(Received for publication, December 23, 1996, and in revised form, April 14, 1997)
From the Division of Biology, California Institute of Technology, Pasadena, California 91125
In vivo and in organello footprinting techniques based on methylation interference have been utilized to investigate protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of human mitochondrial DNA (mtDNA) in functionally active mitochondria. In particular, the changes in methylation reactivity of these regions in response to treatment of the organelles with ATP or ethidium bromide, which affects differentially the rates of mitochondrial rRNA and mRNA synthesis, have been analyzed. Two major sites of protein-DNA interactions have been identified in the main control region of mtDNA, both in vivo and in organello, which correspond to the regions of the light-strand promoter and heavy-strand rRNA-specific promoter. The in organello footprinting of the latter showed ATP- and ethidium bromide-dependent modifications that could be correlated with changes in the rate of rRNA but not of mRNA synthesis. By contrast, no ATP effects were observed on the in organello footprinting pattern of the termination region and on in vitro transcription termination, strongly suggesting that ATP control of rRNA synthesis occurs at the initiation level. Several methylation interference sites were found upstream of the whole H-strand transcription unit, pointing to possible protein-DNA interactions related to the activity of this unit. In vivo footprinting of the rDNA transcription termination region of human mtDNA has revealed a very strong protection pattern, indicating a high degree of occupancy of the termination site by mitochondrial transcription termination factor (~80%).
One of the hallmarks of the compact gene organization of mammalian
mtDNA is the location of most of the regulatory cis elements in a restricted segment adjacent to the D-loop, which contains the
promoters for the transcription of the heavy
(H)1- and light (L)-strands and the origin of
replication for the H-strand (1). Mapping and kinetic analysis of
in vivo synthesized H-strand transcripts in HeLa cells
(2-5) and in organello studies (6-8) have indicated that
the mechanism of H-strand transcription involves the activity of two
overlapping, independently controlled transcription units starting at
closely located sites in the main control region, one covering the rDNA
region and the other the whole H-strand (9). The differential activity
of the two H-strand transcription units and an H-strand transcription
attenuation event at the 3-end of the 16 S rRNA gene account for the
fact that the rRNA genes and adjacent tRNA genes are transcribed to a
20 to 50 times higher rate than the downstream protein coding and tRNA
genes (10). A central role in the attenuation phenomenon mentioned
above is played by the mitochondrial transcription termination factor
(mTERF), a DNA-binding protein that protects a 28-base pair DNA segment
immediately adjacent and downstream of the 16 S rRNA/tRNALeu(UUR) boundary (11-13), which comprises a
tridecamer sequence critical for directing accurate termination
(14).
Protein factors that bind to mtDNA in the main control region (15, 16) and at the rDNA transcription termination site (11-13) have been identified, and their interactions with DNA have been investigated by DNase I protection footprinting assays using purified components (11, 15) and, more recently, by applying on isolated organelles (17-20) and on intact cells (21, 22) the footprinting method of methylation interference (23, 24).
Previous work carried out in our laboratory with a highly efficient RNA-synthesizing system utilizing isolated HeLa cell mitochondria (6-8) has shown that, in this system, the relative transcription rates of rRNA and mRNA can be modulated independently by ATP (8) and by the intercalating drug EtBr (6). In the present work, the changes in the relative rates of rRNA and mRNA synthesis produced in response to ATP or EtBr have been correlated with the patterns of protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of mtDNA, as detected by analysis of methylation interference in cultured human cells or in organelles isolated from HeLa cells. In particular, the significant ATP- and EtBr-related modifications observed in the footprint of the rRNA-specific promoter, which were specifically correlated with changes in the rate of rRNA synthesis, have given support to the model of two independently controlled overlapping H-strand transcription units (9). Furthermore, no significant ATP effect on rDNA transcription termination has been detected.
HeLa S3 cells were grown in
suspension in high phosphate-containing Dulbecco's modified Eagle's
medium with 5% calf serum. The human cell line 143B.TK
(25) was grown on solid substrate in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
Samples of 107 to 108 HeLa S3 cells
or 3 × 106 143B.TK cells were routinely
used. The methylation reaction, cell lysis, and total nucleic acid
extraction were carried out as described previously (21). The yield of
total nucleic acids was 0.4 to 4.0 mg from each HeLa cell sample and
~200 µg from each 143B.TK
cell sample.
The
mitochondrial fraction was isolated from 1.5 to 3.0 × 108 HeLa cells, essentially as described previously (8),
resuspended in incubation buffer (40 mM Tris-HCl, pH 7.4, 25 mM NaCl, 5 mM MgCl2, 10%
glycerol) and washed three times; the final pellet was then
resuspended, at ~2 mg/ml, in incubation buffer containing 1 mg/ml
bovine serum albumin, 10 mM
NaH2PO4, 2.5 mM pyruvate, and the
desired ATP or EtBr concentration. For each incubation condition
tested, two 500-µl samples of the mitochondrial suspension were
transferred into Eppendorf tubes, and to one of them, destined for
analysis of RNA synthesis, 10 µCi of [-32P]UTP (400 Ci/mmol) was added. One of the two tubes was incubated at 37 °C
under shaking for 20 min and the other for 30 min, and then DMS
treatment or, respectively, RNA extraction was performed on the two
samples, as detailed below. In particular, for DMS treatment, 26 µl
of a freshly prepared 2% DMS solution in water was added to each
mitochondrial suspension incubated for 20 min and allowed to react for
2 min at 37 °C, and then 900 µl of ice-cold phosphate-buffered
saline was added, and the mitochondrial fraction was pelleted at
12,000 × g for 1 min. The DMS-treated mitochondrial fraction was washed three times by centrifugation and resuspension in 1 ml of ice-cold phosphate-buffered saline, resuspended in 400 µl of
proteinase K buffer (10 mM Tris-HCl, pH 7.5 (25 °C), 0.2 M NaCl, 0.1% SDS, 0.1 mg/ml proteinase K) by vortexing,
and incubated at room temperature for 30 min. The sample was then extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and the
nucleic acids were ethanol-precipitated as previously detailed for
in vivo DMS-treated samples (21). For each set of in
organello footprinting experiments, a sample of the mitochondrial
fraction was treated identically, except for the omission of DMS
treatment, and DNA extracted from it for subsequent in vitro
DMS treatment.
For analysis of the in organello transcription products, after the 30 min incubation, the mitochondrial samples were pelleted at 12,000 × g for 1 min, washed, dissolved in proteinase K buffer, and digested for 10 min at 37 °C. The samples were then phenol-extracted and ethanol-precipitated; the RNA was electrophoresed on a 2.2 M formaldehyde, 1.4% agarose gel using MOPS buffer (20 mM MOPS, 10 mM sodium acetate, 1 mM EDTA, pH 7.0, at 25 °C), and the RNA bands were quantified, after drying the gel, using a PhosphorImager screen and ImageQuant software (Molecular Dynamics).
In Vitro DMS Treatment of Naked DNAThe DNA methylation was carried out on pellets containing about 200 µg of total nucleic acids deriving from non-DMS-treated cells or 5-10 µg of nucleic acids from untreated mitochondrial fractions as described elsewhere (21).
Piperidine Cleavage of DNAThis was performed on pellets containing approximately 200 µg of total nucleic acids from DMS-treated cells or 5-10 µg of total nucleic acids from DMS-treated mitochondrial fractions and on equivalent samples of in vitro DMS-treated total nucleic acids from cells or isolated mitochondria, as described previously (21).
Primer Extension of DMS-treated DNAThe following
oligodeoxynucleotides, designated according to the numbering system of
the Cambridge sequence (26), were used for primer extension of
DMS-treated DNA in the assay as follows: L315,
5-CGCTTCTGGCCACAGCAC-3
; L476, 5
-CTAATCTCATCAATACAACC-3
; H495,
5
-GGTTGTATTGATGAGATTAG-3
; and H719, 5
-CTCACTGGAACGGGGATG3
for the fingerprinting of the initiation region; and L3064,
5
-ATCTGAGTTCAGACCGG-3
; and H3360, 5
-TGCCATTGCGATTAGAATGG3
for
the fingerprinting of the rDNA transcription termination region. The
oligodeoxynucleotides were 5
-end-labeled with
[
-32P]ATP as detailed previously (21).
Five µg of DMS-treated and piperidine-cleaved total DNA from cells (containing approximately 0.005 pmol of mtDNA) or 0.2-0.4 µg of similarly treated DNA from the isolated mitochondrial fractions were used as a template for primer extension analysis. Polymerase chain reaction amplification and electrophoresis of the products through 6% polyacrylamide, 7.7 M urea sequencing gels in Tris borate/EDTA buffer were carried out as described previously (21).
DNA Binding AssaysThe DNA binding activity of mTERF was determined by mobility shift assays using the double-stranded 44-mer oligodeoxynucleotide probe described previously (11). A constant amount of mitochondrial lysate protein was incubated with 10 fmol of probe in the presence of the indicated concentrations of ATP, and the samples were then run on a native 5% polyacrylamide gel, as detailed elsewhere (27).
In Vitro Transcription Termination and S1 Protection AssaysThe pTER plasmid, used as a template for the transcription termination assays, has been previously described (11), as has been the clone BSAND, utilized to synthesize the RNA probe employed in the S1 protection assays (12, 27). The transcription termination reactions and the S1 protection assays were performed as detailed previously (12, 27); quantification of the S1-resistant products was carried out by laser densitometry of the autoradiogram.
Mitochondria isolated from HeLa cells were incubated
in the presence of [-32P]UTP in the appropriate
buffer, as detailed under "Experimental Procedures." It has been
previously shown that HeLa cell mitochondria, under these conditions,
are able to carry out RNA synthesis in a way closely resembling the
in vivo process (6-8). In particular, transcription has
been shown to start at the two H-strand initiation sites
(H1 for the rDNA transcription unit, and H2 for
the whole H-strand transcription unit) and at the single L-strand
initiation site, and the transcripts have been shown to be processed in
a way reproducing the in vivo patterns; however, the
relative rates of rRNA synthesis and polyadenylation are somewhat
reduced in this in vitro system, as compared with the
in vivo situation (7).
To determine the optimum time of DMS treatment for in
organello footprinting during mitochondria incubation, time course
and time interval pulse-labeling experiments were carried out. Fig. 1a shows the electrophoretic patterns in an
agarose-formaldehyde gel of RNA labeled with [-32P]UTP
in isolated HeLa cell mitochondria, in the presence of 1 mM
ATP, after different times of incubation or after different time
interval pulses. Fig. 1, b and c, shows the
radioactivity incorporated into total mitochondrial RNA, measured as
overall signal from gel-separated RNA bands scanned with a
PhosphorImager, in the continuous incorporation and, respectively, time
interval pulse-labeling experiments. From Fig. 1b it appears
that the accumulation of label into total mitochondrial RNA proceeds at
a fairly constant rate for 30 min at 37 °C. On the other hand, the
time interval pulse-labeling experiments, which tend to exclude a
possible role in the observed kinetics of changes in RNA turnover rate
during in vitro incubation, indicate that the maximum rate
of incorporation of radioactivity occurs between 10 and 30 min after
the beginning of incubation and suggest a fairly constant rate of
incorporation up to 45 min. On the basis of these experiments, DMS
treatment for in organello footprinting was carried out 20 min from the start of incubation, and the synthesized RNA was isolated
at 30 min.
In Vivo and in Organello Footprinting of the L-Strand and H-Strand Transcription Promoter Regions of HeLa Cell and 143B.TK
Fig. 2a shows the in
vivo methylation patterns of the H-strand in the L-strand
transcription promoter region of HeLa cell and 143B.TK
cell mtDNA and the in organello methylation pattern of the
H-strand in the same region of HeLa cell mtDNA (lanes E). By
comparing these patterns with those generated by DMS treatment of naked DNA isolated from total cells or mitochondria (lanes N), one
recognizes regions of altered reactivity to DMS. In particular, in the
region around the L-strand initiation site (at position ~407), one
H-strand residue (nucleotide 409), exhibits hypermethylation in the
in vivo footprinting pattern, whereas five H-strand residues
(nucleotides 401, 403, 408, 409, and 410) exhibit methylation
protection or hypermethylation in the in organello
footprinting pattern. In the region spanning nucleotides 418 to 447 upstream of the L-strand transcription initiation site, which
corresponds to the binding site of the mitochondrial transcription
factor A (mTFA) (15), three H-strand residues (nucleotides 427, 437, and 438) (Fig. 2a) and one L-strand residue (nucleotide 429)
(not shown) exhibit methylation protection, and one L-strand residue
(nucleotide 443) (not shown) hypermethylation in the in vivo
footprinting pattern. In the in organello footprinting
pattern, eight H-strand residues (420, 427, 437, 438, 441, 444, 445, 446, and 447) (Fig. 2a) and two L-strand residues (429 and
443) (not shown) exhibit methylation protection or hypermethylation in
the same region. In both the sequence immediately surrounding the
L-strand transcription initiation site and in the mTFA binding domain
there is a reasonable correspondence between the residues exhibiting
protection or hypermethylation in the in vivo and in the
in organello footprinting (~30 and 50% correspondence of
residues showing methylation interference in the two regions).
Particularly noteworthy is the partial correspondence of
hypermethylated nucleotides in vivo and in
organello immediately upstream of the L-strand transcription
initiation site.
Fig. 2b shows the in vivo methylation patterns of
the L-strand in the regions of the H1 and H2 H-strand transcription
initiation sites of HeLa cell and 143B.TK cell mtDNA and
the in organello methylation pattern of the L-strand in the
same regions of HeLa cell mtDNA. The in vivo and in
organello methylation interference patterns in the L-strand of the
region of the rRNA-specific H-strand transcription initiation site H1 (at position ~559) and of the adjacent regions is shown with a greater resolution in Fig. 2c after a longer electrophoretic
run of the same samples used in the experiment of Fig. 2b.
In particular, in the segment immediately surrounding the H1 initiation
site, one L-strand residue (nucleotide 560) exhibits a hypermethylation in the in vivo footprinting pattern (Fig. 2, b
and c); in the in organello footprinting pattern,
the same residue exhibits a much stronger hypermethylation (Fig. 2,
b and c), whereas one H-strand residue
(nucleotide 557) shows methylation protection (not shown). In the mtDNA
segment spanning nucleotides 524-546 upstream of the H1
transcription initiation site, which corresponds to the mTFA binding
site (15), a single L-strand residue (nucleotide 545) exhibits
methylation protection in the in organello footprinting pattern (Fig. 2, b and c). Within the segment
between the mTFA binding site and the H1 initiation site,
two additional L-strand residues (nucleotides 551 and 552) exhibit
hypermethylation in the in organello pattern (Fig. 2,
b and c). Furthermore, in the segment immediately
downstream of the H1 start site, three L-strand residues
(nucleotides 577, 583, and 586) (Fig. 2, b and c)
and two H-strand residues (nucleotides 568 and 575) (not shown) exhibit methylation protection in the in organello pattern.
The protection or hypermethylation phenomena described above appear to
occur at positions of known protein-DNA interactions (like those
involving mTFA) or at potential sites of similar interactions (involving RNA polymerase or transcription factor(s)) at or near the
RNA synthesis start positions. An additional pattern of altered methylation reactivity was observed in the L-strand in the segment spanning nucleotides 601-621 within the tRNAPhe gene,
located about 40 nucleotides downstream of the H1
transcription initiation site at position ~559. This mtDNA segment
lies 27 nucleotides upstream of the second H-strand transcription
initiation site (H2 in Fig. 3)
near the 5-end of the 12 S rRNA gene. In particular, two L-strand
residues (nucleotides 620 and 621) are hypermethylated in the in
vivo footprinting (Fig. 2b), and three L-strand
residues (nucleotides 601, 602, and 611) (Fig. 2b) and two
H-strand residues (nucleotides 602 and 609) (not shown) exhibit altered
methylation reactivity in the in organello pattern. Fig. 3
summarizes the in organello and in vivo
methylation reactivity pattern of the L-strand and H-strand
transcription promoter regions.
Effect of ATP and EtBr on in Vivo and/or in Organello Footprinting of the Promoter Regions
It has been previously shown that the ATP
requirements for rRNA synthesis and mtDNA L-strand transcription in
isolated HeLa mitochondria are markedly different from those for
mRNA synthesis (8). To investigate whether ATP had any effect on
the in organello footprinting patterns, which could be
correlated with changes in transcription, isolated HeLa cell
mitochondria were incubated in different concentrations of ATP, ranging
from 0 to 10 mM, and DMS treatment was performed 20 min
after the beginning of incubation. With due account for some
differences in the amount of sample loaded, no significant change in
the in organello footprinting pattern corresponding to the
H-strand (Fig. 4a) or L-strand (not shown) of
the L-strand promoter was observed in mtDNA from mitochondria incubated
in different ATP concentrations. By contrast, a clear-cut ATP-dependent change was detected in the footprinting
pattern of the H-strand rDNA-specific promoter. In particular, the
hypermethylated L560 band exhibited a marked increase in intensity
after addition of 0.25 mM ATP to the medium, reaching a
maximum between 1 and 5 mM, and declining at higher ATP
concentrations (Fig. 4b). Since the L560 band showed the
most evident change in intensity in relationship to ATP concentration,
it was utilized as an indicator of modifications in putative
protein-DNA interactions in subsequent experiments. It should be
noticed that the in organello methylation patterns obtained
in the presence of 1 mM ATP for the L-strand of the
rDNA-specific H-strand promoter and the H-strand of the L-strand
promoter, which are shown in Fig. 4, a and b, are
substantially identical to those observed in the independent experiment
illustrated in Fig. 2, a and b, indicating the
reproducibility of the in organello patterns.
Fig. 4c shows the patterns in a formaldehyde-agarose gel of
RNA labeled in isolated HeLa cell mitochondria in the presence of
different concentrations of ATP. It is clear that, in the absence of
added ATP, the mRNA species are labeled to a substantial extent, whereas there is only marginal labeling of the rRNA species; by contrast, addition of ATP at 0.25 mM strongly stimulates
rRNA synthesis, which reaches a maximum (including the rRNA precursors) at about 1 mM, with only a moderate increase in mRNA
synthesis. Fig. 4d correlates the ATP-related changes in
intensity of the L560 band with the variations of the in
organello rate of synthesis of 16 S rRNA and mitochondrial
mRNAs (ND5, ND4/4L, COI, Cytb, A6/8, COIII, and COII). It is clear
that the changes in level of methylation of the L560 band follow much
more closely the modifications in rate of synthesis in
organello of rRNA than the changes in mRNA synthesis.
It has been previously shown (6) that, in isolated HeLa cell mitochondria, intercalating drugs, like EtBr, inhibit preferentially rRNA synthesis over mRNA synthesis. Such behavior is clearly shown in the experiment illustrated in Fig. 4e. To investigate the possible occurrence of EtBr-induced changes in the in organello footprinting patterns of the L-strand and H-strand transcription promoter regions, isolated HeLa cell mitochondria were incubated in the appropriate buffer containing 1 mM ATP in the presence of different concentrations of the drug and then DMS-treated 20 min after the beginning of incubation. A progressive decrease in methylation interference of the H-strand footprinting pattern of the L-strand transcription promoter (at the sites indicated by arrows in Fig. 4a) and of the L-strand footprinting pattern of the same promoter (not shown) was observed with increasing EtBr concentrations. In the H-strand transcription promoter region, the hypermethylated band L560 again exhibited the most pronounced changes, in relationship to the EtBr concentration, decreasing progressively in intensity with increasing doses of the drug (Fig. 4b). That these changes reflected a phenomenon occurring in vivo was shown by the finding that a clear decrease in the hypermethylation of L560 was also observed by in vivo footprinting in HeLa cells exposed to 0.125 µM EtBr (Fig. 2c). Fig. 4f correlates the changes in intensity of the L560 band in the footprinting patterns obtained from isolated HeLa cell mitochondria incubated in the presence of different EtBr concentrations with the variations in labeling of 16 S rRNA and mRNAs (ND5, ND4/4L, COI, Cytb, A6/8, COIII, and COII) synthesized in organello under the same conditions. It is apparent that the intensity of the L560 band and the amount of labeled 16 S rRNA decrease in a parallel way over the range of EtBr concentrations utilized, whereas the labeling of the mRNAs remains essentially constant in the presence of EtBr up to 1 µM and decreases moderately at 1.5 µM.
In Vivo and in Organello Footprinting of the Mitochondrial rDNA Transcription Termination Region of HeLa and 143B.TKFig. 5, a and
b (panels labeled HeLa), shows the in
vivo methylation patterns of the L-strand and, respectively,
H-strand of the HeLa cell mitochondrial rDNA transcription termination region, which is the site of interaction of mTERF with DNA (11, 12).
The mtDNA segment extending from nucleotide 3235 to nucleotide 3250, which is entirely comprised within the region protected by mTERF from
DNase I digestion, exhibits several sites of methylation protection or
hypermethylation in the in vivo footprinting pattern. In
particular, three L-strand residues (nucleotides 3235, 3236, and 3244)
are hypermethylated, and five L-strand residues (nucleotides 3238, 3239, 3242, 3248, and 3249) and three H-strand residues (nucleotides
3245, 3246, and 3247) exhibit methylation protection. Fig.
5c shows the in vivo footprinting pattern of the
L-strand of the termination region in HeLa cells, in a repeat
independent methylation experiment, and in 143B.TKcells.
It is apparent that the in vivo footprinting pattern is quite reproducible.
To analyze the effect of EtBr on the in vivo footprinting of the mitochondrial rDNA transcription termination region, HeLa cells were pretreated overnight with the drug at 0.125 µM. As shown in Fig. 5, a and b (panels labeled HeLa/EtBr), the methylation interference pattern of the binding site of mTERF changed dramatically under these conditions. Among the L-strand residues which were affected in their methylation in the absence of the drug (Fig. 5a, HeLa), only three exhibited methylation interference in the ethidium bromide-treated cells. In particular, L-strand residues 3235 and 3244 remained hypermethylated, and the protected L-strand nucleotide 3242 became hypermethylated (Fig. 5a, HeLa/EtBr). The protection of H-strand residues 3245, 3246, and 3247 (Fig. 5b, HeLa) all but disappeared in the presence of EtBr. By contrast, the H-strand residue 3240 became strongly hypermethylated (Fig. 5b, HeLa/EtBr). It appears that the in vivo methylation interference pattern of the rDNA transcription termination region, after treatment of the cells with a low concentration of EtBr, became in general more similar to the pattern obtained with naked DNA. This effect pointed to a decrease, and probably a modification, in the binding of mTERF to mtDNA, which presumably caused a reduction in its transcription termination activity.
To investigate the effect of EtBr on the in organello footprinting of the HeLa cell rDNA transcription termination region, isolated organelles were exposed to various concentrations of the drug. Fig. 5d shows the in organello methylation patterns of the L-strand of that region. The naked DNA methylation pattern in the segment between nucleotides 3230 and 3250 shows a general similarity to that obtained for DNA extracted from whole cells (Fig. 5, a and c). However, there are some differences in the identity, type of methylation interference, and relative abundance of the methylated residues, as compared with the pattern obtained for naked DNA extracted from whole cells. These differences are possibly related to changes in the structure of mtDNA (conversion of supercoiled to open circular form?), that may have occurred during the isolation and incubation of the organelles and thereby affected the methylation efficiency of DMS or possibly the pausing of Taq polymerase (20). Despite these differences in the reference DNA pattern, the in organello methylation interference pattern obtained after incubation of the organelles in the absence of EtBr (i.e. under the standard conditions for in organello footprinting) (Fig. 5d) shows a substantial similarity to the in vivo pattern, with ~50% correspondence of the residues exhibiting protection or hypermethylation. Likewise, the footprinting pattern obtained in the absence of EtBr for the H-strand of the termination region is reasonably similar to the in vivo pattern (not shown).
Fig. 5e summarizes the in vivo DMS reactivity pattern, as well as the in organello footprinting pattern, of the rDNA transcription termination region. The percentage of protection at several nucleotides, after correction for the difference in loading of the lanes, ranged from 60% (nucleotide 3242) to 82% (nucleotides 3248 and 3250).
After exposure of the isolated mitochondria to 0.5 and 1.0 µM EtBr, there is a progressive decrease in the hypermethylation of L-strand residues 3235, 3236, and 3245 and a progressive decrease in the protection of L-strand residues 3238, 3242, 3243, 3248, and 3250 (Fig. 5d). As a result of these changes, the methylation pattern of the L-strand tends to become more similar to that of the naked DNA, as already observed for the in vivo footprinting in the presence of EtBr (Fig. 5a). This tendency, therefore, also points to a decreased binding of mTERF to mtDNA in the presence of EtBr.
Lack of ATP Effects on in Organello Footprinting of HeLa Cell Mitochondrial rDNA Transcription Termination RegionThe
experiments described in a previous section, which correlated the
effects of ATP on the in organello footprinting of the H-strand transcription promoter region with those on the synthesis of
RNA in isolated organelles (Fig. 4d), did not exclude the
possibility that ATP acted at the level of termination of mitochondrial
rDNA transcription. To obtain evidence on this possibility, the ATP effects on in organello footprinting of the termination
region were analyzed. Fig. 6, a and
b, illustrates the in organello methylation patterns of the L-strand and, respectively, H-strand of the termination region after incubation of the organelles in the presence of various concentrations of ATP. It should be noted that the L-strand pattern obtained in the presence of 1 mM ATP (i.e. under
our standard conditions of in organello footprinting) is
almost identical to that observed in the independent methylation
experiment in the absence of EtBr shown in Fig. 5d, pointing
again to the reproducibility of the in organello methylation
interference pattern. The most significant result of the experiments
shown in Fig. 6, a and b, is the absolute
identity of the methylation patterns obtained for both the L- and
H-strands after incubation of the organelles in the presence of
concentrations of ATP varying between 0 and 8-10 mM. These
observations argue against the possibility of an ATP effect at the
level of transcription termination.
Lack of ATP Effects on in Vitro mTERF Binding to DNA and Transcription Termination
Further evidence against a role of ATP in transcription termination was provided by experiments in which the effects of ATP on in vitro mTERF binding to DNA (Fig. 6c) and on in vitro transcription termination (Fig. 6d) were analyzed. As shown in Fig. 6c, mTERF binding to DNA, as measured by band shift assays, was only marginally increased in the presence of ATP concentrations from 0.5 to 5.0 mM, as compared with no ATP, and slightly more at 10 mM ATP. As illustrated in Fig. 6d, the proportion of terminated transcripts in an in vitro reaction remained about 10% over the whole range of ATP concentrations from 0.1 to 4 mM. Concentrations higher than 4 mM inhibited almost completely the transcription activity in our in vitro system (42).
DMS is known to methylate guanine residues at the N-7 position and, with a lower frequency, adenine at positions N-3 and N-7. It can also methylate adenine at position N-1 and cytosine at position N-3 in single-stranded DNA (28). Thymine can also be methylated at N-3 in single-stranded DNA, but, being a much weaker base than cytosine, its alkylation would be expected to occur in alkaline solution; however, there are reports of alkylation of thymine at neutral pH (28).
In the present work, although the majority of methylation reactivity changes were observed at purine sites, a few pyrimidines also showed an altered methylation pattern, in particular, in almost all cases, a hypermethylation. The methylation of cytosine residues in vivo or in organello could be explained by the occurrence of single-stranded DNA segments at those sites as a result of helical distortions caused by protein binding (20, 29-31) or by RNA polymerase pauses (32, 33). However, since piperidine does not cleave effectively the DNA chains at the methylated cytosine sites, it is likely that Taq polymerase pauses created fragments ending at those sites. Effective termination of primer extension by Taq polymerase at modified bases has been recently demonstrated (20). In the case of thymine, it seems possible that the alkaline environment of the mitochondrial matrix would favor the methylation of this base. In previous work on in organello footprinting of mammalian mtDNA by DMS interference, evidence was reported for the presence of methylated thymine residues in single-stranded mtDNA segments (20), as well as for methylated cytosine residues (18).
In the present study, the in organello footprinting patterns of the transcription initiation and rDNA transcription termination regions of human mtDNA showed a substantial similarity to the in vivo footprinting patterns, as concerns the location of the mtDNA segments exhibiting methylation interference. These included, in particular, the L-strand and H1 H-strand transcription start sites, the mtDNA regions upstream of the L-strand and of the H2 H-strand transcription start sites, and the transcription termination region. However, in each segment, there were differences in the identity, type of methylation interference, and relative abundance of residues exhibiting protection or hypermethylation. The reason for these differences is not known, but they were quite reproducible in independent methylation experiments. They probably reflect small variations in the protein-DNA contact sites due to DNA or protein conformational changes. These may occur during the isolation or incubation of mitochondria as a result of modifications in the internal environment of the organelles or of the presence of additional components interacting with DNA or with the DNA-binding factors (11, 34). Despite these variations, it is reasonable to assume that the fundamental protein-DNA interactions underlying the methylation reactivity of mtDNA in isolated organelles reflect those occurring in vivo. This conclusion is supported by the close similarity between the RNA synthesis pattern obtained, under the same conditions utilized for in organello footprinting, in mitochondria isolated from HeLa cells, and the in vivo pattern (6-8, and the present work), and by the comparable EtBr effects on the methylation interference patterns in vivo and in organello in the mtDNA transcription initiation and termination regions.
Evidence for Protein-DNA Interactions at the L-strand and H1 H-strand Transcription Start SitesIn previous work, the in organello footprinting patterns, as determined by methylation interference, of the promoter regions of mtDNA have been analyzed in mitochondria from bovine brain (17), human placenta (18), and rat liver (20). Methylation reactivity changes were observed in the mtDNA region upstream of the L-strand transcription start site and other changes, although considerably weaker, in the region upstream of the H1 H-strand transcription start site; these regions correspond to the mTFA binding domains (15). The observations reported here on the in vivo and in organello footprinting of the L-strand promoter region agree with the above-mentioned findings. By contrast, almost no nucleotide with altered methylation reactivity was found in the mTFA binding region of the H1 H-strand promoter. However, it should be noted that the present results are consistent with the evidence indicating a much lower affinity of mTFA for binding in vitro at the H1 H-strand promoter, as compared with the L-strand promoter (15).
A significant observation made in the present work is the presence of hypermethylated nucleotides, both in vivo and in organello, at or very near the L-strand and the H1 H-strand transcription start sites. It is interesting that, at both sites, the hypermethylated nucleotides occur in the non-template strand. In previous in organello footprinting studies, a weak hypermethylation of the non-template strand had also been observed at the L-strand and H1 H-strand transcription initiation sites in human placenta mtDNA (18). No such DMS hypersensitivity near the RNA start sites had, on the contrary, been reported in the in organello footprinting analysis of bovine brain (17) and rat liver mtDNA (20). A possible explanation of this discrepancy is the difference in nucleotide context of the transcription start sites in human versus bovine or rat mtDNA. Earlier investigations utilizing deletion analysis and site-specific mutagenesis had identified sequence elements absolutely necessary for transcription around the transcription start sites in human mtDNA (35, 36). It should be mentioned, in this connection, that the L-strand and H1 H-strand transcription start sites are surrounded by the octanucleotide consensus sequence ACC-CCAAA (underlined in Fig. 3) (35, 37) and that the homologous sites in Xenopus laevis mtDNA are also surrounded by an octanucleotide consensus sequence, although different (38). These findings had suggested possible protein-DNA interactions at these sites. However, the present observations provide the first clear evidence of local DNA conformational changes or increases in reagent concentration in hydrophobic pockets, due to protein-DNA interactions, at these sites (39). Since the in vivo and in organello hypermethylated residues at both the L-strand and the H1 H-strand transcription start sites were exclusively (in vivo) or mostly (in organello) Cs, it is possible that these sites contain single-stranded DNA segments as a result of helical distortions caused by protein binding to DNA, a situation required for transcription activity.
Correlation of L-560 DMS Reactivity with ATP- and EtBr-dependent Changes in Rate of 16 S rRNA Synthesis in Isolated MitochondriaThe main observation reported in the present work is that the changes in degree of in organello hypermethylation of L-560 at the H1 H-strand transcription initiation site detected in response to either ATP or EtBr followed closely the variation in rate of 16 S rRNA synthesis but not the variation in rate of mRNA synthesis. This observation supported the earlier strong evidence indicating that rRNA and mRNA synthesis are independently controlled in mammalian mitochondria (7, 8, 40). Furthermore, in view of the lack of influence of ATP on transcription termination, which has been observed in the present work, the results obtained on the ATP effects corroborated the idea that the regulation by ATP of rRNA synthesis occurs mainly, if not exclusively, at the level of initiation. It is particularly relevant to mention here that a specific requirement for ATP at an early step of in vitro transcription of the rDNA of human mtDNA has been previously documented (41). A regulatory role of ATP in mitochondrial RNA synthesis in mammalian cells has been recently proposed (42).
Methylation Interference Upstream of the H2 H-strand Transcription Start SiteAnother significant finding made in the
present work is the occurrence of in vivo hypermethylated
nucleotides and in organello protected or hypermethylated
residues in a ~20-base pair segment within the tRNAPhe
gene, which was not previously analyzed by others for in
organello footprinting and which is located 27 base pairs upstream
of the second in vivo H-strand transcription start site
(H2 in Fig. 3). This site has been identified in
this laboratory at the 5-end of the 12 S rRNA gene (3, 9), whereas
others have positioned it within the ACCCC at nucleotides 636-640
(37). It is particularly interesting that the latter assigned position
for the second H-strand transcription start site lies within the
octanucleotide ACCCCATA (underlined in Fig. 3), which
exhibits a seven of eight nucleotide match to the consensus sequence
mentioned above (36). The results obtained here, which point to
protein-DNA interactions upstream of this transcription initiation
site, provide support to the strong evidence from in vivo
and in organello studies that indicate the existence of two
overlapping, independently controlled H-strand transcription units in
human mtDNA (3, 4, 6-9).
The in
vivo footprinting pattern of the mitochondrial rDNA transcription
termination region observed in the present work in HeLa cells was
nearly identical to the in organello footprinting pattern of
the same region previously obtained from bovine brain (17) and rat
liver (20). In this pattern, the nucleotides with altered reactivity
are within the tridecamer sequence previously shown to be required for
accurate transcription termination (14), with the exception of the two
hypermethylated G residues which flank this tridecamer on the 5-end
side of the L-strand. In view of the fact that the tridecamer sequence
is a recognition site for mTERF and is completely comprised within the
DNA binding domain for this factor (11), it is reasonable to assume
that the observed footprint of the rDNA transcription termination
region is caused by the binding of mTERF. Remarkable is the pronounced
asymmetry in the distribution between the two strands of the residues
with altered DMS reactivity. These occur predominantly in the
non-template L-strand, pointing to a higher affinity of mTERF for this
strand. It is possible that this asymmetry is significant for the
function of mTERF, in connection with the modulation of its activity
that is required for the transcription of the downstream genes (11). The high proportion of in vivo occupancy of binding sites
for mTERF in HeLa cell mtDNA, as estimated from the percentage of protection from methylation by DMS of the residues in the footprinting pattern (up to about 80% for several nucleotides), is in agreement with the observations made by in organello footprinting of
the rDNA transcription termination region in bovine brain and rat liver
mitochondria (17, 20).
We are very grateful to Giovanni Lesa for collaboration in the early stages of the investigations and to Carlo Ausenda for help in the experiments involving ethidium bromide treatment of HeLa cells. We also thank A. Drew and L. Tefo for expert technical assistance.