molecular weight RNA synthesis by exogenous RNA polymerases might not also be due to the action of these enzymes on chromatin fragments leaking out of the nuclei. We compared the total incorporation into RNA by the endogenous polymerases with and without added E. coli RNA polymerase under a variety of salt conditions (Table 1). The salt used to adjust the ionic strength of the incubation mixture was a mixture of potassium chloride and sodium chloride in a ratio of 3:l. The ionic composition of nuclei has been reported to contain approximately this ratio of the two monovalent ions (Siebert and Larrgendorf, Naturwissenschaften 57, 119-124, 1970). Erythroblast nuclei at low salt concentrations gave a high rate of incorporation with E. coli RNA polymerase which, as expected, was insensitive to cy-amanitin. Transcription by the enzyme at high salt was much lower and was mostly accounted for by the endogenous high salt transcription. Preincubation of the erythroblast nuclei in 0.1 M salt at 37°C for 15 minutes in the usual incubation mixture containing the usual amount of unlabeled UTP, but no radioactive tracer, produced a supernatant fraction which was highly active when E. coli RNA
polymerase was added and incubation was continued for another 30 minutes with labeled UTP. The nuclei themselves were inactive when incubated with the polymerase. We conclude that E. coli polymerase in this system acts on something, presumably chromatin fragments, released from the nuclei. When myoblast-fibroblast nuclei were incubated with E. coli RNA polymerase at 0.1 M salt, a high rate of synthesis was obtained. The product consisted almost entirely ‘of material of about 5S, which end-group analysis proved to be approximately 100 nucleotides long. Preincubation of the myoblast-fibroblast nuclei under the usual incubation conditions in 0.1 M salt for 15 minutes at 37°C gave rise to a supernatant fraction which was active in transcription with added E. coli RNA polymerase during 30 minutes (Table 1). In comparison, the nuclei were much less active, although not as inactive as the corresponding erythroblast Table 1. Endogenous coli Polymerase
Transcription
of Nuclei
Stimulated
by E.
+ Polymerase (pmoles LIMP Per Pi! DNA?
(pmoles UMP per pg DNA”)
5.0 (4.4)C 2.7 (1 .I) NTe
0.2 (O.l)C 1.5 (0.1) 1 .I (0.2)
13.6 0
NT NT
Untreated 0.1 M (K. Na)CI 0.7 M (K, Na)CI 0.2 M (NH&SO,
18.1 NT NT
0.2 2.0 (0.1) 2.0
Preincubated,” Supernatant Nuclei
10.1 8.2
NT NT
Erythroblast 5 Day Chick
Nuclei (from Embryos)
Untreated 0.1 M (K, Na)Clb 0.7 M (K, Na)CI 0.2 M (NH&SO, Preincubated,dO.l Supernatant Nuclei
M (K, Na)CI
MyoblastlFibroblast (from 11 Day Chick Embryos)
0.1 M (K, Na)CI
a Units are pmoles of 3H-UMP per pg “chromatin DNA’ incorporated in 30 min at 3PC (Pomerai, Chesterton and Butterworth, Eur. J. Biochem. 48, 461-471, 1974). E. coli RNA polymerase is at 32 units per ml. Erythroblast nuclei were prepared in 0.25 M sucrose 50 mM Tris, 25 mM KCI, 5 mM MgCh (pH 7.0), to which 0.2% Triton X-100 and IO-’ M PMSF were added. Nuclei were washed in the same buffer without Triton and resuspended in the same buffer without Triton or PMSF. Mixed myoblast/ fibroblast cells were obtained by trypsinization of thigh tissue from 11 day chick embryos. Nuclei were obtained by the above procedure. b (K. Na)CI is a 3:l mixture of KCI and NaCl. ’ Numbers in parentheses are incorporation in the presence of 0.4 pglml of a-amanitin. d Preincubated in transcription mixture without “H-UTP tracer, but with cold UTP. for 15 min at 3PC. * Not tested.
cell 214
nuclei. Myoblast-fibroblast nuclei prelabeled with 14C-thymidine lost almost 1% of their TCA-precipitable radioactivity into the supernatant in the usual incubation mix without incubation. An additional 1.8% of total radioactive precipitable DNA was released during preincubation at 37°C for 15 minutes. In our system at least, the pronounced stimulation of synthesis of low molecular weight RNA by the addition of E. coli RNA polymerase to nuclei might be largely or entirely accounted for by action of the enzyme on released chromatin, particularly in low salt. The possibility of obtaining mostly external transcription with exogenously added polymerases is a real one and must be considered in experiments involving these enzymes. Helen K. Hagopian and Vernon M. Ingram Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139
In a previous report (Sklar and Roeder, Cell 70, 405-414, 1977), we demonstrated greatly increased rates of synthesis of 5s RNA and 4.5s RNAs when isolated mouse plasmacytoma nuclear templates were incubated with purified class III RNA polymerases. In the accompanying letter, Hagopian and Ingram report that an exogenous bacterial RNA polymerase stimulates total RNA synthesis when incubated with isolated erythroblast or myoblast nuclei. They further speculate that the increased level of RNA synthesis results from the transcription of chromatin released from nuclei during the incubation. As a result of these findings, they suggest that our earlier results could reflect transcription of released chromatin by exogenous RNA polymerase III rather than the transcription of intranuclear genes as was implied in the title of our manuscript. Although the point raised by Hagopian and Ingram is indeed a valid and important one, there are several reasons why their results and conclusions might not be directly comparable to our own. First, they examined the synthesis of undefined RNAs, whereas we analyzed the transcription of specific genes known to be active in vivo. Second, the nature of the presumed template for the E. coli RNA polymerase was not established in the erythroblast-myoblast studies. It is relevant to note that the bacterial RNA polymerase may exhibit aberrant reactions in vitro, including the use of RNAs as primers (Zazloff and Felsenfeld, Biochemistry, in press). In our studies, the synthesis of the low molecular weight RNAs has been shown to be sensitive to DNAase and to require all four ribonu-
cleoside triphosphates (unpublished observations). Third, when incubated with isolated nuclei, E. coli RNA polymerase stimulates RNA synthesis much more extensively than do eucaryotic RNA polymerases. Moreover, the E. coli enzyme transcripts are very heterogeneous in size (Jaehning and Roeder, J. Biol. Chem. 252, 8752-8781,1977) and appear to result from random transcription events. Although the question raised has not been addressed directly by us, it seems most probable that the templates for 5s and 4.5s synthesis by class III RNA polymerases are intranuclear or nucleus-associated. This hypothesis is based in part on analogous studies of the transcription of specific genes (those encoding cellular 5s and 4.5s and adenovirus 5.5s RNAs) by purified class III RNA polymerases in another nuclear system (Jaehning and Roeder, J. Biol. Chem. 252, 8752-8781, 1977). In this system, control nuclei and nuclei preincubated under synthesis conditions and recovered by centrifugation were equally active as templates for specific gene transcription by the exogenous class III RNA polymerases (Jaehning and Roeder, unpublished observations). It is, of course, highly probable that some transcription of released DNA or chromatin fragments by the class III polymerases occurs in these nuclear systems. If the fragments result from a random degradation of intranuclear chromatin, however, then a random array of transcripts would be expected. Such transcripts need not necessarily complicate the analysis of discrete sized transcripts of specific genes. Finally, it should be mentioned that the major objective of our isolated nuclei transcription studies has been to develop reconstructed systems which exhibit faithful gene transcription by exogenous eucaryotic RNA polymerases. Whether or not the actual templates are intranuclear or extranuclear (soluble) in no way detracts from the significance of these studies or the general applicability of this approach. In fact, since the next major goal of these studies is to simplify the nuclear template systems (first to total chromatin and then to chromatin subfractions), it would be extremely fortunate if the active templates in these systems were in a soluble extranuclear form. R. G. Roeder, J. A. Jaehning and V. E. F. Sklar Division of Biology and Biomedical Sciences Department of Biological Chemistry Washington University St. Louis, Missouri 63110
