From the Institute of Clinical Molecular Biology and Tumor
Genetics and Department for Protein Chemistry,
Research Centre for Environment and Health (GSF),
Marchioninistrasse 25, D-81377 München, Germany and the
§ McArdle Laboratory for Cancer Research, University of
Wisconsin Medical School, Madison, Wisconsin 53706
Received for publication, January 11, 2001, and in revised form, February 12, 2001
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ABSTRACT |
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The proto-oncogene c-myc is
transcribed from a dual promoter P1/P2, with transcription initiation
sites 160 base pairs apart. Here we have studied the transcriptional
activation of both promoters on chromatin templates. c-myc
chromatin was reconstituted on stably transfected, episomal,
Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2
promoters showed only basal activity but were strongly inducible by
histone deacetylase inhibitors. The effect of promoter mutations on
c-myc activity, chromatin structure, and E2F binding was
studied. The ME1a1 binding site between P1 and P2 was required for the
maintenance of an open chromatin configuration of the dual
c-myc promoters. Mutation of this site strongly reduced the
sensitivity of the core promoter region of P1/P2 to micrococcal
nuclease and prevented binding of polymerase II (pol II) at the P2
promoter. In contrast, mutation of the P2 TATA box also abolished
binding of pol II at the P2 promoter but did not affect the chromatin
structure of the P1/P2 core promoter region. The E2F binding site
adjacent to ME1a1 is required for repression of the P2 promoter but not
the P1 promoter, likely by recruitment of histone deacetylase
activity. Chromatin precipitation experiments with E2F-specific
antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site
of the c-myc promoter in vivo if the E2F site
was intact. Taken together, the analyses support a model with a
functional hierarchy for regulatory elements in the c-myc
promoter region; binding of proteins to the ME1a1 site provides a
nucleosome-free region of chromatin near the P2 start site, binding of
E2F results in transcriptional repression without affecting polymerase
recruitment, and the TATA box is required for polymerase recruitment.
The nucleosomal structure of promoter regions constitutes an
essential regulatory mechanism of eukaryotic gene repression. Gene
activation is accompanied by perturbations or alterations of the
nucleosomal structure like remodeling and acetylation of chromatin (1,
2). A first critical step for activating a gene locus seems to be
liberating the promoter region from histone-mediated repression (1, 2).
The transcription complex seems not to be recruited to the
promoter until positioned nucleosomes have been disrupted by the
concerted action of invading chromatin-remodeling and
trans-acting factors (1, 2). A large number of chromatin remodeling activities have been isolated and characterized
biochemically. The properties of remodeled nucleosomes include
increased accessibility of DNA to DNA-binding proteins throughout the
nucleosome (3-5), rotational phasing of DNA on the histone octamer
(6), reduction of the total length of DNA per nucleosome (7), octamer
susceptibility to displacement in trans (8), nucleosome
movement without disruption or trans-displacement of histone
octamer (9), and other effects.
Chromatin remodeling activities transfer promoters into a configuration
that is accessible for sequence-specific binding of transcription
factors and the recruitment of the transcription machinery. After
initiation, the transcriptional machinery requires further activation
signals for processive transcription. An increasing number of
genes, including heat-shock genes (10, 11), c-fos (12),
immunoglobulin The c-myc gene is transcribed from the dual P1 and P2
promoters that are located 160 bp apart. Both promoters have been
studied intensively in in vitro and transient transfection
experiments as well as in Xenopus oocytes and transgenic
mice (for reviews see Refs. 17-19). In normal cells, c-myc
is transcribed predominantly from the P2 promoter. In addition to the
TATA box, two further elements have been identified required for P2
activity: a ME1a1 site at position In the present study we aimed at extending the aforementioned findings
on c-myc regulation by mutagenizing promoter elements of the
episomal genes. We deleted the P2 TATA box and mutated the ME1a1 and
E2F site. We asked to what extent deletion of the TATA box abolishes
binding and pausing of pol II at the P2 promoter, and if so, how does
the absence of pol II affect the chromatin structure of the promoter?
We further asked whether mutation of the ME1a1 and E2F sites affects
c-myc expression at the same regulatory level. The results
were quite unexpected and gave deeper insight into the architecture and
regulation of the c-myc promoter in vivo.
Cell Lines and Cell Culture--
Cell lines were obtained by
stable transfection of Raji cells with the DNA containing the
8.1-kilobase pair HindIII-EcoRI c-myc gene locus on the episomal, self-replicating
Epstein-Barr virus-derived vector, pHEBOPL. Raji is an Epstein-Barr
virus-positive Burkitt's lymphoma cell line with a t(8;14)
translocation expressing a functional Rb protein. wt cells contained a
construct with c-myc germline sequences. Preparation of Total Cellular RNA--
Approximately 1 × 108 cells were washed with ice-cold phosphate-buffered
saline and spun down at 1200 rpm for 10 min at 4 °C. The pellet was
resuspended in 20 ml of a 4 M guanidinium isothiocyanate solution (Sigma) and sheared by drawing the suspension into a syringe
and expelling it through a 23-gauge needle several times until the
preparation was no longer viscous. RNA was pelleted through a CsCl
cushion as described (29).
Preparation of Nuclei--
Isolation of the nuclei was carried
out as described (14). Briefly, cells were spun down at 1200 rpm for 10 min at 4 °C and washed twice with ice-cold phosphate-buffered
saline, and the pellets of 1 × 108 cells were
resuspended in 10 mM Tris/HCl, pH 7.4, 10 mM
NaCl, 3 mM MgCl2, and 0.5% (v/v) Nonidet P-40.
After incubation on ice for 5 min, the lysate was spun down at 1500 rpm
for 15 min at 4 °C. The pelleted nuclei were resuspended in storage
buffer (50 mM Tris/HCl, pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA) and
immediately frozen in liquid nitrogen in portions of 100 µl corresponding to 2 × 107 nuclei.
Nuclease S1 Mapping--
A single-stranded uniformly labeled DNA
probe was prepared by primer extension of a M13 clone. Labeling of the
probe and hybridization of labeled DNA fragments to RNA was carried out
according to Berk and Sharp (30). Hybridization mixtures of 30 µl
containing 1 × 105 cpm of the probe (specific
activity 108 cpm/µg), 30 µg RNA, 90% (v/v) formamide,
400 mM NaCl, 40 mM Pipes, pH 6.4, and 1 mM EDTA were denatured at 90 °C for 5 min and
immediately transferred to 56 °C. After at least 12 h the
hybridization process was terminated by the addition of 180 µl of
ice-cold stop buffer containing 250 mM NaCl, 30 mM sodium acetate, pH 4.2, 2 mM zinc acetate,
5% (v/v) glycerol, and 400 units of nuclease S1 (Roche Molecular
Biochemicals). The samples were incubated at 25 °C for 1 h, extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), and precipitated with ethanol. Protected DNA fragments were
separated on 5% (w/v) polyacrylamide gels.
Mapping of Nucleosomes by Micrococcal Nuclease
(MNase)--
Nuclei were isolated essentially as described above.
2 × 107 nuclei in 200 µl of buffer (30 mM Tris-HCl, pH 8.3, 150 mM KCl, 10 mM CaCl2, 5 mM MgCl2,
20% glycerol, 0.05 mM EDTA) were incubated for increasing
periods of times with 3 units of MNase (Sigma) at room temperature.
Chromatin was cut to various extents in MNase digestion kinetics. The
reaction was stopped by the addition of 10 µl 0.5 M EDTA,
and DNA was purified as described (26). For further analysis, DNA
samples were chosen in which ~5-10% and 30-40% of nucleosomal
spacers had been cut by MNase. In addition, nuclease-digested DNA was
cut with either restriction endonuclease XbaI,
AccI, or HindIII in conditions recommended by the
manufacturer (New England Biolabs). DNA fragments were separated in a
2% (w/v) agarose gel, denatured by alkali treatment, transferred to a
nylon membrane (Hybond N+, Amersham Pharmacia Biotech), and hybridized with multi-prime labeled PCR probes as indicated. Hybridization probes
A (nt 111-315), L (nt 1865-2065), and T (nt 2881-3085) were
generated by PCR using specific c-myc primers and
multi-prime labeled with [ Nuclear Run-on Assay--
Isolation of nuclei, purification, and
hybridization of labeled RNA to membrane-bound oligonucleotides and the
washing procedure of membranes including the digestion of
single-stranded RNA with RNase A have been described in detail
elsewhere (14, 53). Briefly, 100 µl corresponding to 2 × 107 isolated nuclei in storage buffer (50 mM
Tris/HCl, pH 8.3, 40% (v/v) glycerol, 5 mM
MgCl2, 0.1 mM EDTA) were thawed on ice and subsequently incubated with 100 µl of reaction buffer (10 mM Tris/HCl, pH 8.0, 5 mM MgCl2,
300 mM KCl, 0.5 mM ATP, GTP, and UTP, and 100 µCi of [ Preparation of in Vitro Transcribed RNA by T7 RNA
Polymerase--
For production of a uniformly labeled c-myc
RNA, DNA fragments encompassing the c-myc region from
positions 2328 to 2880 and from positions 1878 to 2638 were generated
by PCR. PCR fragments were reamplified with primers carrying the T7 RNA
polymerase promoter for in vitro transcription by T7 RNA
polymerase (Roche Molecular Biochemicals). In vitro
transcription was done essentially as recommended by the manufacturer
in the presence of [ In Vitro Transcription--
Jurkat cells were grown to a density
of 3.5 × 105/ml and harvested. Cells were resuspended
in 4 cell volumes of buffer A (10 mM Tris-HCl, pH 7.3 (room
temperature), 1.5 mM MgCl, 10 mM KCl), incubated for 10 min on ice, and spun down at 2000 rpm in a Heraeus Varifuge 3.0R. Cell pellets were resuspended in 2 volumes of buffer A
and homogenized 10 times using a glass Dounce homogenizer with a
type B pestle. The nuclei were spun down for 15 min at 3000 rpm at
4 °C, resuspended in 1 volume of buffer C (20 mM
Tris-HCl pH 7.3 (room temperature), 50% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) and homogenized 10 times in a Dounce homogenizer using a B pestle. Nuclei were extracted for 60 min at 4 °C and centrifuged for 30 min at 18,000 rpm in a SS34 rotor. The extract was
dialyzed against buffer C (20 mM Tris-HCl pH 7.3 (room
temperature), 20% glycerol, 100 mM KCl, 0.2 mM
EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol). For in vitro transcription reactions, 1.5 µg of the indicated DNA reporters were transcribed in
the presence of 4 mM MgCl2, 25 mM
Hepes, pH 8.2, 5 mM dithiothreitol, 200 ng/µl bovine
serum albumin (Roche Molecular Biochemicals), 1 mM
phenylmethylsulfonyl fluoride, 100 µM ATP, GTP, and UTP, respectively, 5 µM CTP, and 50 µCi of
[ Formaldehyde Cross-linking and Immunoprecipitation--
Cells
were formaldehyde-cross-linked and immunoprecipitated as previously
described (32). Cells were swelled in RSB buffer (3 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.1% ICEPAL CA-330
(Sigma)) instead of the reported buffer, in order to isolate cross-linked nuclei. Antibodies against E2F and Ets proteins, E2F-1
(sc-193), E2F-2 (sc-633), E2F-3 (sc-879), E2F-4 (sc-866), E2F-5 (a
mixture of sc-1083 and sc-999) and Ets 1/2 (sc-112), were purchased
from Santa Cruz Biotechnology. As controls, we included a reaction
lacking primary antibody (No Ab) and a reaction that lacked chromatin
(Mock). Each of the antibodies was shown by Western blot
analysis to detect its cognate protein in NIH3T3 nuclear extracts (data
not shown). Antibody-protein-DNA complexes were isolated by
immunoprecipitation with blocked protein A-positive Staph A cells.
Following extensive washing, bound DNA fragments were eluted and
analyzed by subsequent PCR.
PCR Analysis--
Immunoprecipitates were dissolved in 30 µl
of water except for input samples, which were diluted in 100 µl and
then further diluted 1:100. Each reaction contained 3 ml of
immunoprecipitated chromatin, 1× Taq reaction buffer
(Promega), 1.5 mM MgCl2, 50 ng of each primer,
1.7 units of Taq polymerase (Promega), 200 µM
each dNTP (Roche Molecular Biochemicals), and 1 M betaine
(Sigma) in a final reaction volume of 20 µl. PCR reactions were
amplified for 1 cycle of 95 °C for 5 min, annealing
temperature of the primers for 5 min, 72 °C for 3 min, and 27 cycles
of 95 °C for 1 min, annealing temperature of the primers for 2 min,
72 °C for 1.5 min. PCR products were separated by electrophoresis
through a 1.5% agarose gel and visualized by ethidium bromide
intercalation. The resulting PCR products were quantitated using
ImageQuant Mac version 1.2 (Molecular Dynamics). Binding to the
c-myc promoter episome in wt cells was analyzed using
primers Myc-2411 and Myc-2857, in E2Fmt cells using primers Myc-E2Fmt
and Myc-2857, and in ME1a1mt cells using primers Myc-ME1a1mt and
Myc-2857. The sequences of the primers used are as follows:
Myc-2411, 5'-GGCTTCTCAGAGGCTTGGCGGG-3'; Myc-2857,
5'-TCCAGCGTCTAAGCAGCTGCAA-3'; Myc-E2Fmt, 5'-GGCTTCTCAGAGGCTTGAATTC-3'; Myc-ME1a1, 5'-GCTTGGCGGGAAAAAGGAATTC-3'.
Activity of c-myc Promoter Mutants--
We have previously shown
that the episomal c-myc promoters P1 and P2 are repressed in
stably transfected Raji cells. Although repression of the P1 promoter
occurs at the level of initiation, the P2 promoter is repressed by
promoter-proximal pausing of pol II (25). Transcription from episomal
P1 and P2 promoters is strongly inducible by inhibitors of deacetylases
(16, 27). In an extension of this work, we studied episomal
c-myc promoter mutants. The E2F binding site at 58 bp and
the ME1a1 site at 40 bp upstream of P2 were mutated by introducing
EcoRI recognition sites (E2Fmt, ME1a1mt); the TATA box at 24 bp upstream of P2 was deleted (
Expression of the wild type promoter and mutants were studied in
the human B cell line Raji, stably transfected with DNA constructs, which contain an 8-kilobase pair fragment of the human c-myc
gene together with the Epstein-Barr virus origin of replication
(oriP), to allow episomal propagation in Epstein-Barr
virus-positive cells (Fig. 1A). To prevent the formation of
a functional c-Myc protein from the transfected constructs, a
frameshift mutation was inserted into the coding part of
c-myc exon 2. Cell batches were selected in the presence of
hygromycin for ~4 weeks. The wt cell line and the four mutant cell
lines
Basal expression of the episomal c-myc P2 promoter was
studied by nuclease S1 analysis (Fig.
2A). The S1 probe, the size of protected fragments, and a quantification of the results are shown in
Fig. 2, B and C. In wt, ME1a1mt, and Effect of Mutations on Binding and Pausing of pol II at the P2
Promoter--
Expression of the c-myc P2 promoter is
regulated mainly by pausing of pol II proximal to the start site (14,
15). To study whether mutations in the P2 promoter affect initiation at
the P2 promoter or pausing of pol II, high resolution nuclear run-on experiments were performed. Hybridization of run-on RNAs to short oligonucleotides specific for c-myc exon 1 sequences allowed
us to refine the resolution of the assay. Using this method we have shown previously that paused polymerases become transcriptionally activated in nuclear run-on reactions and transcribe a short piece of
chromosomal and episomal c-myc RNA. The region transcribed after activation of these polymerases extends to ~100 bp downstream of the c-myc P2 promoter (16).
Nuclei of wt, E2Fmt, ME1a1mt,
The E2F and ME1a1 sites in the c-myc promoter have been
reported previously to be positive elements for the activity of the P2
promoter in transient transfection and in in vitro
transcription experiments (20). We tested the activity of the mutant
c-myc promoters in nuclear extracts from Jurkat cells (see
"Materials and Methods"). Labeled transcripts were hybridized to
c-myc exon 1 antisense oligonucleotides. A plasmid with the
wt P2 promoter was able to produce large amounts of transcripts in
nuclear extracts. However, similarly to nuclei, most of these
transcripts were not full size, indicating that transcription was
paused or prematurely terminated on the wt construct (Fig.
3C, lane 1). Transcription in extracts was
entirely sensitive to The Mutants ME1a1mt and
Differences in c-myc chromatin could be confirmed by
hybridization with probe L upstream of P2. Probe L detected two
nucleosomes downstream of the AccI site (nucleosomes 1 and
2) in the chromatin of wt and E2Fmt cells (Fig. 4, F and
G, lane 2). A hypersensitive site beyond
nucleosome 2 led to an interruption of the ladder. The chromatin of
ME1a1mt cells was less sensitive to MNase, and additional nucleosomes
(nucleosomes 3 and 4) were detectable (Fig. 4H, lane
2). A significant change was also observed for the chromatin of
Occupancy of the E2F Site at the c-myc Promoter in Vivo--
E2F
activity consists of a heterodimer containing one of six factors
(E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, and E2F-6) that pairs with a second
subunit (DP-1 or DP-2) (for review see Ref. 33). The transcriptional
activation potential of E2F is counterbalanced by pocket proteins,
which tightly associate with E2F in a cell cycle-dependent
manner (34, 35) and can recruit histone deacetylase activity to
E2F sites (36). Because mutation of the E2F binding site resulted in
such striking differences in c-myc P2 promoter activity
in vitro and in living cells, we wanted to determine which
members of the E2F factor family bind to the c-myc P2
promoter in vivo. To this end, we employed chromatin
immunoprecipitation experiments to study transcription factor binding
to the P2 upstream regulatory region in Raji cells (32, 35).
Cross-linked chromatin from equivalent numbers of cells was
immunoprecipitated using antibodies against E2F-1, E2F-2, E2F-3, E2F-4,
and E2F-5. After immunoprecipitation and reversal of the cross-links,
enrichment of the endogenous c-myc-promoter fragment in each
sample was monitored by PCR amplification using gene specific primers.
As shown in Fig. 5, the pattern of E2F
binding differed on the c-myc wt promoter and the ME1a1mt
and E2Fmt promoters. Anti-E2F-1, E2F-2, and E2F-4 immunoprecipitates
from wt chromatin contain high levels of c-myc-promoter fragment. In contrast, the E2F-3- and E2F-5-specific antibodies failed
to immunoprecipitate significant amounts of c-myc promoter fragment. Anti-E2F-1, E2F-2, and E2F-4 failed to immunoprecipitate significant amounts of the c-myc promoter fragment from
cross-linked E2Fmt cells, indicating that E2F-1, E2F-2, and E2F-4 bind
to the E2F site of the episomal c-myc promoter in
vivo. Interestingly, mutation of the E2F binding site correlated
with increased binding of Ets to the c-myc
promoter; this is in agreement with the finding of Roussel et
al. (37) that the binding of E2F and Ets to the c-myc
promoter excludes each other. The notion that members of the E2F family
might control binding of Ets to the c-myc promoter deserves further analysis. Inversely, we found evidence that binding of
E2Fs to the c-myc promoter is controlled by factor binding in the neighborhood. Mutation of the ME1a1 site affected binding of
E2F-1 and E2F-4 to the c-myc promoter but apparently had no effect on binding of E2F-2. Because of the lack of specific antibodies, the factors binding to the ME1a1 site in Raji cells have not been identified. In summary, several members of the E2F family bind to the
c-myc promoter in vivo, and their binding is
probably influenced or even specified by factors binding adjacent to
them.
Pol II Binding to the c-myc P2 Promoter Can Be Dissected from
Chromatin Opening--
Previous studies have shown that the
c-myc promoter region showed an open chromatin configuration
if the gene was repressed. In addition, the repressed c-myc
harbored a paused pol II proximal to P2 (16, 26-27). This observation
raised the question of whether pol II itself, or pol II-associated
factors, might be essential to inducing and keeping the open promoter
configuration. The mutants analyzed in this study allowed us to answer
this question. The ME1a1 Site Is Required for Chromatin Remodeling--
Mutation of
the ME1a1 site had the most severe effect on the activity of the P1/P2
promoter. This mutation abolished inducibility of the P1 as
well as the P2 promoter. Consistently, paused pol II was no longer
detectable at P2. Because ME1a1mt cells showed an altered nucleosomal
structure for the P1/P2 promoter region compared with wt, it is likely
that the ME1a1 binding factors are required for the opening of the
c-myc promoter region. So far, two binding factors for the
ME1a1 site have been described. MAZ (MYC-associated zinc finger
protein) is a ubiquitously expressed protein able to activate
c-myc reporter constructs in transient transfection assays
(21). MAZ is essential for the ME1a1-mediated expression of the
c-myc gene during neuroectodermal differentiation in P19
cells (40). Binding of MAZ to the ME1a1 motif in the c-myc
core promoter is likely also to play an important role in the control
of developmental expression of the CD4 gene (41) and of CLC-K1 and
CLC-K2, two kidney-specific CLC chloride channel genes (42). The second
known ME1a1 binding factor is hu-CUT, the human homolog of the
Drosophila CUT homeodomain protein (43). hu-CUT represses a
c-myc reporter gene in transient transfection assays. Our
data suggest that the ME1a1 binding factors contribute to
c-myc activation and that pol II cannot bind to the P2
promoter before c-myc chromatin has been opened by a
ME1a1-dependent activity. This opening step cannot
be substituted by inhibition of histone deacetylases, because SoB
treatment was unable to induce transcription from the P1/P2 promoter in
ME1a1mt cells. However, this does not rule out a function of ME1a1
binding factors in acetylation-dependent activation steps
after opening of the promoter. Finally, the effect of the mutation of
the ME1a1 site on c-myc chromatin is restricted locally and
does not influence a HSI located 2 kilobase pairs upstream.
The E2F Site Negatively Regulates the P2 Promoter--
The E2F
site in the c-myc P2 promoter was first described as a
positive element for c-myc expression. In fact, mutation of this site reduced P2 activity in transient transfection assays (20, 44)
as well as in a pol II-dependent in vitro
transcription assay (this work). Activation of a c-myc P2
reporter construct by E2F-1 overexpression is abrogated by Rb
co-expression (44). This suggested that E2F sites can act as positive
and negative elements for gene activity. Our results indicate that the
binding of E2F factors is not required for activation of the P2
promoter. Inhibition of pocket protein recruitment to the E2F site
appears to be sufficient for P2 activation. This finding is in line
with observations in transgenic mice either overexpressing or having knocked-out E2F-1. Both types of transgenics establish tumors (45, 46).
Although overexpression of E2F-1 probably titrates and thereby
inactivates pocket proteins, loss of E2F-1 inhibits the recruitment of
Rb to c-myc and other E2F-regulated promoters. Therefore,
both overexpression and knockout of E2F-1 could lead to deregulation of
c-myc. Can other members of the E2F family replace E2F-1 in
c-myc repression? Chromatin immunoprecipitation experiments
indicate that E2F-2 and E2F-4, in addition to E2F-1, bind to the
c-myc promoter. Because E2F-2 is able to recruit Rb, E2F-1 and E2F-2 might be involved in the negative regulation of the
episomal c-myc in Raji cells.
Similar to the c-myc P2 promoter, the cyclin E promoter is
negatively regulated by Rb. The repressive role of Rb in cyclin E
transcription has been mapped recently to its control of the acetylation status of a single
nucleosome.2 We have mapped
the first nucleosomes next to the E2F site in the c-myc
promoter at positions ~130 bp downstream (nucleosome 5) and ~450 bp
upstream (nucleosome 2). Whether acetylation of these nucleosomes is
controlled by the E2F site in the c-myc promoter remains to
be demonstrated. However, other targets must be considered. Finally, in
addition to HDAC activities recruited by the E2F site to the P2
promoter, additional HDAC activity probably contributes to P2
repression. Mutation of the E2F site leads to only ~50% promoter
activation as compared with activation by SoB. A potential additional
candidate is CTCF (CCCTC-binding protein), which has been shown
to bind to the P2 core promoter and to recruit HDAC activity (47,
48).
Mutation of the E2F Site Does Not Activate the P1
Promoter--
The E2F site is centered almost exactly between the
transcription start sites of P1 and P2. Although mutation of this site strongly induced transcription from the P2 promoter, the adjacent P1
promoter was not activated. Interestingly, the P1 promoter is inducible
by SoB to the same extent as the P2 promoter. From this observation we
can draw the following conclusions: (i) the HDAC recruited to the E2F
site is not able to repress transcription from the P1 promoter; and
(ii) the activity of the P1 promoter is also controlled by a HDAC, but
recruitment of this activity occurs by a separate element. The
c-myc P1 promoter harbors several SP1 sites essential for
its activity (49, 50). SP1 sites have recently been shown to be
involved in PCAF (P300/CBP-associated factor)-mediated
stimulation of transcription (51), as well as HDAC1-mediated repression
of transcription (52).
Conclusions--
This study has unraveled the hierarchical
structures in the regulation of the dual c-myc promoters P1
and P2 (Fig. 6). Both promoters are under
the common control of ME1a1 binding factors. This site controls the
global chromatin structure in the promoter region of a wt
c-myc gene and thereby probably the accessibility of the
transcriptional machinery to each promoter. Once the c-myc promoter region is brought into an open chromatin configuration, pol II
can initiate at P2 and subsequently pauses downstream thereof. The
c-myc promoter region contains one single E2F site. This
site negatively controls P2 activity by recruitment of HDAC activity. P1 promoter activity is controlled by HDAC in an E2F-independent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(13), and c-myc (14-16), has been shown to be regulated by promoter proximal pausing of RNA polymerase II (pol
II).1
40 bp and a E2F-binding site at
58 bp relative to the P2 transcription start (20-23). Mutation of
either or both sites strongly reduced P2 activity in transient
transfection assays. Stable transfection of c-myc promoter
constructs in cell lines or transgenic mice consistently led to an
inactivation of the dual c-myc promoter P1/P2 (24).
Repression of c-myc was also observed after stably
introducing the gene on episomal vectors in human B cell lines. The
episomal c-myc established a chromatin structure
indistinguishable to the chromosomal c-myc with identical positions for nucleosomes and DNase I-hypersensitive sites. Moreover, a
paused pol II is detectable downstream of the P2 start site. Transcription from the episomal c-myc promoters was strongly
inducible by inhibitors of histone deacetylases (16, 25-27).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
P2TATA, E2Fmt,
ME1a1mt, and
P1/P2TATA (Fig. 1) were constructed starting from
previously described deletions and EcoRI scanner mutants
(28), which were introduced into the wt construct by PCR technology.
Cells were grown to a density of 1 × 106 cells/ml in
10% fetal calf serum, RPMI 1640 medium (Life Technologies, Inc.)
supplemented with penicillin, streptomycin, and
L-glutamine. Medium for the c-myc transfectants
contained additionally 300 µg/ml hygromycin B. Treatment of the cells
with sodium butyrate was for 16 h at a final concentration of 3 mM.
-32P]dCTP. Hybridization was
carried out for 24 h in Church buffer (7% (w/v) SDS, 0.5 M sodium phosphate, 1 mM EDTA, pH 7.1) at
65 °C. Membranes were washed at room temperature with 1% SDS, 0.1× SSC (0.15 M NaCl, 15 mM sodium citrate, 1 mM EDTA, pH 7.5) and at 50 °C to achieve higher
stringency. After drying, filters were exposed to Kodak X-Omat AR film
at
80 °C with intensifying screens.
-32P]CTP (800 Ci/mmol, Amersham Pharmacia
Biotech)) for 15 min at 28 °C. Nuclear transcripts were isolated,
and labeled RNA was hybridized to DNA oligonucleotides immobilized on a
nylon membrane (Hybond+, Amersham Pharmacia Biotech) at 65°C for at
least 48 h in 5 ml of Church buffer. After being washed and dried,
the membranes were exposed to Kodak X-Omat AR film at
80 °C with intensifying screens. The intensities of the hybridization signals were
determined with a BAS 1000 phosphorimaging system (Fuji) and calculated
relatively to signals obtained with a homogeneously labeled RNA
transcribed by T7 RNA polymerase in the presence of [
-32P]CTP. Oligonucleotides complementary to the human
antisense strand, 50 nt long each, were synthesized according to the
sequence described by Gazin et al. (31) and have been
described elsewhere (14).
-32P]CTP. Full-length transcripts
were isolated by preparative polyacrylamide gel electrophoresis and
used for hybridization to DNA oligonucleotides as described above.
-32P]CTP (Amersham Pharmacia Biotech, 3000 Ci/mmol)
in a reaction volume of 100 µl and otherwise under standard
conditions (54). If indicated,
-amanitin was added to a final
concentration of 0.5 µM. Nuclear extracts of Jurkat cells
(50 µl or 250 µg OF nuclear extract) or a partially purified
transcription system served as a source for general transcription
factors and ubiquitous activators. To obtain the latter, nuclear
extracts were loaded on phosphocellulose (P11 at 10 mg/ml beads) and
step-eluted with 300, 500, and 850 mM KCl in buffer C. The
P11 system consisted of 15 µg of the fraction eluting at 850 mM KCl and 40 µg of the fraction eluting at 500 mM KCl. Reactions were incubated for 1 h at 28 °C
and processed as described below.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
P2TATA) (Fig.
1,A and B). As a
fourth mutant the sequence between the TATA box of P1 and the TATA box
of the P2 promoter was deleted leaving the TATA box of P1 intact
(
P1/P2, Fig. 1A).
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Fig. 1.
Episomal c-myc genes in
stably transfected B cell lines. A, episomal
pHEBO-derived c-myc constructs used for stable
transfections. Open boxes correspond to c-myc exons 1 to 3. All constructs carry the origin for episomal replication of the
Epstein-Barr virus (EBV-oriP), the ampicillin resistance
gene (amp), and the gene for hygromycin resistance
(Hyg). Constructs contained the c-myc wild type
promoter (wt), a deletion from the TATA box of the P1
promoter up to the TATA box of the P2 promoter
( P1/P2), a deletion of the TATA box of the P2
promoter (
P2TATA), an E2F site mutation, or an
ME1a1 site mutation. B, detailed description of mutations.
C, Southern blot analysis of episomal constructs in
transfectants. D, determination of copy numbers.
P1/P2,
P2TATA, E2Fmt, and ME1a1mt carried ~25 copies of
the respective constructs (Fig. 1, C and D).
P2TATA
cells, only low levels of P2-specific RNA were detectable (Fig.
2A, lanes 1, 5, and 7). In
E2Fmt cells, the levels of P2-specific RNA were increased ~4-fold
(lane 3). The mutations affected the basal transcription from the P1 promoter to various extents. Although E2Fmt and ME1a1mt did
not significantly affect P1 activity (Fig. 2A, lanes
3 and 5),
P2TATA showed strongly increased basal
activity of the P1 promoter (lane 7). Basal activity was
also significantly increased for the chimeric
P1/P2 (lane
9). In wt cells, the treatment with an inhibitor of histone
deacetylases, sodium butyrate (SoB), strongly induced transcription
from the P1 and P2 promoter (lane 2), whereas the endogenous
P1t promoter, in which expression is driven by Ig enhancers on
the translocated c-myc allele, was repressed (lane 2). The basis for repression of P1t is unclear but probably is linked to the Ig locus because transcription of the non-translocated Ig
heavy chain µ-gene in Raji cells is repressed also by SoB (data not
shown). SoB did not induce the P2 promoter in ME1a1mt and
P2TATA
cells (lanes 6 and 8), whereas expression of P2 in E2Fmt cells was increased up to the levels seen in wt cells (lane
4). SoB was also able to induce the P1 promoter in E2Fmt cells
(lane 4) but not in ME1a1mt cells (lane 6). In
P2TATA cells, the high basal levels of P1 RNA were further elevated
by SoB up to levels as seen in wt cells (lane 8). Taken
together, mutation of the TATA box and the ME1a1 site severely affected
the inducibility of the P2 promoter. In addition, mutation of the ME1a1
site abolished the inducibility of P1. In contrast, mutation of the E2F
site induced constitutive transcription from the P2 promoter but had only a minor effect on P1. For quantitative evaluation of signals, see
Fig. 2, C and D.
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Fig. 2.
Analysis of RNAs expressed from stably
transfected c-myc constructs in cell lines wt, E2Fmt,
ME1a1, P2TATA, and
P1/P2. A, cells were cultivated in
the absence (
) or presence (+) of 3 mM sodium butyrate
(SoB) for 16 h. Total RNA was isolated, and
c-myc RNA was studied by nuclease S1 analysis. The probe
discriminated RNAs derived from the transfected constructs
(P1 and P2) and RNA from the active endogenous
c-myc allele (P1t). A probe specific for
glyceraldehyde-3-phosphate dehydrogenase (gapdh) RNA
served as internal control. B, description of the probe and
of protected fragments. C and D, evalution of
signal intensities obtained for the P1 (C) and P2
(D) promoters in the absence and presence of sodium
butyrate, respectively.
P2TATA, and
P1/P2 cells were
subjected to run-on reactions, and labeled RNAs were purified and hybridized to membrane-bound oligonucleotides A-L complementary to
the entire c-myc exon 1 (Fig.
3A). The strong signals seen for oligonucleotides E and F are indicative of paused pol II, which
becomes activated in the run-on reaction and transcribes a short
stretch of RNA. We took the signals on oligonucleotides E and F as a
measure of density of paused pol II. The signal strength for both
oligonucleotides was high in wt cells (Fig. 3A, lane 1) but strongly reduced in ME1a1mt and
P2TATA cells
(lanes 3 and 4). In contrast, the density of pol
II at the P2 promoter in E2Fmt cells was increased compared with wt
cells (lane 2). Although the P1 wt promoter showed almost no
pausing (Fig. 3A, lane 1, oligonucleotides B and
C), significant pausing was seen at the chimeric
P1/P2 promoter
(lane 5). All signals were calculated relatively to the
signal obtained for the 7SK gene (Fig. 3B). These results
support the hypothesis that the TATA box and the ME1a1 binding site are
positive regulatory elements.
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Fig. 3.
Pol II distribution within promoter-proximal
sequences of c-myc genes. A, the
nuclei of wt, E2Fmt, ME1a1, P2TATA, and
P1/P2 cells were
isolated. Nuclear run-on reactions were performed in the presence of
[32P]CTP. Labeled RNAs were isolated and hybridized to
membrane-fixed oligonucleotides. Oligonucleotides A-L correspond to
the antisense strand of c-myc exon 1 with the promoters P1
and P2. Signals obtained with uniformly labeled c-myc RNA
transcribed in vitro by T7 RNA polymerase
(T7-RNA) served as a control for hybridization efficiency of
the different oligonucleotides. The 7SK oligonucleotide served as the
pol III transcription probe. B, run-on transcription signals
were measured with a phosphorimager BAS 1000 system (Fuji). The
transcriptional activities for oligonucleotides E and F of one
representative experiment were determined relative to the corresponding
signals in wt cells. C, constructs carrying wt
c-myc and the mutants E2Fmt, ME1a1, and
P2TATA were
subjected to an in vitro transcription experiment in the
presence of [
-32P]CTP. D, labeled
RNAs were hybridized to oligonucleotides, and intensities of signals
were evaluated as described above.
-amanitin (lane 2), indicating the
complete dependence of c-myc transcription in these extracts on pol II. The mutant constructs E2Fmt and ME1a1mt showed 2.5- and
2-fold reduced transcriptional activity compared with the wt construct
(Fig. 3C, lanes 3 and 4); the activity
of
P2TATA was reduced >10-fold (lane 5). Taken
together, a striking discrepancy for the transcriptional activity of
E2Fmt and ME1a1mt is observed in nuclei versus in
vitro transcription experiments. Both mutants have similar
activity in in vitro transcription experiments. However, in
the context of chromatin in isolated nuclei, they display striking differences; E2Fmt is transcriptionally active and has high amounts of
pol II bound, whereas ME1a1mt is transcriptionally inactive and is not
bound by significant amounts of pol II.
P1/P2 Have an Altered Chromatin
Structure--
The observed differences of transcription experiments
in vitro and in nuclei suggested that altered chromatin
structures in c-myc mutants might affect the binding of pol
II to the P2 promoter and inducibility of the P1/P2 promoters by SoB.
Therefore, we studied the nucleosomal structure of the P1/P2 promoter
region after MNase digestion. The nuclei from the various cell
lines were treated with MNase for increasing periods of time, and the DNA was purified and separated by gel electrophoresis. The resulting DNA fragments displayed the characteristic nucleosomal ladder (Fig.
4L). A digest of 20 min
produced mostly mono-, di-, and trinucleosomal fragments (Fig.
4L, lane 6) with decreased fragment length
compared with fragments from a 3-min digest (lane 5). The same pattern emerged when DNA was cut, in addition to MNase, with a
restriction endonuclease (lanes 2 and 3). DNA fragments were hybridized with the radioactively labeled probe T homologous to sequences downstream of the P2 promoter region. Using this probe, more
than five nucleosomes were detected in all five cell lines (Fig. 4,
A, B, D, and E, lane
5). This pattern was restricted to five nucleosomes if DNA was cut
with XbaI (Fig. 4, A-E, lane 2),
indicating that the region 70-100 bp downstream of the P2 cap site was
hypersensitive to MNase in cell lines wt, E2Fmt,
P2TATA, and
P1/P2. This region was less sensitive to MNase digestion in ME1a1mt
cells (Fig. 4C, lane 2). To exclude the
possibility that MNase might have cut chromatin in the nuclei of
ME1a1mt cells to a lesser extent than in the nuclei of other cells, we
compared the hypersensitivity of HSI (DNase I-hypersensitive site I) to MNase in nuclei of E2Fmt and ME1a1 cells (Fig. 4, M and
N). DNA was cut with HindIII and hybridized with
probe A. Two nucleosomes, a and b, were detectable, indicating that HSI
showed a similar sensitivity to MNase in the nuclei of both cell lines.
Partial digestion of naked cellular DNA with MNase did not produce the pattern of a nucleosomal ladder after hybridization with probe T (data
not shown).
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Fig. 4.
Nucleosomal patterns upstream and downstream
of the c-myc P2 promoter on episomal constructs.
A-K, M, and N, Southern blot analysis of
MNase-digested DNA of wt, E2Fmt, ME1a1, DP2TATA, and P1/P2 cell
lines. L, ethidium bromide staining of nucleosomal DNA
corresponding to the Southern blot shown in N. Nuclei were
isolated and incubated with MNase for different periods of time.
Subsequently, DNA was purified and subjected to Southern analysis
either uncut (lanes 4-6) or cut with restriction
endonucleases AccI, XbaI, and HindIII
as indicated (lanes 1-3). The PCR fragments A, L, and T
were used as hybridization probes for nucleosomal patterns upstream
(probes A and L) or downstream (probe T) of the c-myc P2
promoter. Lanes 1 and 4, DNA treated with no
MNase; lanes 2 and 5, DNA treated with MNase for
3 min; lanes 3 and 6, DNA treated with MNase for
20 min; M, labeled fX174 DNA-HaeIII digest (New
England Biolabs) serving as the molecular weight standard. Nucleosomes
detected after restriction enzyme digest are shown schematically on the
left side of each panel. O, scheme
depicting the relative locations of nucleosomes and probes A, L, and T
within the c-myc promoter region. The positions of
restriction endonuclease recognition sites refer to the previously
published c-myc sequence (31). Nucleosomes are numbered from
1 to 10; stippled nucleosomes are not detectable in all cell
lines.
P1/P2 cells. These cells clearly established the nucleosome 3 (Fig.
4K, lane 2), which was only marginally detectable
in chromatin of wt cells. If DNA was cut with the restriction enzyme
PstI (Fig. 4O), the nucleosomal ladders seen in
lanes 2 and 3 (Fig. 4, F-K) were
shortened by exactly one nucleosome (data not shown). Taken together,
clear differences were detectable in the c-myc chromatin of
ME1a1mt and
P1/P2 cells compared with wt cells. The differences are
depicted schematically in Fig. 4O.
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Fig. 5.
E2F-1, E2F-2, and E2F-4 bind to the
c-myc promoter in vivo.
A, formaldehyde-cross-linked chromatin was prepared from wt,
E2Fmt, and ME1a1mt cells, sonicated, and immunoprecipitated
(IP) with specific antibodies as indicated.
Immunoprecipitates from each antibody were aliquotted and subsequently
analyzed by PCR with primers specific for the c-myc
promoter. To verify that at each time point an equivalent amount of
chromatin was used in the immunoprecipitations, a sample representing
0.02% of the total input chromatin (Input) was included in
the PCR reactions. No Ab, no antibody. B,
quantification of PCR results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
P2TATA mutant abolished initiation of pol II, and
pausing of pol II was greatly reduced, as revealed by nuclease S1 and
run-on experiments. However, the chromatin of this mutant still
displayed hypersensitivity to MNase in the P1/P2 promoter region
indicating that opening of c-myc in the promoter region is
not linked to recruitment of pol II to the P2 promoter. Other elements
in the promoter region, as described in the discussion of the ME1a1
site below, are critical for chromatin opening. The
P2TATA mutant also sheds light on the complexity of the dual c-myc
promoters. Unexpectedly, the inhibition of pol II binding to the P2
promoter activates the P1 promoter, suggesting that P2 is a negative
regulator of P1 transcription. We postulate a mechanism associated with the TATA box of P2 that negatively regulates the activity of P1. This
idea has already been discussed for Burkitt lymphoma cells with t(8;14)
translocations. In these cells c-myc is under the control of
immunoglobulin enhancers, which constitutively activate the paused pol
II at the P2 promoter. In the context of the dual promoter, liberation
of the P2 pause site appears to be a prerequisite for P1 activation by
immunoglobulin enhancers (25). In wt cells, the episomal P1 is strongly
inducible by SoB. Because the activation of P1 in
P2TATA cells is
not increased further by SoB, but rather replaces the action of SoB, P2
may regulate the P1 activity by a mechanism involving acetylation.
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Fig. 6.
Model of c-myc
regulation. Although ME1a1 binding factors affect the global
chromatin structure at the dual c-myc P1/P2 promoter, the
E2F binding site is a negative site for P2 activity and probably
recruits HDAC activity. P1 recruits HDAC activity by a yet unknown
element.
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ACKNOWLEDGEMENTS |
---|
We thank T. Meulia, A. Krumm, and M. Groudine for providing c-myc mutants.
![]() |
FOOTNOTES |
---|
* This work was supported by Die Deutsche Forschungsgemeinschaft (SFB190) and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Lab. for Physiological Chemistry and Centre for
Biomedical Genetics, Utrecht University, P. O. Box 80042, 3508 TA
Utrecht, The Netherlands.
¶ Present address: Lab. of Molecular Tumor Biology, Dept. of Dermatology, University of Erlangen, 91052 Erlangen, Germany.
** To whom correspondence should be addressed: Institute of Clinical Molecular Biology and Tumor Genetics, GSF Research Centre, Marchioninistr. 25, D-81377 München, Germany. Tel.: +49-89-7099512; Fax: +49-89-7099500; E-mail: eick@gsf.de.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100265200
2 A. J. Morrison and R. E. Herrera, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol, polymerase; bp, base pair(s); wt, wild type; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; MNase, micrococcal nuclease; nt, nucleotide(s); SoB, sodium butyrate; MAZ, MYC-associated zinc finger protein; HSI, DNase I-hypersensitive site I; HDAC, histone deacetylase; Rb, retinoblastoma tumor suppressor.
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