In the case of the c-MYC promoter, it was shown that the purine-rich strand of DNA in the nuclease-hypersensitive region of the promoter can adopt different intramolecular G-quadruplex c
Trang 1of human thymidine kinase 1
Richa Basundra1,*, Akinchan Kumar1,*, Samir Amrane2,*, Anjali Verma1, Anh Tuaˆn Phan2 and Shantanu Chowdhury1,3
1 Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Delhi, India
2 Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore
3 G N Ramachandran Knowledge Centre for Genome Informatics, Institute of Genomics and Integrative Biology, CSIR, Delhi, India
Introduction
Nucleotide sequences are established as regulatory
ele-ments [1] However, DNA conformation(s) is relatively
unexplored in a regulatory context Non-B DNA
struc-tures have been implicated in recombination,
replica-tion and regulareplica-tion of gene expression [2–6], in both
prokaryotes [7] and eukaryotes [2,8] A particular type
of non-B DNA structure, the G-quadruplex motif, has attracted interest in the context of gene regulation, owing to reports indicating the prevalence of such motifs in promoters [9,10] G-quadruplex motifs are
Keywords
G-quadruplex; NMR; thymidine kinase 1
Correspondence
S Chowdhury, G N Ramachandran
Knowledge Centre for Genome Informatics,
Institute of Genomics and Integrative
Biology, CSIR, Mall Road, Delhi 110 007,
India
Fax: +91 011 27667471
Tel: +91 011 27666157 ext 144
E-mail: shantanuc@igib.res.in
A T Phan, Division of Physics and Applied
Physics, School of Physical and
Mathematical Sciences, Nanyang
Technological University, Singapore 637371,
Singapore
Fax: 6795 7981
Tel: 6514 1915
E-mail: phantuan@ntu.edu.sg
*These authors contributed equally to this
work
(Received 10 October 2009, revised 8 July
2010, accepted 16 August 2010)
doi:10.1111/j.1742-4658.2010.07814.x
G-quadruplex motifs constitute unusual DNA secondary structures formed
by stacking of planar hydrogen-bonded G-tetrads Recent genome-wide bioinformatics and experimental analyses have suggested the interesting possibility that G-quadruplex motifs could be cis-regulatory elements Here, we identified a characteristic potential G-quadruplex-forming sequence element within the promoter of human thymidine kinase 1 (TK1) Our NMR, UV and CD spectroscopy and gel electrophoresis data sug-gested that this sequence forms a novel intramolecular G-quadruplex with two G-tetrads in K+solution The results presented here indicate the role
of this G-quadruplex motif in transcription of TK1 in cell-based reporter assays Specific nucleotide substitutions designed to destabilize the G-quad-ruplex motif resulted in increased promoter activity, supporting direct involvement of the G-quadruplex motif in transcription of TK1 These studies suggest that the G-quadruplex motif may be an important target for controlling critical biological processes, such as DNA synthesis, medi-ated by TK1
Abbreviations
TDS, thermal difference spectrum; TK1, thymidine kinase 1; TSS, transcription start site.
Trang 2structural conformations formed by consecutive
stack-ing of coplanar arrays of four cyclic hydrogen-bonded
guanines [11–15]
G-quadruplex conformations were first reported in
telomeres [16], and subsequently in other genomic
regions, i.e immunoglobin heavy chain switch regions
[17], G-rich minisatellites [18] and rDNA [19] Recently,
genome-wide analysis of recombination-prone regions
showed the enrichment of potential
G-quadruplex-forming sequences within human hot spots or short
re-combinogenic regions [20] In another recent study, it
was proposed that G-quadruplex motifs may act as
nucleosome exclusion signals [21] Moreover, several
gene promoters, such as b-globin [22], retinoblastoma
susceptibility genes [23], the insulin gene [24],
adenovi-rus serotype 2 [25], PDGF [26], c-KIT [27],
hypoxia-inducible factor 1a [28], BCL-2 [29] and c-MYC
[10,30,31], harbor G-quadruplex motifs In
genome-wide studies, enrichment of G-quadruplex-forming
motifs in promoters of several bacterial [32], chicken
[33] and mammalian genomes, including the human
genome [9,34–36], has been observed, suggesting a
wide-spread regulatory influence of G-quadruplexes Further
support comes from reports showing that more than
700 orthologous promoters conserve putative
G-quad-ruplex sequences in human, mouse and rat [37], and a
recent genome-wide gene expression study showing that
the expression of many genes, whose promoters harbor
putative G-quadruplex forming sequences, had changes
in the presence of G-quadruplex-binding ligands in two
different human cell lines [36]
A role of G-quadruplex motifs in the transcription
of specific genes has been experimentally
demon-strated In the case of the c-MYC promoter, it was
shown that the purine-rich strand of DNA in the
nuclease-hypersensitive region of the promoter can adopt different intramolecular G-quadruplex confor-mations It was further demonstrated that destabiliza-tion or stabilizadestabiliza-tion of a G-quadruplex motif resulted
in increased or decreased promoter activity, respec-tively, in a luciferase reporter assay [30] Similarly, it was shown in KRAS [38] and PDGF-A [39] that stabil-ization of a G-quadruplex motif in the promoter region with a quadruplex-specific ligand resulted in decreased promoter activity
We note that all of the above studies considered G-quadruplex motifs formed by sequences containing at least four tracts of three or more consecutive guanines, which, in principle, can fold into G-quadruplexes with three stacked G-tetrads The regulatory role of G-quadruplex motifs comprising two G-tetrads (Fig 1A), where the core involves only two stacked G-tetrads instead of three, has not been studied A possible rea-son for this could be that a stack of two G-tetrads would confer less stability than a G-quadruplex with three stacked G-tetrads Although it has not been stud-ied in a regulatory context, the existence and biological role of G-quadruplexes with two G-tetrads has been reported in multiple cases [40–46] In the retinoblas-toma susceptibility gene, it was shown that a potential two-G-tetrad structure at the 5¢-end of the gene acts as
a barrier to DNA polymerase activity [46] Likewise,
in an in vitro study, the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) was reported to form a unimolecular stable G-quadruplex motif with two G-tetrads connected by two TT loops and a TGT loop, which inhibits thrombin-induced fibrin clot formation [43] Recently, it was found that human and Giardia telomeric DNA sequences containing four tracts
of three consecutive guanines can form intramolecular
AAATCTCCCGCCAGGTCAGCGGCCGGGCGCTGATTGGCCCCATGGCGGCGGGGCCGGC
TCGTGATTGGCCAGCACGCCGTGGTTTAAAGCGGTCGGCGCGGGAACCAGGGGCTTAC
TGCGGGACGGCCTTGGAGAGTACTCGGGTTCGTGAACTTCCCGGAGGCGCAATGAGCT
A
B
Loop3
5 ′ 3 ′
Loop2 Loop1
G G
G G
Fig 1 G-quadruplex motif and TK1
pro-moter (A) Schematic representation of a
G-quadruplex motif with two stacks of
G-tetrad in the core connected by three
loops (B) The TK1 promoter [62] showing
two potential G-quadruplex-forming
sequences: TKQ1 (bold) and TKQ2
(underlined) The TSS is indicated by the
arrowhead.
Trang 3G-quadruplexes comprising only two G-tetrads in K+
solution, and, interestingly, these structures can be
more stable than other G-quadruplex conformations
comprising three G-tetrads [47–50]
Here, we have identified a characteristic potential
G-quadruplex-forming sequence element, containing
several tracts of two consecutive guanines, within the
promoter of human thymidine kinase 1 (TK1)
Thymi-dine kinase is a critical enzyme required for the
production of TTP during DNA synthesis, and is
therefore ubiquitously conserved in prokaryotes and
eukaryotes It is tightly regulated during the cell cycle,
and has been shown to increase protein promoter
activity more than 10-fold during S-phase, to meet
demands for increased TTP synthesis; enzymatic
activ-ity remains high until about the time of cell division,
and then decreases rapidly [51] We show, by a series
of biophysical and biochemical experiments, including
NMR, UV and CD spectroscopy and gel
electrophore-sis, that this sequence forms a novel intramolecular
G-quadruplex with two G-tetrads in K+ solution
Using intracellular reporter experiments, we observed
that the promoter activity of TK1 is directly influenced
by specific nucleotide substitutions that disrupt the
G-quadruplex motif
Results
Identification of potential G-quadruplex-forming
sequences within the TK1 promoter
We used a customized perl program to search for
potential G-quadruplex-forming motifs G2–5L1–7G2–
5L1–7G2–5L1–7G2–5, which contained at least four runs
of two to five guanines separated by linkers of one to
seven nucleotides We identified two such motifs within
the functional promoter of TK1, spanning from)89 to
+58 of the transcription start site (TSS) [52] The two
identified sequences were designated TKQ1 ()13 to
+8) and TKQ2 ()47 to )68) (Table 1) Their locations
within the TK1 promoter are shown in Fig 1B TKQ1
harbors two tracts of two guanines, one of three
gua-nines and one of four guagua-nines The G-tracts are
sepa-rated by linkers composed of two, three and five
nucleotides, respectively TKQ2 comprises four tracts
of two guanines and one of four guanines, separated
by linkers of one, two or six nucleotides
TKQ1 forms a G-quadruplex structure – NMR study
In order to determine whether TKQ1 and TKQ2 form G-quadruplex structures, we recorded their NMR spectra in K+solution The imino proton spectrum of TKQ1 (Fig 2A, top) displayed sharp peaks between
10 and 12 p.p.m., which were characteristic of G-quad-ruplex formation, whereas the imino proton spectrum
of TKQ2 (Fig 2B, top) displayed peaks only at
13 p.p.m., which probably resulted from Watson– Crick base pairing For the spectrum of TKQ1, the observation of eight major sharp imino proton peaks between 10 and 12 p.p.m was consistent with forma-tion of a major G-quadruplex structure involving two G-tetrad layers; three major sharp peaks between 12 and 14 p.p.m might come from other base pairing alignments, e.g Watson–Crick or Hoogsteen base pairs
in the loops of the G-quadruplex; minor sharp peaks between 10 and 12 p.p.m should represent minor G-quadruplex conformation(s); a rather big hump in this region reflected yet other conformation(s) adopted
by TKQ1 For the spectrum of TKQ2, very little signal
Table 1 Oligonucleotides used in the study (5¢- to 3¢) G-tracts are
underlined One or two guanines were replaced by the same
num-ber of adenines in the modified oligonucleotides (bold).
A
B
Fig 2 NMR spectroscopy Imino proton spectra of: (A) TKQ1 (top) and TKQ1m (bottom); and (B) TKQ2 (top) and TKQ2m (bottom) Experimental conditions: DNA concentration, 0.5 m M ; temperature,
25 C; 70 m M KCl; 20 m M potassium phosphate (pH 7.0).
Trang 4between 10 and 12 p.p.m was observed, indicating the
presence of insignificant populations of
G-quadru-plex(es) Imino protons at 13 p.p.m might suggest
the formation of DNA duplex(es) or hairpin(s) involving
Watson–Crick base pairs, consistent with the presence
of complementary fragments G1–G2–C2–C4–C5–C6
and G15–G16–G17–G18–C19–C20 in TKQ2, which
would participate in the formation of the stem of a
hairpin or a duplex
To further probe the G-quadruplex-forming
poten-tial of TKQ1 and TKQ2, we designed two modified
sequences, TKQ1m and TKQ2m (Table 1), in which a
G-tract was disrupted by G-to-A substitutions For
TKQ1m, no sharp imino proton peaks between 10 and
12 p.p.m were observed (Fig 2A, bottom), indicating
that the TKQ1 G-quadruplex(es) were disrupted; imino
protons at 12–13 p.p.m suggested the formation of a
few base pairs of a residual structure such as a hairpin
(see gel electrophoresis data below) The imino proton
spectrum of TKQ2m (Fig 2B, bottom), like that of
TKQ2, exhibited peaks only at 13 p.p.m., indicative
of other types of base pairing alignments (e.g
Wat-son–Crick base pairs), rather than those in G-tetrads
In order to test the effect of flanking bases on the
G-quadruplex formation of TKQ1, we analyzed the
NMR spectrum of a DNA sequence containing four
additional bases (two on each side) Although the
imino proton spectrum of the new sequence was now
not well-resolved, and peaks at 13 p.p.m were
observed, the presence of peaks at 10–12 p.p.m
indi-cated the existence of G-quadruplexes (Fig S1)
A novel G-quadruplex motif of TKQ1 – TDS and
CD signatures
Thermal difference spectrum (TDS) signatures of
TKQ1, TKQ1m, TKQ2 and TKQ2m are shown in
Fig 3 Only the TDS profile of TKQ1 exhibited a
sig-nature compatible with G-quadruplex structures [53]
TDS of TKQ1 displayed two positive maxima at 245 and 275 nm and one negative minimum at 295 nm In contrast, TDS profiles of TKQ1m, TKQ2 and TKQ2m did not present any negative peak at 295 nm, but only major positive peaks at 250–275 nm, consistent with NMR observations that these sequences did not adopt G-quadruplex structures The G-to-A mutations com-pletely disrupted the G-quadruplex TDS signature of TKQ1, but showed little effect on the TDS of TKQ2 The CD spectra of TKQ1, TKQ1m, TKQ2 and TKQ2m are presented in Fig 4 They show negative peaks at 240 nm and positive peaks from 265 to
290 nm It is difficult to confirm or disprove the for-mation of G-quadruplexes based solely on CD signa-tures, particularly if multiple structures coexist [49] It has been reported that parallel-stranded
G-quadruplex-es give a positive peak at 260 nm and a negative peak
at 240 nm, whereas antiparallel-stranded G-quadru-plexes give a positive peak at 290–295 nm and a nega-tive peak at 265 nm [54] The CD spectrum of TKQ1 showed a positive peak at 290 nm, a positive shoulder
at 260 nm, and a negative peak at 240 nm This spec-trum could correspond to a mixture of different G-quadruplex conformations or a mixed parallel⁄ anti-parallel G-quadruplex [47,49,55] The CD profile of TKQ1 was significantly different from that of the modi-fied sequence TKQ1m (Fig 4A), whereas modification
of the TKQ2 sequence resulted in only a small spectral change (Fig 4B) This observation is consistent with the NMR and TDS data shown above, supporting the observation that, among the four sequences, only TKQ1 forms a significant population of G-quadru-plex(es)
Thermal stability of TKQ1 – UV melting experiments
To assess the thermal stability of the structures of TKQ1, TKQ1m, TKQ2 and TKQ2m, we performed
–0.4 –0.2 0 0.2 0.4 0.6 0.8 1
Wavelength (nm)
–0.4 –0.2 0 0.2 0.4 0.6 0.8 1
Wavelength (nm)
Fig 3 Normalized UV absorbance TDS of:
(A) TKQ1 (continuous line) and TKQ1m (red
dotted line); and (B) TKQ2 (continuous line)
and TKQ2m (red dotted line) Experimental
conditions: DNA concentration, 4 l M ;
70 m M KCl; 20 m M potassium phosphate
(pH 7.0).
Trang 5melting experiments Folding⁄ unfolding processes of
G-quadruplexes can be monitored by the change in
UV absorption at 295 nm as a function of temperature
[56] Typical denaturation profiles of TKQ1, TKQ1m,
TKQ2 and TKQ2m, as measured by the 295 nm
absorbance, are presented in Fig 5A,B At heating
and cooling rates of 0.5CÆmin)1, the melting and
folding profiles were superimposable, indicating
equi-librium processes Only TKQ1 exhibited a
characteris-tic profile of G-quadruplex melting curves, with a
decrease in the 295 nm absorbance upon increasing
temperature The G-quadruplex melting⁄ folding
transi-tion of TKQ1 was more evident in the presence of
higher K+ concentrations (Fig 5C) The stability of
the structure increased as the K+ concentration
increased, and its melting temperature reached 50C
at 500 mm K+ (Fig 5D) In the cases of TKQ1m, TKQ2 and TKQ2m, the increase in the 295 nm absor-bance when the temperature increased and⁄ or the absence of significant cooperative transitions showed that these sequences did not form G-quadruplexes
Native gel electrophoresis Native PAGE was performed to assess the molecular sizes and shapes of the structures formed by TKQ1 and TKQ2 A 21-nucleotide oligonucleotide (dT21) for TKQ1 and a 22-nucleotide oligonucleotide (dT22) for TKQ2 were used as controls to check relative mobility TKQ1 migrated faster than dT21 of the same length (Fig 6A), consistent with the formation of a mono-meric intramolecular G-quadruplex structure of
–1
–0.5
0
0.5
1
1.5
2
2.5
3 deg·
Wavelength (nm)
–1 –0.5 0 0.5 1 1.5 2 2.5
3 deg·
Wavelength (nm)
Fig 4 CD spectra of: (A) TKQ1 (continuous line) and TKQ1m (red dotted line); and (B) TKQ2 (continuous line) and TKQ2m (red dot-ted line) Experimental conditions: DNA concentration, 4 l M ; temperature, 20 C;
70 m M KCl; 20 m M potassium phosphate (pH 7.0).
0.16
0.168
0.176
0.184
0.192
0.2
0.208
0.14 0.15 0.16 0.17 0.18 0.19
35 40 45 50 55
Tm
0 100 200 300 400 500
0.155
0.16
0.165
0.17
0.175
0.18
0.185
0.19
0.13 0.14 0.15 0.16 0.17 0.18
Temperature (°C)
0.155 0.16 0.165 0.17
0.155 0.16 0.165 0.17 0.175 0.18 0.185
Fig 5 UV melting curves recorded at
295 nm, with a DNA concentration of 4 l M (A) TKQ1 (filled circles, left axis) and TKQ1m (red open circles, right axis) The buffer con-tained 70 m M KCl and 20 m M potassium phosphate (pH 7.0) (B) TKQ2 (filled circles, left axis) and TKQ2m (red open circles, right axis) The buffer contained 70 m M KCl and
20 m M potassium phosphate (pH 7.0) (C) Melting profiles of TKQ1 recorded at differ-ent KCl concdiffer-entrations: 70 m M (continuous line), 300 m M (dotted line), 400 m M (green squares) and 500 m M (red open circles) All experiments were performed in the pres-ence of 20 m M potassium phosphate (pH 7.0) Right axis for 300, 400, 500 m M KCl; left axis for 70 m M KCl (D) Plot of the melting temperature (T m ) of TKQ1 as a function of KCl concentration.
Trang 6TKQ1 TKQ1m, on the other hand, migrated more
slowly than TKQ1 but faster than dT21 The
migra-tion profile of TKQ1m is consistent with the
disrup-tion of the TKQ1 G-quadruplex structure, resulting in
a less compact intramolecular structure (e.g a hairpin,
as suggested by NMR data), induced by G-to-A
sub-stitutions
In contrast, migrations of TKQ2 and TKQ2m were
similar and both faster than that of dT22 (Fig 6B)
This result was in agreement with NMR, UV and CD
data indicating the absence of quadruplex formation
by TKQ2: the G-to-A mutation that was designed to
specifically disrupt potential quadruplex formation did
not induce a significant conformational change A
slightly faster migration of TKQ2 and TKQ2m than of
dT22 could be explained by the formation of an
intra-molecular structure, such as a hairpin involving
Watson–Crick base pairs
Base substitutions in TKQ1 result in increased
promoter activity
Previously, it has been reported that stabilization of a
G-quadruplex motif in the c-MYC promoter by the
specific ligand TMPyP4 results in decreased promoter
activity On the other hand, substitution of a single
nucleotide (G to A) that was critical for the
G-quadru-plex motif gave approximately three-fold increased
promoter activity [30] Another such example is
PDGF-A, where a stable parallel G-quadruplex motif
in the promoter was shown to regulate PDGF-A
expression [39] Regulation of transcription by a
G-quadruplex motif has also been demonstrated in the
case of the KRAS proto-oncogene, where, in the
pres-ence of the cationic porphyrin TMPyP4, promoter
activity is reduced to 20% of the normal value [38]
We hypothesized that formation of a G-quadruplex
structure by TKQ1 could be of significance in the
transcription of TK1 To test this hypothesis, the func-tional promoter of TK1 [52] (Fig 1B) was cloned upstream of the firefly luciferase gene in the pGL2 pro-moter (pTK1), and the propro-moter activity of pTK1 was measured 24 and 48 h after transfection in A549 cells (see Experimental procedures) The difference in trans-fection efficiency was normalized with Renilla lucifer-ase expression in each clucifer-ase As discussed above,
in vitro, TKQ1 forms an intramolecular G-quadruplex, whereas TKQ1m does not We anticipated that if the G-quadruplex adopted by TKQ1 was involved in the transcription of TK1, specific nucleotide substitutions that disrupted this G-quadruplex (e.g TKQ1m) would alter the luciferase activity We found 2-fold and 2.7-fold increases in promoter activity at 24 and 48 h, respectively, for promoter pTKQ1m (carrying the TKQ1m modification) relative to pTK1 (Fig 7) Fur-thermore, as an additional control, we studied the sequence TKQ2 (Table 1) Although TKQ2 contains several G-tracts, it was shown that this sequence does not form G-quadruplexes in vitro Therefore, in con-trast the situation with TKQ1, we expected that a mutation within TKQ2 would not affect TK1 moter activity Indeed, we noted no change in the pro-moter activity of pTKQ2m (carrying the TKQ2m modification) relative to that of pTK1 at 24 h, whereas there was a marginal decrease at 48 h (Fig 7) Taken together, these results suggested that the G-quadruplex adopted by TKQ1 may be involved in suppression of TK1promoter activity
Fig 6 Native gel electrophoresis PAGE of potential
G-quadruplex-forming sequences and their modified variants under nondenaturing
(native) conditions (A) dT21 marker, TKQ1, and TKQ1m (B) dT22
marker, TKQ2, and TKQ2m Experimental conditions: 15%
nonde-naturing polyacrylamide gel, 70 m M KCl in 1· TAE.
0 0.5 1 1.5 2 2.5 3
24 h
48 h
Fig 7 G-quadruplex motif alters the activity of the TK1 promoter Normalized luciferase activity of pTKQ1m and pTKQ2m relative to pTK1 at 24 and 48 h following transfection in human lung cancer cells A549 Error bars in all experiments denote standard deviation observed across three independent experiments.
Trang 7We identified two potential G-quadruplex-forming
sequences, TKQ1 and TKQ2 (Fig 1B), within the
min-imal functional promoter of human TK1, which was
taken from )89 to +58 with reference to the TSS In
a study by Arcot et al [52], it was shown that
progres-sive deletion of upstream regions from the promoter
()457 to +34 with respect to the TSS) of human TK1
resulted in decreased chloramphenicol acetyltransferase
activity, wherein the minimal promoter region from
)88 to +34 was shown to have an activity of 28
(nor-malized chloramphenicol acetyltransferase activity)
We were interested in deciphering the effect of two
potential G-quadruplex-forming sequences found in
this minimal promoter region on promoter activity
Of these, TKQ1 showed characteristics of a novel
G-quadruplex motif in vitro, whereas TKQ2 did not
This was confirmed by incorporating specific
nucleo-tide substitutions within TKQ1 and TKQ2 (TKQ1m
and TKQ2m, respectively) that were intended to
dis-rupt the G-quadruplex motif TKQ1m showed clear
signs of losing secondary structure; in contrast,
TKQ2m did not show any noticeable change These
findings were supported by biophysical and
biochemi-cal experiments, including NMR, UV and CD
spec-troscopy and gel electrophoresis Interestingly, we
observed that TKQ1, but not TKQ2, appeared to
affect the promoter activity of TK1 in A549 cells The
fact that disruption of TKQ1, but not TKQ2, leads to
an appreciable change in the promoter activity of
pTK1 suggests involvement of the G-quadruplex motif
formed by TKQ1 in regulating TK1 expression
Inter-estingly, to the best of our knowledge, TKQ1 is the
first G-quadruplex motif overlapping a TSS to be
reported, and it is therefore possible that it functions
independently of any transcription factor binding In
line with this, we did not find any transcription
factor-binding site overlapping the TKQ1 sequence (searched
for with transfac 2.1)
The characteristic nature of the TK1 G-quadruplex
motif with tracts of two guanines is noteworthy
Although this is the first time that it has been studied
in a regulatory context, examples of its biological role
have been reported Apart from the retinoblastoma
susceptibility gene and the thrombin-binding aptamer
(see above), GGA triplet repeats that may adopt
G-quadruplex motifs with a core of two G-tetrads [44]
are widely dispersed throughout eukaryotic genomes,
and are frequently located within biologically
impor-tant gene regulatory regions and recombination hot
spots The Bombyx mori telomere repeat d(TTAGG)
[42,57] and the yeast telomeric repeat d(TGGTGGC)
[45] were also shown to form stable G-quadruplex motifs Interestingly, the nick site for adenoassociated virus type 2 on human chromosome 19 was observed
to fold into a quadruplex structure The sequence, GGCGGCGGTTGGGGCTCG, indicates a quadru-plex motif comprising two G-tetrads in the core [41]
In addition to this, RNA sequences containing runs of two guanines could also form quadruplex motifs and
be physiological targets of the fragile X mental retar-dation protein [40] However, we note that the pres-ence of several tracts of two guanines or three guanines does not necessarily imply the formation of two-G-tetrad or three-G-tetrad structures, respectively Recent structural studies of G-quadruplexes [58,59] revealed various unusual folding patterns: for a G-quadruplex formed in the c-MYC promoter [58], a guanine in a continuous G-tract is not involved in the G-tetrad core, but is displaced by a ‘snap-back’ guan-ine further downstream in the sequence; for a G-quad-ruplex in the c-KIT promoter [59], an isolated guanine
is involved in G-tetrad core formation, despite the presence of four-three-guanine tracts In an analogous way, it is possible that the G-quadruplex motif adopted by the TKQ1 sequence involves not only G-tracts but also one or more isolated guanines in the tetrad formation
Consistent with the expectation that a G-quadruplex motif with only two stacked G-tetrads would be of low stability, the melting temperature of the G-quadru-plex motif adopted by TKQ1 was 35–40 C at
90 mm K+, and increased at higher K+ concentra-tions In contrast to the general belief that such struc-tures may be of limited significance, we observed a significant change in promoter activity that was influ-enced by the presence⁄ absence of the G-quadruplex motif formed by TKQ1 Furthermore, one must con-sider that the formation of such structures in vivo may
be facilitated by various cellular factors, proteins or other intracellular ligands Consistent with this, several proteins that bind G-quadruplex motifs have been reported [60], supporting the possibility that quadru-plex motifs are sequestered by proteins inside cells, in which case protein recognition would be critical rela-tive to stability per se It can also be argued that motifs of moderate⁄ low stability could be useful, in relation to stable ones, in potential regulatory roles where the contextual presence⁄ absence of the structure could be significant However, more evidence is required to distinguish between these possibilities
In conclusion, our results identify a novel G-quadru-plex motif in the promoter of TK1 and suggest its role
as a ‘repressor’ element in the transcription of TK1, as specific disruption of the quadruplex motif resulted in
Trang 8increased promoter activity A role for a G-quadruplex
motif constituting two stacks of G-tetrads in gene
tran-scription has not been reported before, and therefore
this study opens yet another avenue for exploration of
the role of quadruplex motifs
Experimental procedures
DNA sample preparation
Oligonucleotides (Table 1) were chemically synthesized at a
1 lmol scale on an ABI 394 synthesizer, and purified with
cartridges (Poly Pack II; Glen Research) as described by
the manufacturer All concentrations were expressed in
strand molarity, using a nearest-neighbor approximation
for the absorption coefficients of the unfolded species [61]
Samples were dialyzed successively against 50 mm KCl
solution and against water Unless otherwise stated,
experi-ments were carried out in a buffer containing 20 mm
potas-sium phosphate (pH 7) and 70 mm KCl
NMR spectroscopy
NMR experiments were performed on 600 MHz and
700 MHz Bruker spectrometers at 25C Proton spectra in
H2O were recorded using JR-type pulse sequences for water
suppression [62,63] The DNA concentration in NMR
sam-ples was typically 0.5 mm The solution contained 90%
H2O and 10% D2O The oligonucleotides were heated at
95C for 10 min, and allowed to slowly cool down to
room temperature overnight
UV melting experiments
The thermal stability of different oligonucleotides was
char-acterized in heating⁄ cooling experiments by recording the
UV absorbance at 295 nm as a function of temperature
[53], with a Cary 300 VARIAN Bio UV⁄ Vis
spectropho-tometer The heating and cooling rates were 0.5CÆmin)1
Experiments were performed with 1 cm pathlength quartz
cuvettes The DNA concentration was 4 lm All melting
profiles were perfectly reversible at the chosen temperature
gradient, indicating that these curves corresponded to the
equilibrium curves
TDSs
The TDS of a nucleic acid is obtained by simply recording
the UV absorbance spectra of the unfolded and folded
states at temperatures, respectively, above and below its
melting temperature (Tm) The difference between these two
spectra is defined as the TDS The TDS can provide specific
signatures of different DNA and RNA structural
confor-mations [50] Spectra were recorded between 220 and
320 nm on a Cary 300 VARIAN Bio UV⁄ Vis spectropho-tometer, using quartz cuvettes with an optical pathlength of
1 cm The DNA concentration was 4 lm
CD
CD spectra were recorded on a JASCO-810 spectropolarim-eter, using a 1 cm pathlength quartz cuvette in a reaction volume of 800 lL The concentration of oligonucleotides was 4 lm They were heated at 95C for 10 min, and allowed to slowly cool down to room temperature over-night Scans were performed at 20C over a wavelength range of 220–320 nm, with a scanning speed of
200 nm min)1 An average of three scans was taken, the spectrum of the buffer was subtracted, and the data were zero-corrected at 320 nm The spectra were finally normal-ized to the concentration of the DNA samples
Nondenaturing gel electrophoresis
Oligonucleotides were end-labeled with [32P]ATP[cP], using T4 polynucleotide kinase Labeled oligonucleotides were purified with Sephadex G25 columns to remove free ATP Corresponding 21-nucleotide and 22-nucleotide marker oli-gonucleotides (dT21 and dT22) were prepared similarly Labeled oligonucleotides were heated at 95C in 20 mm potassium phosphate buffer (pH 7.0) containing 70 mm KCl for 10 min, and then gradually cooled to room temper-ature overnight The samples were run on nondenaturing 15% polyacrylamide gel containing 70 mm KCl in 1· TAE buffer at 100 V for 4 h
Plasmid construction and site-directed mutagenesis
The promoter sequence of 151 base pairs, including the minimal functional promoter region based on previous experimental characterization of TK1 [61], was amplified from genomic DNA with forward (5¢-AAATCTCCCCTC GAGTCAGCGG-3¢) and reverse (5¢-AGCTCATTAAGCT TCCGGGAAGTTC-3¢) primers harboring restriction sites for XhoI and HindIII, respectively The amplified product was purified from gel, and then subjected to restriction diges-tion with both enzymes The digested product was cloned upstream of the firefly luciferase gene in the pGL2 (basic) vector from Promega (Madison, WI, USA), which was also digested with XhoI and HindIII The clones obtained were then screened by restriction digestion with XhoI and HindIII and further confirmed by sequencing Two independent site-directed mutants were made, representing: (a) TKQ1m (with a GG to AA substitution in TKQ1); and (b) TKQ2m (with a G to A substitution in TKQ2; see Table 1) Substitu-tions were incorporated by the use of primers containing the desired mutation, with the Quick Change Site-Directed mutagenesis Kit from Stratagene, according to the
Trang 9manufacturer’s protocol Plasmids were screened by
sequencing to obtain the desired mutant
Transfection and luciferase assay
Human lung cancer cell line A549 was cultured in DMEM
One day prior to transfection 6 · 105cells per well were
seeded in a six-well plate to obtain 90–95% confluent cells
before transfection Transfection was performed with
Lipo-fectamine 2000 (Invitrogen, Invitrogen BioServices India
Pvt Ltd, Whitefield, Bangalore), according to the
manufac-turer’s protocol For normalization of transfection efficiency,
Renilla luciferase plasmid (pGL4 from Promega) was
co-transfected in each well Cells were lysed either 24 or 48 h
after transfection, and luciferase assays were performed for
both firefly (pGL2) and Renilla (pGL4) luciferase in each
sample, with the dual luciferase assay kit from Promega,
according to the manufacturer’s protocol Firefly luciferase
counts were normalized with Renilla luciferase counts Three
independent experiments were performed in triplicate, and
the results were used for the measurement of standard
devia-tion All reporter assays were conducted at 25C
Acknowledgements
We thank members of the Chowdhury laboratory for
helpful discussion and comments on the manuscript,
and V Yadav for assistance with making some of the
figures This research was supported by fellowships
from CSIR (A Kumar and A Verma) and research
grants to S Chowdhury from the Department of
Science and Technology (DST⁄ SJF ⁄ LS-03) R Basundra
is in receipt of a project fellowship from CMM 0017
(CSIR Task Force Project) Research performed in the
Phan laboratory was supported by Singapore Ministry
of Education grant ARC30⁄ 07, Nanyang
Technologi-cal University (NTU) grant RG62⁄ 07 and Singapore
Biomedical Research Council grant 07⁄ 1 ⁄ 22 ⁄ 19 ⁄ 542 to
A T Phan We thank the Division of Chemistry and
Biological Chemistry (NTU School of Physical and
Mathematical Sciences) and the NTU School of
Bio-logical Sciences for granting us access to their CD
spectropolarimeter and NMR spectrometers We thank
Professor L Nordenskio¨ld (NTU School of Biological
Sciences) for allowing us to use the UV
spectropho-tometer of his laboratory
References
1 Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V,
Lindblad-Toh K, Lander ES & Kellis M (2005)
System-atic discovery of regulatory motifs in human promoters
and 3¢ UTRs by comparison of several mammals
Nature 434, 338–345
2 Bacolla A & Wells RD (2004) Non-B DNA conforma-tions, genomic rearrangements, and human disease
J Biol Chem 279, 47411–47414
3 Pedersen AG, Jensen LJ, Brunak S, Staerfeldt HH & Ussery DW (2000) A DNA structural atlas for Escheri-chia coli J Mol Biol 299, 907–930
4 Perez-Martin J & de Lorenzo V (1997) Clues and conse-quences of DNA bending in transcription Annu Rev Microbiol 51, 593–628
5 Sinden RR (1994) DNA: Structure and Function Academic Press, San Diego
6 Wells RD (2007) Non-B DNA conformations, mutagen-esis and disease Trends Biochem Sci 32, 271–278
7 Hatfield GW & Benham CJ (2002) DNA topology-med-iated control of global gene expression in Escherichia coli Annu Rev Genet 36, 175–203
8 Rich A & Zhang S (2003) Timeline: Z-DNA: the long road to biological function Nat Rev Genet 4, 566–572
9 Huppert JL & Balasubramanian S (2007) G-quadru-plexes in promoters throughout the human genome Nucleic Acids Res 35, 406–413
10 Simonsson T, Pecinka P & Kubista M (1998) DNA tetraplex formation in the control region of c-myc Nucleic Acids Res 26, 1167–1172
11 Burge S, Parkinson GN, Hazel P, Todd AK & Neidle S (2006) Quadruplex DNA: sequence, topology and struc-ture Nucleic Acids Res 34, 5402–5415
12 Davis JT (2004) G-quartets 40 years later: from 5¢-GMP to molecular biology and supramolecular chemistry Angew Chem Int Ed Engl 43, 668–698
13 Gellert M, Lipsett MN & Davies DR (1962) Helix formation by guanylic acid Proc Natl Acad Sci USA
48, 2013–2018
14 Patel DJ, Phan AT & Kuryavyi V (2007) Human telo-mere, oncogenic promoter and 5¢-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics Nucleic Acids Res 35, 7429–7455
15 Sen D & Gilbert W (1988) Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis Nature 334, 364–366
16 Williamson JR, Raghuraman MK & Cech TR (1989) Monovalent cation-induced structure of telomeric DNA: the G-quartet model Cell 59, 871–880
17 Dunnick W, Hertz GZ, Scappino L & Gritzmacher C (1993) DNA sequences at immunoglobulin switch region recombination sites Nucleic Acids Res 21, 365– 372
18 Weitzmann MN, Woodford KJ & Usdin K (1997) DNA secondary structures and the evolution of hyper-variable tandem arrays J Biol Chem 272, 9517–9523
19 Hanakahi LA, Sun H & Maizels N (1999) High affinity interactions of nucleolin with G-G-paired rDNA J Biol Chem 274, 15908–15912
20 Mani P, Yadav VK, Das SK & Chowdhury S (2009) Genome-wide analyses of recombination prone regions
Trang 10predict role of DNA structural motif in recombination.
PLoS ONE 4, e4399
21 Halder K, Halder R & Chowdhury S (2009)
Genome-wide analysis predicts DNA structural motifs as
nucleo-some exclusion signals Mol Biosyst 5, 1703–1712
22 Howell RM, Woodford KJ, Weitzmann MN & Usdin
K (1996) The chicken beta-globin gene promoter forms
a novel ‘cinched’ tetrahelical structure J Biol Chem
271, 5208–5214
23 Murchie AI & Lilley DM (1992) Retinoblastoma
sus-ceptibility genes contain 5¢ sequences with a high
pro-pensity to form guanine-tetrad structures Nucleic Acids
Res 20, 49–53
24 Catasti P, Chen X, Moyzis RK, Bradbury EM &
Gupta G (1996) Structure–function correlations of the
insulin-linked polymorphic region J Mol Biol 264,
534–545
25 Kilpatrick MW, Torri A, Kang DS, Engler JA & Wells
RD (1986) Unusual DNA structures in the adenovirus
genome J Biol Chem 261, 11350–11354
26 Ma D, Xing Z, Liu B, Pedigo NG, Zimmer SG, Bai Z,
Postel EH & Kaetzel DM (2002) H1 and
NM23-H2 repress transcriptional activities of
nuclease-hyper-sensitive elements in the platelet-derived growth
factor-A promoter J Biol Chem 277, 1560–1567
27 Rankin S, Reszka AP, Huppert J, Zloh M, Parkinson
GN, Todd AK, Ladame S, Balasubramanian S &
Neidle S (2005) Putative DNA quadruplex formation
within the human c-kit oncogene J Am Chem Soc 127,
10584–10589
28 De AR, Wood S, Sun D, Hurley LH & Ebbinghaus
SW (2005) Evidence for the presence of a guanine
quad-ruplex forming region within a polypurine tract of the
hypoxia inducible factor 1alpha promoter Biochemistry
44, 16341–16350
29 Dexheimer TS, Sun D & Hurley LH (2006)
Deconvolut-ing the structural and drug-recognition complexity of
the G-quadruplex-forming region upstream of the bcl-2
P1 promoter J Am Chem Soc 128, 5404–5415
30 Siddiqui-Jain A, Grand CL, Bearss DJ & Hurley LH
(2002) Direct evidence for a G-quadruplex in a
pro-moter region and its targeting with a small molecule to
repress c-MYC transcription Proc Natl Acad Sci USA
99, 11593–11598
31 Thakur RK, Kumar P, Halder K, Verma A, Kar A,
Parent JL, Basundra R, Kumar A & Chowdhury S
(2009) Metastases suppressor NM23-H2 interaction
with G-quadruplex DNA within c-MYC promoter
nuclease hypersensitive element induces c-MYC
expres-sion Nucleic Acids Res 37, 172–183
32 Rawal P, Kummarasetti VB, Ravindran J, Kumar N,
Halder K, Sharma R, Mukerji M, Das SK &
Chowdhu-ry S (2006) Genome-wide prediction of G4 DNA as
regulatory motifs: role in Escherichia coli global
regulation Genome Res 16, 644–655
33 Du Z, Kong P, Gao Y & Li N (2007) Enrichment of G4 DNA motif in transcriptional regulatory region of chicken genome Biochem Biophys Res Commun 354, 1067–1070
34 Yadav VK, Abraham JK, Mani P, Kulshrestha R & Chowdhury S (2008) QuadBase: genome-wide database
of G4 DNA – occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes Nucleic Acids Res 36, D381–D385
35 Zhao Y, Du Z & Li N (2007) Extensive selection for the enrichment of G4 DNA motifs in transcriptional regulatory regions of warm blooded animals FEBS Lett
581, 1951–1956
36 Verma A, Yadav VK, Basundra R, Kumar A & Chowdhury S (2009) Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells Nucleic Acids Res 37, 4194–4204
37 Verma A, Halder K, Halder R, Yadav VK, Rawal P, Thakur RK, Mohd F, Sharma A & Chowdhury S (2008) Genome-wide computational and expression analyses reveal G-quadruplex DNA motifs as conserved cis-regulatory elements in human and related species
J Med Chem 51, 5641–5649
38 Cogoi S & Xodo LE (2006) G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription Nucleic Acids Res 34, 2536– 2549
39 Qin Y, Rezler EM, Gokhale V, Sun D & Hurley LH (2007) Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4 Nucleic Acids Res 35, 7698–7713
40 Darnell JC, Jensen KB, Jin P, Brown V, Warren ST & Darnell RB (2001) Fragile X mental retardation protein targets G quartet mRNAs important for neuronal func-tion Cell 107, 489–499
41 Jang MY, Yarborough OH III, Conyers GB, McPhie
P & Owens RA (2005) Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19 J Virol 79, 3544– 3556
42 Kettani A, Bouaziz S, Wang W, Jones RA & Patel DJ (1997) Bombyx mori single repeat telomeric DNA sequence forms a G-quadruplex capped by base triads Nat Struct Biol 4, 382–389
43 Macaya RF, Schultze P, Smith FW, Roe JA & Feigon
J (1993) Thrombin-binding DNA aptamer forms a uni-molecular quadruplex structure in solution Proc Natl Acad Sci USA 90, 3745–3749
44 Matsugami A, Okuizumi T, Uesugi S & Katahira M (2003) Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA J Biol Chem 278, 28147–28153