Dec 5, 1989 - stream enhancer element (8, 24). The activity of the c-fos enhancer is rapidly induced by growth factors in serum and autorepressed by the Fos ...
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1990, p. 1126-1133 0270-7306/90/031126-08$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 10, No. 3
Serum Stimulation of the c-fos Enhancer Induces Reversible Changes in c-fos Chromatin Structure BRYANT VILLEPONTEAUl 2* Department of Biological Chemistry2 and Institute of Gerontology,' University of Michigan, Ann Arbor, Michigan 48109-2007 JUNLI FENG'
AND
Received 24 August 1989/Accepted
5
December 1989
Transcription of the proto-oncogene c-fos is known to be activated by growth factors in serum and subsequently repressed by the Fos protein. We show that generalized DNase I sensitivity of c-fos chromatin correlates closely with enhancer activity during induction, repression, and superinduction of the c-fos gene. Within 90 s of serum stimulation, proximal DNA sequences on both sides of the enhancer exhibit increased DNase I sensitivity. Within 5 min, elevated DNase I sensitivity spreads to chromatin at the distal 3' end of the c-fos gene. These results suggest that an open state of chromatin is propagated in both directions from the enhancer. The induced alterations in chromatin structure precede the increased transcriptional activity of the c-fos gene, suggesting that these changes in chromatin structure potentiate transcription.
Chromatin displays two types of DNase I sensitivity. The first, DNase I hypersensitivity, is localized to short, 100- to 400-base-pair stretches of chromatin (12), while the second, domain DNase I sensitivity (23, 30, 32), extends over 12 to 100 kilobases (kb) of chromatin and includes both nontranscribed and transcribed DNA sequences (1, 14, 15). An increase in domain DNase I sensitivity has been reported to be an early event in the activation of genes during development (23) and may be an obligatory step in the initiation of RNA transcription (29). Although DNA elements which serve to establish DNase I-sensitive domains have not been identified, one study (34) has suggested that transcriptional enhancers may induce the formation of active chromatin structure. Enhancers are cis-acting regulatory elements which can stimulate transcription from promoters thousands of base pairs away. One hypothesis about this effect at a distance states that enhancers trigger a change in chromatin structure that propagates along the chromatin fiber and stimulates promoter initiation (6, 20, 33). Support for this hypothesis comes from the finding that some enhancers can override chromosomalposition effects (3, 13) and thus may play a role in decondensing heterochromatin domains. To investigate the role of enhancers in altering chromatin structure, we have examined the DNase I sensitivity of the proto-oncogene c-fos (for a review, see reference 25). The transcription of c-fos is known to be regulated by an upstream enhancer element (8, 24). The activity of the c-fos enhancer is rapidly induced by growth factors in serum and autorepressed by the Fos protein (18, 19). Since the c-fos enhancer is both inducible and repressible, it is possible to test for the effects of the c-fos enhancer on the chromatin structure of the c-fos gene by monitoring changes in domain DNase I sensitivity as a function of enhancer activity. Using this approach, we found that c-fos chromatin becomes three- to fourfold more sensitive to DNase I within five min of stimulation with serum. We show also that subsequent autorepression led to a rapid reversal of domain DNase I sensitivity in c-fos chromatin. *
MATERIALS AND METHODS Recombinant DNA. The human c-fos gene [denoted pc-
fos(human)-1I (7) was obtained from Inder Verma as a 9-kb EcoRI insert in pBR322. A plasmid containing the argininosuccinate synthetase (AS)-coding region (2) was obtained from Svend Freytag. Cell culture, serum induction, and superinduction. HeLa cells were grown in monolayer cultures in a 1:1 mixture of Dulbecco modified Eagle medium and Ham Nutrient F-12 medium (no. 8900; Sigma Chemical Co.) supplemented with 10% bovine calf serum (Hazelton Laboratories). To prepare serum-starved cells, HeLa cells were grown to 70% confluency in this medium plus 10% serum, transferred to medium plus 0.5% serum for 20 h, and finally transferred to medium with 0% serum for 30 min. For normal induction experiments, medium plus 15% serum was added and the cells were incubated at 37°C for various times before being harvested. For superinduction experiments, serum-starved cells were transferred for 30 min to medium supplemented with 0% serum plus 100 ,uM anisomycin. Medium supplemented with 15% serum and 100 F.M anisomycin was then added for various incubation times. The cells were washed, scraped from the plate, and collected in ice-cold phosphatebuffered saline. Isolation of nuclei and DNase I digestions. The cells were pelleted and suspended in cold RSB (10 mM Tris hydrochloride, 10 mM NaCl, 3 mM MgCl2) containing 1 mM pchloromercuriphenylsulfonic acid as an inhibitor of nuclease and protease activity. After 5 min, 1% Nonidet P-40 was added, and the resulting lysate was centrifuged at 2,000 x g for 5 min to pellet the nuclei. The nuclei were resuspended in RSB at a concentration of 16 optical density units (at 260 nm) per ml (assayed by dilution into 2 M NaCl and 5 M urea) and stored on ice briefly. Each sample was split into five aliquots, each containing 5.7 A260 units of nuclei, and the aliquots were preincubated at 37°C for 5 min. The nuclei were digested for 3 min at 37°C with 0, 0.11, 0.33, 1, or 3 ,ug of DNase I (no. D 5025; Sigma). Reactions were stopped by adding sodium dodecyl sulfate and EDTA to final concentrations of 0.4% sodium dodecyl sulfate and 20 mM EDTA.
Corresponding author. 1126
ENHANCER-INDUCED CHANGES IN DNase I SENSITIVITY
VOL. 10, 1990
A Probe C _
E
Probe
A
-
Prote
_
Probe
D-
a
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-
Tr-,Primery
ntiscript
Bam Hi Eco RI
B 2.2 kb
0
1
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let
18
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FIG. 1. Transcription map and Northern blot analIysis of c-fos. (A) Map of human c-fos. Coding regions are shown ars solid boxes. The c-fos enhancer and promoter are depicted as open boxes. Fragments from plasmid pc-fos(human)-1 were suibcloned into pBR322 and used to prepare purified probes A to E (sI hown as thick bars above map) for Northern blot and indirect end-laibeling Southern blot experiments. (B) Northern blot analysis of c-j fos induction, Total RNA was purified from serum-starved HeLa with medium plus 15% serum for 0, 3, 5, 15, or 60 m The RNA was then denatured by glyoxal, fractionated by agarosse gel electrophoresis, transferred to nitrocellulose, and hybridi2zed to a-32p_ labeled probe A from the c-fos coding region. (C) Northern blot analysis of c-fos superinduction. Anisomycin-trez ated, serumstarved HeLa cells were incubated in medium with 15'% serum plus 100 ,uM anisomycin for 0, 15, 60, or 180 min. After t he cells were harvested, total RNA was purified and analyzed by Northern blotting as described for panel B.
cin.
DNA isolation, Southern blotting, hybridizatio: and band integration. DNA was purified from the nuclei a: s previously described (27) and digested overnight at 370C wit o of various restriction enzymes per ,ug of DN A. Te re-e stricted DNA was fractionated on 1% agaror se transferred to nitrocellulose by Southern blottin ig(22.Blot hybridization to a-32P-labeled probes was done 2as described previously (27). After autoradiography at -80°C for 5 to 10 days, the autoradiographs were scanned with a d (Hoefer Scientific Instruments) and the bands ; were integrated with a microcomputer. RNA isolation, blotting, and hybridization. T cellular RNA was purified by guanidine-thiocyanate ex (5), denatured with glyoxal and dimethyl sulfoxid le (16), and electrophoresed on a 1% agarose gel. Transfer tI nitrocellulose and subsequent hybridization were carries out under the same conditions as those used for Southern i blots. As a check on the quality and quantity of loaded RI} agarose gels were stained with ethidium bromide and th A) blotting. visualized under UV light before Northern (RN A)bloting,
sn, hA2 ThU
(2e2)s Balnd
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RESULTS Induction and superinduction of c-fos expressi ion in HeLa cells. The kinetics of c-fos expression have beer previously studied (10, 11, 17) in mouse BALB/c-3T3 or NI] H 3T3 cells. Our experiments were carried out with the humain c-fos gene in HeLa cells. Figure 1A shows a map of the hiuman c-fos gene and the probes used in this study. The e 6xperiments
1127
detailed below establish that the regulation of c-fos expression during induction, repression, and superinduction in HeLa cells is similar to that found in mouse 3T3 cells. The expression of c-fos in serum-starved 3T3 cells becomes highly elevated within 15 min after stimulation with serum and then returns to basal levels 30 to 30 min later because of autorepression (10, 11, 17). To verify that the
same kinetics of c-fos induction and repression occurs for HeLa cells under our culture conditions, we monitored HeLa c-fos expression by Northern blot analysis. c-fos expression is highest 15 min after induction by serum and returns to a low basal level by 60 min (Fig. 1B), as in mouse 3T3 cells. In 3T3 cells stimulated with growth factors, c-fos gene expression is superinduced when inhibitors of protein synthesis such as anisomycin or cycloheximide are added to the culture medium (10, 17). Inhibition of Fos protein synthesis delays Fos-mediated autorepression so that c-fos expression is prolonged for at least 180 min after stimulation. To verify that HeLa cells undergo similar superinduction kinetics, serum-starved HeLa cells were treated with 100 ,uM anisomycin for 30 min and then induced with medium containing 15% serum plus anisomycin. At various times after induction, the cells were harvested and total RNA was extracted. Figure 1C shows a Northern blot of total RNA challenged with c-fos probe A. As with superinduced mouse 3T3 cells, the steady-state level of c-fos RNA increases and remains elevated for at least 180 min after stimulation. These data show that the expression of the HeLa c-fos gene has the same kinetics during induction, repression, and superinduction as the expression of the gene in mouse 3T3 cells. Although posttranscriptional regulation of mRNA halflife plays a role in determining the final levels of c-fos mRNA, nuclear run-on assays in mouse 3T3 cells have shown that the changes in c-fos mRNA levels largely reflect induction, repression, or superinduction of c-fos transcription (10, 11). Moreover, c-fos transcription is reported to be controlled by the c-fos enhancer in both HeLa and mouse 3T3 cells (8, 18, 19, 24). Taken together, these findings indicate that the human c-fos enhancer regulates induction, repression, and superinduction of c-fos transcription in HeLa cells with the same kinetics as is found in mouse 3T3 cells. Inducing reversible changes in domain DNase I sensitivity by activating the c-fos enhancer. To examine the chromatin structure of the c-fos gene during induction in serum, we monitored the sensitivity of c-fos chromatin to DNase I digestion as a function of time after stimulation with serum. Nuclei were isolated from serum-starved HeLa cells induced with 15% serum for 0, 15, or 60 min and then digested with increasing concentrations of DNase I. DNA was purified from the nuclei, cleaved with restriction enzymes, and analyzed by Southern blotting. The blots were hybridized to c-fos probes, and the DNase I sensitivities of c-fos chromatin regions were estimated by the rates of disappearance of their specific restriction fragments with increasing DNase I
digestion.
To estimate the relative DNase I sensitivities of various c-fos regions, the blots were washed free of c-fos probes and rehybridized to an AS probe as an inactive control. The AS probe (2) cross-hybridizes with a family of nontranscribed AS pseudogenes which have the same DNase I sensitivity as bulk chromatin (unpublished data). The inactive AS pseudogenes thus function as an internal standard for the extent of DNase I digestion and for estimating the quantity of HeLa DNA on the blots.
1128
FENG AND VILLEPONTEAU
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FIG. 2. DNase I sensitivity of c-fos chromatin during induction. Nuclei were isolated from serum-starved HeLa cells and samples containing 5.7 A260 units of DNA were digested for 3 min at 37°C with 0, 0.11, 0.33, or 1 ,ug of DNase I. DNA was purified from the nuclei and cleaved with BamHI (A) or PvuII (B). The cleaved DNA was fractionated by agarose gel electrophoresis, transferred to nitrocellulose, and hybridized to c-fos probe A or B. After autoradiography to visualize the c-fos bands (bottom autoradiograph in each panel), the c-fos probes were washed off and the blots were rehybridized to the AS probe (top autoradiograph in each panel). The hybridization probes are shown as thick solid boxes below each genetic map, and the corresponding genomic restriction fragments are depicted as thin lines bounded by restriction enzyme cuts. c-fos HS sites are DNase I-hypersensitive sites that appear as subbands of 2.7 kb or less.
Digested DNA samples were restricted with BamHI, fractionated on an agarose gel, Southern blotted, and hybridized to c-fos probe B (Fig. 2A, lower blot) and to the AS probe (upper blot). A 5.7-kb BamHI fragment containing the c-fos transcription unit was more sensitive than AS fragments to DNase I at the 15-min point (Fig. 2A, lanes 2). In contrast, in the case of the c-fos gene, little preferential sensitivity was observed at 0 or 60 min. Thus, 15 min after stimulation with serum, c-fos chromatin had a transiently higher level of DNase I sensitivity that reverted to a less sensitive state by 60 min. The observed changes in c-fos DNase I sensitivity (Fig. 2A) might be the result of DNase I hypersensitivity rather than domain DNase I sensitivity. To check for this possibility, HeLa genomic DNA from the induction experiment described above was cleaved with PvuII, fractionated on an agarose gel, Southern blotted, and hybridized to probe A (Fig. 2B, lower blot). This end labeled a 3.7-kb PvuII fragment containing the 5' half of the c-fos transcription unit in addition to 5' upstream sequences. The blot was then washed and rehybridized to the AS probe (Fig. 2B, upper blot). The 5' c-fos region includes two DNase I-hypersensitive sites that have been mapped to the enhancer and the promoter of the c-fos gene (8). The 3' end labeling of the 3.7-kb PvuII fragment detected these hypersensitive sites as a pair of subfragments (2.7 and 2.4 kb) that first appeared in lanes 1 of Fig. 2B (lower blot). In lanes 2, another subband appeared because of cutting at a third hypersensitive site (not depicted in the map) inside the c-fos gene. All hypersensitive subfragments in lanes 2 were degraded in the 15-min samples but not in the 0- and 60-min samples. Thus, transient increases in DNase I sensitivity were also observed in indirect end-labeling experiments with the 5' portion of the c-fos gene (Fig. 2B). DNase I sensitivity in different regions of the c-fos gene. Having established that the c-fos gene assumes a more DNase I-sensitive chromatin structure upon activation, we went on to quantitate the DNase I sensitivities of various regions within the c-fos gene. To quantitate DNase I sensi-
tivity in the 5' half of c-fos, lanes C and 2 from the autoradiogram in Fig. 2B were scanned with a densitometer. Results with control and digested samples hybridized to AS or 5' c-fos probes are shown on the lefthand side of Fig. 3. We also examined DNase I sensitivity in the 3' half of c-fos. A Southern blot containing BamHI-StuI-restricted DNA hybridized to probe B to label the middle 0.7-kb StuI-StuI and 2.1-kb StuI-BamHI fragments of the c-fos gene was subjected to densitometry scanning (Fig. 3B). The same lanes were also hybridized to the AS probe (Fig. 3A). To calculate rates of c-fos DNase I sensitivity, the areas under the peaks of the digested c-fos samples were integrated and divided by the control peak values. This process was repeated for the AS scans, and the c-fos DNase I sensitivity was normalized by dividing the percent sensitivity by the rate of digestion for an equivalently sized AS band. These data were used to prepare a histogram (Fig. 3C) of the relative DNase I sensitivities of the various c-fos fragments as a function of stimulation with serum. In the histogram, a ratio of more than 1 denotes increased DNase I sensitivity relative to AS chromatin. When the c-fos enhancer was weak or repressed (as was the case at the 0- and 60-min points), a gradient of declining DNase I sensitivity formed over the c-fos gene, with peak sensitivity occurring near the 5' end (Fig. 3C). In contrast, at 15 min DNase I sensitivity was high over the entire gene, with less than a 15% difference in DNase I sensitivity between various c-fos regions. Figure 3C also quantitates the increases in c-fos sensitivity with induction. All three regions of the c-fos gene were more DNase sensitive at 15 than at 0 or 60 min. The increased DNase I sensitivity of c-fos at 15 min of induction was not dependent on the choice of a particular restriction fragment or probe (Fig. 3C). We conclude that the c-fos gene becomes more DNase I sensitive throughout its length at the time of maximum c-fos transcription. Increase in DNase I sensitivity upstream of the c-fos enhancer. If the c-fos enhancer functions by generating an altered chromatin structure in the downstream c-fos transcription unit, then the region upstream of the enhancer
ENHANCER-INDUCED CHANGES IN DNase I SENSITIVITY
VOL. 10, 1990
C
1129
Normal induction
A
AS
AS
.6
c
cn
co
co
co
z
0 ,1
I
I
1 5 min.
I
0 min. 15 min. 60 min. Minutes after serum stimulation 5' fos
Middle fos
II 3' fos FIG. 3. Relative DNase I sensitivities of various regions of c-fos during induction. Relative losses of c-fos fragments were determined by densitometry of c-fos bands and normalization to the band intensities of the same blot rehybridized to AS. (A and B, left side) Densitometry scans of control samples in lanes C ( ) and II ~ i. digested samples in lanes 2 (-----) from the PvuII-digested DNA in 'I I Fig. 2B are shown. Panel B contains scans of the blot hybridized to I I' ~f probe A, which labels 5' c-fos region. A map of the 5' region identified by probe A is shown in the bottom left of panel B. Open arrows denote DNase I-hypersensitive sites (HS sites). Panel A contains scans of the same blot hybridized to the AS probe. (A and Pvull digest Stul-BamHl digest B, right side) A Southern blot containing densitometry scans of 3.7 BamHI-StuI-digested DNA was hybridized to probe B to label the 5' Fos 3' Fos middle (0.7-kb fragment) and 3' third (2.1-kb fragment) of c-fos. A B map of the 3' region identified by probe B is shown in the bottom 0.7 and ----- denote scans of control or digested right of panel B. samples, respectively. (C) The areas under the peaks were integrated. DNase I sensitivities of the 5' c-fos regions were calculated 2.1 by adding the intensities of the two hypersensitive bands (-----) and I "A~~~~~~~~~~dividing 2.4 by the integrated values of the parent 3.7-kb bands ( ). / \ For the middle and 3' regions, the 0.7 and 2.1-kb bands of the 0min. digested samples (-----) were integrated and the intensities were divided by the values of the undigested lanes ( ). All c-fos intensities were divided by the rate of digestion of similarly sized AS bands to normalize for inactive gene digestion rates. The data are displayed as a histogram of relative DNase I sensitivities. Symbols: -, 5' c-fos region; M , middle c-fos region; E3 , 3' c-fos region.
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- 3'
Proximal
1
Distal
Histogram of relative DNase I sensitivities for nontran-
4.
scribed c-fos regions. DNA from the DNase I-digested nuclei of the normal induction experiment described in the legend to Fig. 2 was restricted with EcoRI-BamHI or BamHI, fractionated on agarose gels, and hybridized to either probe C (5' upstream c-fos) or probe D (3' downstream c-fos). The blots were washed and rehybridized to
the
AS
probe.
Losses
in
band
intensities
for
the
proximal
5'
fragment (1.4-kb EcoRI-BamHI band) or for the 3' distal fragment
(3.3-kb BamHI band) were determined and normalized to AS band intensities as described in the legend to Fig.
_,
3. Symbols:
5'
~, 3' distal fragment.
proximal fragment;
during c-fos induction, indicating that the induced changes in DNase I sensitivity extend less than 7 kb downstream from the enhancer. Kinetics of induction of altered chromatin structure. It is known that c-fos transcription reaches a half-maximal level
within 5
min
enhancer
of stimulation with serum (11).
activates
the
c-fos
promoter
by
If the c-fos
an
generating
altered chromatin structure, then changes in c-fos chromatin should
be
established
within
5
min
of
stimulation
with
0
2
8 10 12 14 16 Minutes after serum stimulation 4
6
FIG. 5. Relative DNase I sensitivities as a function of stimulation time. Nuclei were isolated from serum-starved HeLa cells stimulated with medium plus 15% serum for 0, 1.5, 3, or 5 min. The nuclei were digested with increasing concentrations of DNase I, and the DNA was purified. DNA aliquots were cleaved with BamHI, PvuII, or BamHI plus StuI, fractionated on an agarose gel, Southern blotted, and hybridized to either probe E or probe B. The c-fos probe was then washed off and the blots were rehybridized to the AS probe. Densitometry and quantitation were performed as described in the legend to Fig. 3. For the 0- to 5-min points, relative DNase I band sensitivities were plotted for the upstream element (2.5-kb BamHI band detected by probe E), the 5' half of c-fos (3.7-kb PvuII band and hypersensitive subbands detected by probe B), middle c-fos (0.7-kb StuI-StuI band detected by probe B), and the 3' third of c-fos (2.1-kb StuI-BamHl band detected by probe B). For the 15-min point, data on the upstream sequence were taken from the rate of disappearance of a 1.4-kb EcoRl-BamHl band as detected with probe C, which is a different restriction fragment and hybridization probe from that used in the 0- to 5-min experiment. Thus, the 15-min value for this point should be regarded as an approximation. Data for the 15-min point for the 5', middle, and 3' c-fos fragments were taken from the experiment shown in Fig. 3.
serum. Moreover, if propagated changes in chromatin structure
originate
at
the
enhancer,
then
the
formation
of a
time-dependent gradient of DNase I sensitivity in the region of the enhancer during the first few minutes of stimulation with serum might be detectable. To test these predictions,
nuclei were isolated from HeLa cells at 0, 1.5, 3, and 5
min
after stimulation with serum. The nuclei were digested with DNase I, and the resultant DNA samples were analyzed by Southern blotting and densitometry as before. Figure 5 shows the quantitated and normalized DNase I
sensitivities for various c-fos regions in the early times after stimulation. During the first 5 fragments
nearest
the
mmn
enhancer
of stimulation, the two
(upstream
region
and
5'
c-fos) nearly reach their maximal DNase I sensitivities in the
first 90 s. By contrast, the distal fragments (middle and 3' c-fos)
reach
less
than
40%
of their
sensitivities in 90 s. Even at 3
min
maximum
DNase
I
after induction, the distal
3' fragment fails to reach the DNase I sensitivity found in the proximal fragments at 90 s.
Thus,
chromatin
structure
is
altered near the enhancer before it is affected at the distal 3' end of c-fos. These data are consistent with a propagated change in chromatin structure which originates at the enhancer.
Figure 5 also contains the DNase I sensitivity data for the
15-mmn points Fig.
of the experiments whose results are shown in
3 and 4. These data were added for comparison and
suggest that the DNase I sensitivities of all fragments are maximal at 5
min
after induction, the exception being the
upstream element, whose sensitivity increases slightly after the for
5-mmn point. the
However, the change in DNase I sensitivity
upstream
element
may
be
artifactual,
since
the
probes and restriction fragments used for the 5- and 15-min points were not identical. We conclude that c-fos chromatin modification is essentially complete within 5 min of induction, while the promoter region changes within the first 90 s. Our kinetic data demonstrate that the establishment of domain DNase I sensitivity occurs long before transcription reaches maximal levels (11; Fig. 1B); moreover, the data are consistent with a role for changes in chromatin structure in the regulation of c-fos transcription. Domain DNase I sensitivity during superinduction of the c-fos enhancer. When serum-starved HeLa cells were treated with the protein synthesis inhibitor anisomycin, the basal level of c-fos expression is elevated and autorepression is delayed (Fig. 1C). As in the normal induction experiments, we expected to see changes in domain DNase I sensitivity that parallel the kinetics of c-fos superinduction. To test this prediction, nuclei were isolated from anisomycin-treated, serum-starved HeLa cells 0, 15, 60, or 180 min after stimulation with 15% serum. The nuclei were digested with DNase I, and the DNA samples were analyzed by Southern blotting and densitometry as before. During superinduction, c-fos chromatin was highly DNase I sensitive 15 and 60 min after stimulation (Fig. 6). The histogram in Fig. 6 reveals that at 15 and 60 min, all three regions (5', middle, and 3') of c-fos chromatin were about six- to ninefold more DNase I sensitive than inactive AS chromatin. At 180 min after stimulation, c-fos chromatin retained a preferential DNase I sensitivity fourfold greater
ENHANCER-INDUCED CHANGES IN DNase I SENSITIVITY
VOL. 10, 1990
9 4"
co3 z
0 min. 15 min. 60 min. 180 min.
Minutes after
serum
stimulation
FIG. 6. Relative DNase I sensitivities during superinduction. Anisomycin-treated, serum-starved HeLa cells were incubated with 15% serum plus 100 FM anisomycin for 0, 15, 60, or 180 min. The nuclei were digested with increasing concentrations of DNase I, and the DNA was purified. DNA aliquots were cleaved with PvuII or BamHI plus StuI, fractionated on an agarose gel, Southern blotted, and hybridized to probe B. Band sensitivities to DNase I (relative to AS controls) were plotted for the 5' half (_) (3.7-kb PvuII band and subbands), the middle ( M ) (0.7-kb StuI-StuI band), and the 3' third (ElZ) (2.1-kb StuI-BamHI band) of c-fos.
than that of inactive AS chromatin. Thus, the reversal of c-fos DNase I sensitivity was only partially complete 180 min after superinduction, which is consistent with previously reported levels of c-fos transcription at that time (10). At the 0-min point, the preferential DNase I sensitivity of the 5' half of c-fos was fourfold greater than that of inactive AS chromatin (Fig. 6), correlating with the high basal level of c-fos expression in the presence of anisomycin (Fig. 1C). In contrast to the 5' end, the middle and the 3' end of the c-fos transcription unit had preferential DNase I sensitivities threefold greater than that of inactive AS chromatin. Thus, as for normal induction at 0 min, a weak gradient of DNase I sensitivity forms over the c-fos gene, with the chromatin nearest the enhancer having the highest sensitivity.
DISCUSSION Role of chromatin structure in hormone induction of c-fos. It is not clear whether the formation of open chromatin actively potentiates gene expression or is merely a structural change which accompanies transcription. For c-fos induction, we have shown that the entire transcription unit becomes at least threefold more sensitive to DNase I within 5 min of serum induction of the c-fos enhancer. Moreover, the promoter region of c-fos achieves this threefold-increased sensitivity within 90 s of induction (Fig. 5). In contrast, the rate of c-fos transcription is only half of the maximal rate (11) or less (Fig. 1B) within 5 min of induction. Thus, these changes in c-fos DNase I sensitivity are not only faster than any reported previously but also rapid enough to allow a regulatory effect on the c-fos promoter. As further evidence for a role of chromatin structure in controlling c-fos transcription, we found that c-fos DNase I sensitivity paralleled c-fos transcription levels during autorepression and superinduction. Thus, c-fos domain DNase I sensitivity correlates closely in all cases with the level of c-fos transcription. Furthermore, it is unlikely that the observed changes in c-fos chromatin structure are indirect effects of enhancer-induced transcription, since nontranscribed DNA sequences located upstream from the enhancer
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also undergo changes in DNase I sensitivity during induction with serum and autorepression. Taken together, these results are consistent with a regulatory role for active chromatin structure in promoting c-fos transcription. Reversibility of the active chromatin structure of c-fos during autorepression of the c-fos enhancer. It has been found that domain DNase I sensitivity remains stable in several systems even after the inducing factor is removed (15, 21, 34). In transfected mouse mammary tumor virus expression vectors, domain DNase I sensitivity is stably propagated for at least 20 cell generations after hormone withdrawal (34). For the ovalbumin gene in chicken oviduct cells, domain DNase I sensitivity persists long after estrogen removal and the subsequent 1,000-fold drop in ovalbumin mRNA levels (15, 21). In contrast, we found that c-fos DNase I sensitivity fell back to a low level within 60 min after stimulation with serum (Fig. 3C). This reversal of domain DNase I sensitivity in c-fos is reminiscent of a similar reversal that occurs in the liver vitellogenin gene after withdrawal of estrogen (4, 9, 31). However, the kinetics of c-fos reversal is much faster than that of the vitellogenin gene and is comparable to the rapid reversal of domain DNase I sensitivity in chicken erythrocytes after gamma irradiation or drug treatment (26, 28). It is not clear why domain DNase I sensitivity is reversible in some systems and not in others. In the case of c-fos DNase I sensitivity, reversibility may be explained by the fact that the c-fos enhancer is directly repressed by the Fos protein. Thus, Fos protein binding to the enhancer may actively induce the bidirectional propagation of inactive chromatin, leading to reversal of DNase I sensitivity. Some support for this possibility comes from our superinduction experiments. Blocking Fos protein synthesis by treatment of HeLa cells with anisomycin leads to higher DNase I sensitivity throughout the c-fos gene at 0, 60, and 180 min after induction (Fig. 6). One interpretation of these results is that the c-fos chromatin retains its active conformation because the Fos protein is not present to induce the inactive chromatin conformation. Alternatively, the Fos protein, while still serving to downregulate the activity of the c-fos enhancer, may play no direct role in destabilizing active chromatin. In this case, the decay of DNase I sensitivity may be a passive process caused by an intrinsic instability of the active chromatin state due to the effects of particular DNA sequences. Other DNA sequences may be stably maintained in the active chromatin state, leading to high constitutive levels of gene expression in the absence of continued enhancer activity. Consistent with this possibility, the insertion of the c-fos enhancer into a CAT expression vector gives rise to CAT expression levels even without induction with serum that are nearly as high as those found using the constitutive simian virus 40 enhancer (8, 18). Induction of such constructs with serum further enhances CAT expression by only 2- to 3-fold instead of by the 20-fold induction seen in the intact c-fos gene (18). Thus, surrounding DNA sequences may be important in the repression of c-fos, though no specific sequence other than the c-fos enhancer has been identified. Do enhancers act to stimulate transcription through propagated changes in chromatin structure? Although it has long been recognized that DNase I sensitivity is associated with active genes, it remains unclear how DNase I-sensitive domains are specified and initiated. The present work implicates the c-fos enhancer as the modulator of c-fos chromatin structure. Induction, autorepression, and superinduction of c-fos transcription are all controlled by the c-fos enhancer. Our finding that changes in c-fos domain DNase I sensitivity
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parallel changes in c-fos transcription during induction, repression, and superinduction of c-fos suggests that the c-fos enhancer initiates these changes in c-fos chromatin structure.
In addition to the correlations we have seen between changes in transcription levels and DNase I sensitivity, the observations of both position- and time-dependent gradients in sensitivity around the c-fos enhancer make up a second line of evidence pointing to the c-fos enhancer as the origin of changes in c-fos chromatin structure. The first gradient is position dependent and forms over the c-fos gene before stimulation with serum and after autorepression (Fig. 3C). The second gradient is time dependent and establishes itself after stimulation with serum so that the proximal chromatin segments on either side of the enhancer reach nearly maximal DNase I sensitivity within 90 s after induction, whereas the distal chromatin segments require 5 min to change completely (Fig. 5). The simplest interpretation of these gradients of sensitivity is that the changes in chromatin structure are dampened as the altered structure propagates away from the enhancer. While the possibility exists that some other upstream DNA sequence serves as an origin, the available data point to the c-fos enhancer as the most likely candidate for the origin of propagated changes in chromatin structure. To what extent can these results be generalized to explain how enhancers function? Enhancers have cis-acting domain effects and can stimulate transcription from promoters located thousands of bases away. Previous work has demonstrated that some enhancers can override chromosomalposition effects (3, 13), which suggests a role for enhancers in altering chromatin structure. Active chromatin, once propagated to a distant promoter, could favor the binding of transcription factors or RNA polymerases and thus could effectively serve to stimulate transcription at a distance (20, 33). Our results provide the first experimental evidence that
enhancers can induce rapid changes in chromatin structure that propagate away from the enhancer. ACKNOWLEDGMENTS We thank Michael Pikaart for critically reading the manuscript. This work was supported by grant CD-385 from the American Cancer Society and grant IN-40-30 from the University of Michigan Cancer Research Institute.
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