Volume 2 number 5 May 1975
Nucleic Acids Research
Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides
C. T. Warnick and H. M. Lazarus
Department of Surgery, Veterans Administration Hospital and the University of Utah, Salt Lake City, Utah 84113, USA Received 2 April 1975 ABSTRACT The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin. INTRODUCTION It has been known for some time that polyanions stimulate RNA synthesis (1-6). It has been shown that heparin stimulates the proliferation of some cells in culture(8), and it has been suggested that heparin, or other sulfated polysaccharides may play some role in initiating RNA synthesis and cellular differentiation in the genome of sea urchin embryos(7). Recently, it has been shown that liver cells stimulated to proliferate by partial hepatectomy show an increase in nuclear acid polysaccharide over resting liver cell nuclei which contain mainly neutral polysaccharides(9). It has also been shown that certain polyamines induce the premature condensation of chromosomes in interphase nuclei of Sendai virus-mediated HeLa cells while heparin prevents this condensation(10). All of these facts indicate that polyanions, such as heparin, play some role in controlling events that occur in the nucleus of the cell. In this study, we investigated the effect of sulfated polysaccharides on the in vitro transcription of mammalian chromatin. MATERIALS AND METHODS Nuclei were isolated from the kidneys of white male mice (Charles River
735
Nucleic Acids Research Laboratories, Wilmington, Md.) by methods published previously(ll). Chromatin was prepared from isolated nuclei as described by Shaw and Huang(12). After swelling overnight in distilled water, the chromatin was sheared by homogenizing with ten strokes of a tight Dounce Homogenizer. RNA polymerase was prepared from F. coli K-12 (obtained from Grain Processing Corp., Muscatine, Iowa) as described by Burgess(13). The enzyme preparations had a specific activity of between 160-450 units/mg and had no activity without added template or all four nucleoside triphosphates. Transcription of the chromatin by the E. coli RNA polymerase was measured as follows. The reaction mixture contained, in a total volume of
0.25 ml, 0.04 M tris-HCl, (pH 7.9), 0.01 M MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.15 M KC1, 0.4 mM potassium phosphate, pH 7.5, 0.5 mg/ml bovine serum albumin, 0.24 mM each of ATP, CTP and GTP and 0.08 mm H-UTP (0.15 mCi per umole or 1.31 x 105 cpm/nmole) plus chromatin, template, and sulfated polysaccharide as indicated. In most instances the reaction mixture contained 1.5 - 2.5 tIg chromatin (as DNA) and 16 units of enzyme. These are conditions of excess enzyme; doubling the amount of enzyme does not increase the amount of RNA produced either in the stimulated or unstimulaced reactions. Unless otherwise stated, the enzyme is added last to initiate the reaction. After incubation at 370 for 10 min., duplicate 50 Lul samples were taken from 2 each reaction mixture and applied to separate paper squares (2 cm , Whatman #3 paper) and dropped directly into a solution of 5% trichloroacetic acid (TCA) containing 17, potassium pyrophosphate (10 ml of solution/paper square) (14). The paper squares were then washed with six changes of 5% TCA (5 ml/ paper square) followed by two washes with 95% ethanol, dried, and the RNA solubilized and counted in a liquid scintillation counter. Duplicate samples were assayed under the same conditions except also containing 48 ug/ml actinomycin-D. Any actinomycin D-insensitive incorporation of UTP into RNA was subtracted from the experimental values. In the unstimulated reaction, actinomycin D-insensitive incorporation was between 0.1 to 5 pmoles UTP incorporated/ug DNA and in PDS or heparin stimulated reactions, it was between 5 to 15 pmoles UTP incorporated/tLg DNA. Chromatin was deproteinized using guanidinium chloride(15). Using this method, approximately 90 percent of the protein (both histones and non-
histones) was removed. Protein was measured by the procedure of Lowry, et al.(16) using crystalline bovine pancreatic ribonuclease as the standard(ll). DNA was measured by the diphenylaLnine procedure of Burton(17) with calf thymus DNA as the
736
standard(ll).
Nucleic Acids Research Dithiothreitol (Cleland's reagent) was purchased from Calbiochem, LaJolla, California; crystallized bovine serum albumin was purchased from Pentex, Kankakee, Illinois; calf thymus DNA and crystalline bovine pancreatic ribonuclease were purchased from Worthington Biochemical, Freehold, New Jersey; ATP, GTP, CTP and UTP were purchased from P-L Biochemicals, Milwaukee, Wisconsin; 3H-UTP (26.9 Ci/mmole) was purchased from New England Nuclear, Boston, Massachusetts; sodium dextran sulfate 500 (polydextran sulfate, PDS) was purchased from Pharmacia, Uppsala, Sweden; and mucosal heparin, dextran and DEAE-dextran were purchased from Sigma Chemical Co., St. Louis, Missouri. Chondroitin sulfate B, heparitin sulfate and the other heparin samples were generous gifts of Dr. A. Linker, Salt Lake City, Utah. RESULTS
Figure 1 shows the stimulation of transcription of chromatin by PDS and heparin. At a concentration of 4 Lug/ml PDS stimulates transcription by more than 100 fold (from 12.1 pmoles UTP incorporated/ug DNA to 1273 pmoles UTP incorporated/ug DNA). At this concentration of PDS, the chromatin was transcribed at a level approaching that of dehistonized chromatin. Heparin at the
,1500-1200 0.
E
*,
PDS (Deproteinized Chromatin as template)
\
z
\.
\ S o~~~~~~~~~~~~
900 9
,-
I \
PDS (Chromatin as template)
%. 600o/
=300
\
Heparin (Chromatin as template) * 4.0
8.0 12.0 Sulfated Polysoccharide Concentration (ipg/ml)
20.0
Figure 1. Stimulation of transcription of DNA by sulfated polysaccharides. The conditions for the reaction are given under Materials and Methods. The reaction mixtures contained 1.5 ug mouse kidney chromatin (as DNA) or 1.79 ug deproteinized chromatin as template, 16 units of E. coli RNA polymerase, and the indicated amount of sulfated polysaccharide (either PDS or heparin). Under these conditions, the unstimulated reaction incorporated 12.1 pmoles UTP/ug DNA into acid insoluble products when chromatin was used as the template and 1457 pmoles UTP/ug DNA when deproteinized chromatin was the template.
737
Nucleic Acids Research concentration stimulates by about 40 fold (from 13.5 pmoles UTP incorporated/ug DNA to 436 pmoles UTP incorporated/ug DNA). In both cases the PDS and same
heparin stimulate maximally cause a
decrease in the
at
4 ug/ml and concentrations greater
amount of stimulation.
than this
Also, when deproteinized
occurs with either PDS chromatin is used as heparin. These compounds are, in fact, inhibitory under these conditions, probably because they prevent the polymerase from binding to the template
the template,
no
stimulation
or
(21,24). 280-
Deproteinized Chromotin
as
template
240-
0~~~\ 200f160-
8
.-Chromatin
as
template
120-
.80-
40
2
4
6
8
10
12
14
16
18
20
PDS CONCENTRATION (/Lg/mI)
Figure 2. PDS stimulation of transcription under conditions in which reinitiation is blocked. The reaction conditions are given under Materials and Methods except that the enzyme is allowed to bind to the template for 15 min in the presence of 3 of the 4 nucleoside triphosphates (CTP was omitted). The reaction is then initiated by adding (NH4) S04 (final concentration, 0.4 M) and CTP; ten minutes later the reaction was sampled as usual. The reaction mixture contained 1.4 ug mouse kidney chromatin or 1.79 ug dehistonized chromatin (as DNA), 16 units of E. coli RNA polymerase, and PDS an indicated. The PDS was added with the enzyme (15 min before addition of the (NH4) S04 and CTP). Under these conditions the unstimulated reaction incorporated 6.9 pmoles UTP/Lg DNA into acid insoluble produce. The stimulation by PDS was then studied under conditions in which reinitiation of the RNA polymerase was blocked in order to better measure enzyme binding sites. High salt, 0.4 M (NH4)2S04, has been shown(23) to inhibit binding of the RNA polymerase to the DNA template but not to interfere with polymerase molecules already bound to the template. The enzyme
738
Nucleic Acids Research allowed to bind by incubating for 15 min with 3 of the 4 nucleotides (CTP was omitted); the reaction was then started by adding CTP and (NH4)2S04 (final concentration 0.4M). Under these conditions, PDS at a concentration of 4 ug/ml stimulates transcription of mouse kidney chromatin to levels (Fig. approaching that of deproteinized chromatin. The stimulation is only about 30 fold, however, compared to the 100 fold stimulation seen when the enzyme 20 ug/ml) return the PDS is allowed to reinitiate. High levels of PDS ( stimulated reaction to unstimulated levels. Also, if the PDS is added after the (NH4)2S04, it has no effect either to stimulate or inhibit the reaction. PDS has no effect on the rate at which RNA is made (Figure 3). In these that the enzyme can experiments, reinitiation of the polymerase is blocked only finish the piece of RNA which it has started. With and without PDS, the reaction reaches completion 4-6 min after the 4th triphosphate has been added to start the reaction.
was
2)
so
WITH
X '
=120$
11%.
I ~~p
~Io
/
E
0
/
I
*r
oa80-i
E
PDS
~~~~~~/
Nucleic Acids Research
Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides
C. T. Warnick and H. M. Lazarus
Department of Surgery, Veterans Administration Hospital and the University of Utah, Salt Lake City, Utah 84113, USA Received 2 April 1975 ABSTRACT The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin. INTRODUCTION It has been known for some time that polyanions stimulate RNA synthesis (1-6). It has been shown that heparin stimulates the proliferation of some cells in culture(8), and it has been suggested that heparin, or other sulfated polysaccharides may play some role in initiating RNA synthesis and cellular differentiation in the genome of sea urchin embryos(7). Recently, it has been shown that liver cells stimulated to proliferate by partial hepatectomy show an increase in nuclear acid polysaccharide over resting liver cell nuclei which contain mainly neutral polysaccharides(9). It has also been shown that certain polyamines induce the premature condensation of chromosomes in interphase nuclei of Sendai virus-mediated HeLa cells while heparin prevents this condensation(10). All of these facts indicate that polyanions, such as heparin, play some role in controlling events that occur in the nucleus of the cell. In this study, we investigated the effect of sulfated polysaccharides on the in vitro transcription of mammalian chromatin. MATERIALS AND METHODS Nuclei were isolated from the kidneys of white male mice (Charles River
735
Nucleic Acids Research Laboratories, Wilmington, Md.) by methods published previously(ll). Chromatin was prepared from isolated nuclei as described by Shaw and Huang(12). After swelling overnight in distilled water, the chromatin was sheared by homogenizing with ten strokes of a tight Dounce Homogenizer. RNA polymerase was prepared from F. coli K-12 (obtained from Grain Processing Corp., Muscatine, Iowa) as described by Burgess(13). The enzyme preparations had a specific activity of between 160-450 units/mg and had no activity without added template or all four nucleoside triphosphates. Transcription of the chromatin by the E. coli RNA polymerase was measured as follows. The reaction mixture contained, in a total volume of
0.25 ml, 0.04 M tris-HCl, (pH 7.9), 0.01 M MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.15 M KC1, 0.4 mM potassium phosphate, pH 7.5, 0.5 mg/ml bovine serum albumin, 0.24 mM each of ATP, CTP and GTP and 0.08 mm H-UTP (0.15 mCi per umole or 1.31 x 105 cpm/nmole) plus chromatin, template, and sulfated polysaccharide as indicated. In most instances the reaction mixture contained 1.5 - 2.5 tIg chromatin (as DNA) and 16 units of enzyme. These are conditions of excess enzyme; doubling the amount of enzyme does not increase the amount of RNA produced either in the stimulated or unstimulaced reactions. Unless otherwise stated, the enzyme is added last to initiate the reaction. After incubation at 370 for 10 min., duplicate 50 Lul samples were taken from 2 each reaction mixture and applied to separate paper squares (2 cm , Whatman #3 paper) and dropped directly into a solution of 5% trichloroacetic acid (TCA) containing 17, potassium pyrophosphate (10 ml of solution/paper square) (14). The paper squares were then washed with six changes of 5% TCA (5 ml/ paper square) followed by two washes with 95% ethanol, dried, and the RNA solubilized and counted in a liquid scintillation counter. Duplicate samples were assayed under the same conditions except also containing 48 ug/ml actinomycin-D. Any actinomycin D-insensitive incorporation of UTP into RNA was subtracted from the experimental values. In the unstimulated reaction, actinomycin D-insensitive incorporation was between 0.1 to 5 pmoles UTP incorporated/ug DNA and in PDS or heparin stimulated reactions, it was between 5 to 15 pmoles UTP incorporated/tLg DNA. Chromatin was deproteinized using guanidinium chloride(15). Using this method, approximately 90 percent of the protein (both histones and non-
histones) was removed. Protein was measured by the procedure of Lowry, et al.(16) using crystalline bovine pancreatic ribonuclease as the standard(ll). DNA was measured by the diphenylaLnine procedure of Burton(17) with calf thymus DNA as the
736
standard(ll).
Nucleic Acids Research Dithiothreitol (Cleland's reagent) was purchased from Calbiochem, LaJolla, California; crystallized bovine serum albumin was purchased from Pentex, Kankakee, Illinois; calf thymus DNA and crystalline bovine pancreatic ribonuclease were purchased from Worthington Biochemical, Freehold, New Jersey; ATP, GTP, CTP and UTP were purchased from P-L Biochemicals, Milwaukee, Wisconsin; 3H-UTP (26.9 Ci/mmole) was purchased from New England Nuclear, Boston, Massachusetts; sodium dextran sulfate 500 (polydextran sulfate, PDS) was purchased from Pharmacia, Uppsala, Sweden; and mucosal heparin, dextran and DEAE-dextran were purchased from Sigma Chemical Co., St. Louis, Missouri. Chondroitin sulfate B, heparitin sulfate and the other heparin samples were generous gifts of Dr. A. Linker, Salt Lake City, Utah. RESULTS
Figure 1 shows the stimulation of transcription of chromatin by PDS and heparin. At a concentration of 4 Lug/ml PDS stimulates transcription by more than 100 fold (from 12.1 pmoles UTP incorporated/ug DNA to 1273 pmoles UTP incorporated/ug DNA). At this concentration of PDS, the chromatin was transcribed at a level approaching that of dehistonized chromatin. Heparin at the
,1500-1200 0.
E
*,
PDS (Deproteinized Chromatin as template)
\
z
\.
\ S o~~~~~~~~~~~~
900 9
,-
I \
PDS (Chromatin as template)
%. 600o/
=300
\
Heparin (Chromatin as template) * 4.0
8.0 12.0 Sulfated Polysoccharide Concentration (ipg/ml)
20.0
Figure 1. Stimulation of transcription of DNA by sulfated polysaccharides. The conditions for the reaction are given under Materials and Methods. The reaction mixtures contained 1.5 ug mouse kidney chromatin (as DNA) or 1.79 ug deproteinized chromatin as template, 16 units of E. coli RNA polymerase, and the indicated amount of sulfated polysaccharide (either PDS or heparin). Under these conditions, the unstimulated reaction incorporated 12.1 pmoles UTP/ug DNA into acid insoluble products when chromatin was used as the template and 1457 pmoles UTP/ug DNA when deproteinized chromatin was the template.
737
Nucleic Acids Research concentration stimulates by about 40 fold (from 13.5 pmoles UTP incorporated/ug DNA to 436 pmoles UTP incorporated/ug DNA). In both cases the PDS and same
heparin stimulate maximally cause a
decrease in the
at
4 ug/ml and concentrations greater
amount of stimulation.
than this
Also, when deproteinized
occurs with either PDS chromatin is used as heparin. These compounds are, in fact, inhibitory under these conditions, probably because they prevent the polymerase from binding to the template
the template,
no
stimulation
or
(21,24). 280-
Deproteinized Chromotin
as
template
240-
0~~~\ 200f160-
8
.-Chromatin
as
template
120-
.80-
40
2
4
6
8
10
12
14
16
18
20
PDS CONCENTRATION (/Lg/mI)
Figure 2. PDS stimulation of transcription under conditions in which reinitiation is blocked. The reaction conditions are given under Materials and Methods except that the enzyme is allowed to bind to the template for 15 min in the presence of 3 of the 4 nucleoside triphosphates (CTP was omitted). The reaction is then initiated by adding (NH4) S04 (final concentration, 0.4 M) and CTP; ten minutes later the reaction was sampled as usual. The reaction mixture contained 1.4 ug mouse kidney chromatin or 1.79 ug dehistonized chromatin (as DNA), 16 units of E. coli RNA polymerase, and PDS an indicated. The PDS was added with the enzyme (15 min before addition of the (NH4) S04 and CTP). Under these conditions the unstimulated reaction incorporated 6.9 pmoles UTP/Lg DNA into acid insoluble produce. The stimulation by PDS was then studied under conditions in which reinitiation of the RNA polymerase was blocked in order to better measure enzyme binding sites. High salt, 0.4 M (NH4)2S04, has been shown(23) to inhibit binding of the RNA polymerase to the DNA template but not to interfere with polymerase molecules already bound to the template. The enzyme
738
Nucleic Acids Research allowed to bind by incubating for 15 min with 3 of the 4 nucleotides (CTP was omitted); the reaction was then started by adding CTP and (NH4)2S04 (final concentration 0.4M). Under these conditions, PDS at a concentration of 4 ug/ml stimulates transcription of mouse kidney chromatin to levels (Fig. approaching that of deproteinized chromatin. The stimulation is only about 30 fold, however, compared to the 100 fold stimulation seen when the enzyme 20 ug/ml) return the PDS is allowed to reinitiate. High levels of PDS ( stimulated reaction to unstimulated levels. Also, if the PDS is added after the (NH4)2S04, it has no effect either to stimulate or inhibit the reaction. PDS has no effect on the rate at which RNA is made (Figure 3). In these that the enzyme can experiments, reinitiation of the polymerase is blocked only finish the piece of RNA which it has started. With and without PDS, the reaction reaches completion 4-6 min after the 4th triphosphate has been added to start the reaction.
was
2)
so
WITH
X '
=120$
11%.
I ~~p
~Io
/
E
0
/
I
*r
oa80-i
E
PDS
~~~~~~/
