I. Bent DNA in biological
reactions
Genomic DNA is a source of
genetic and functional information in the form of nucleotide sequences. DNA has
a relatively simple composition of purine or pyrimidine bases attached to a
common phosphate backbone which would not give rise much local structural
variation. However, recent studies have revealed that non-B DNA or
"unusual" DNA structures are actively involved in biologically
important reactions as functional elements (Crothers et al., 1990). For example, Z-DNA is known to activate
transcription and recombination presumably by exposing bases on the outside of
the phosphate backbone, thereby increasing the chance of interaction with
proteins or other bases (Rich et al.,
1984; Blaho and Wells, 1989). Other structures such as triplex DNA and
unisomorphic DNA have been discussed in studies of transcriptional activation
and recombination mechanisms (Crothers et al., 1990; Soyfer and Potaman, 1996). Although their
mechanisms of action are quite different, these non-B DNA structures seem to
act as signals for recognition by protein factors in a way different from
searching nucleotide bases.
Bent DNA was first discovered
as anomalous migration of DNA fragments in gels, and has been extensively
studied due to its potential involvement as a transcriptional modulator
(Travers, 1989; Hagerman, 1990; Crothers et al., 1990; Trifonov, 1991; Ioshikhes et al., 1996; Werner et al., 1996). Such structures also play important roles in
activation of recombination (Nash, 1990). Binding of proteins to DNA can cause
DNA bending, which would further enhance the recognition by other proteins of
the site of the protein-DNA complex (Khan and Crothers, 1992). Therefore, DNA
bending formed by the intrinsic nature of the DNA or by protein binding, as
well as sequence information, would be a good signal for structural
recognition. Note that the non-B DNA structures themselves are the result of
nucleotide sequence information, although the sequence-structure relationship
is not simple.
II. Assays for DNA bend sites
The presence and the location
of DNA bend sites can be analyzed by several assays. Among these, the circular
permutation assay (Wu and Crothers, 1984) has been commonly utilized for
mapping the bend sites in DNA fragments of several hundred bp to 1 kb in
length. The assay procedure is schematically illustrated in Figure 1. Plasmids containing the tandem dimers of the
fragment of interest are cloned, and after digestion of the plasmid DNA with
the restriction enzymes that cut the fragment once the plasmid DNA samples are
resolved by electrophoresis. We routinely use 8% polyacrylamide (mono: bis =
29: 1) gels, which can resolve bands up to 1 kb in size (Wada-Kiyama and
Kiyama, 1994). Electrophoresis should be performed at 4˚C twice or three
times overnight to obtain better resolution of the bands. Cloning of the tandem
dimers can be performed by direct cloning of two identical fragments into the
multiple cloning site of the vector, or cloning them into two different sites
one after another. Most of the clones could be obtained by the former method
under conditions where the fragment (0.1 to 1 mg) is present
in excess over the amount of vector DNA (ten times or more) in a small-volume
reaction mixture (5 to 10 ml). After transformation of E.
coli, only direct repeats of the
fragments, but not
Fig. 1. Assay for bent DNA.
inverted repeats, can be obtained in dimeric form.
The circular permutation assay
is a very simple and reliable method to identify and roughly map DNA bend
sites. The results of mapping are reproducible under identical electrophoresis
conditions and generally reproducible among different subclones containing the
same site, and the patterns can be interpreted without complicate calculations.
However, this method does have some technical limitations. Firstly, the assay
is totally dependent upon the availability of suitable restriction sites. If
there are no appropriate restriction sites, the bend sites cannot be localized
to a small region of DNA. Secondly, the DNA structure of the other part of the
same fragment could influence the mobility. As a result of this effect, mapping
a bend site to a very small region by this method would not always give a
precise location. Although rare, we observed a slight difference in the
location of a site in the e-globin gene region between
clones of different sizes used for mapping. Therefore, the lower limit of
resolution would be 50 to 100 bp.
The more precise location of
the bend sites could be achieved by several other methods. For a relatively
large region, sites can be examined with deletion constructs. When the bend
center is completely deleted from the construct, all restricted fragments have
the same mobility. Meanwhile, mapping the site in regions of approximately 100
bp or less would be achieved by using concatenated oligonucleotides of 20 or 30
bp (Wada-Kiyama and Kiyama, 1995). When the oligonucleotide contains a bend
site, the concatemers exhibit retardation on polyacrylamide gel
electrophoresis. The effect of bending is greater as the length of the oligonucleotide
increases. The bend angle can be estimated by comigration of standards such as
A3N7
(0.63˚/ base) (Calladine et al.,
1988). The bend angle could also be determined by the assay based on ring
closure of concatenated oligonucleotides (Zahn and Blattner, 1987).
III. The human b-globin locus
Using the circular permutation
assay, we mapped the DNA bend sites in the human b-globin locus
which is located on chromosome 11 and contains five (e-, Gg-, Ag-, d- and b-) active genes and one (yb-) pseudogene (Figure 2). This locus is ideal for mapping the sites because
the nucleotide sequence of over 70 kb has been reported. Furthermore, since
most of the locus is intergenic, the influence of the coding region could be
excluded. The similarity of the exon-intron structure and the sequences of the
flanking region would be ideal for evolutional study of the sites. The
chromatin structure in this locus has been extensively characterized in that
switching of globin gene expression is paralleled by alterations of chromatin
structure as revealed by DNase I-hypersensitivity (reviewed by
Stamatoyannopoulos and Nienhuis, 1993; Evans et al., 1990).
The periodicity of the bend
sites at intervals of 680 bp on average was first identified in the e-globin region (Wada-Kiyama and Kiyama, 1994). Further studies of the
sites in the regions of other globin genes revealed that relative locations of
the sites to their cap sites were conserved among most of the members of this
family which were separated as much as 200 million years ago.
The duplication of the two g-globin genes, which occurred most recently, was immediately followed
by diversification of the non-coding region by as much as 70%, while all of the
bend sites were conserved (Slightom et al., 1980). Insertion of an Alu
sequence might have disturbed the periodicity, although as observed in the
region upstream of the e-globin gene, the interval seemed to have returned to
the average after a long period of molecular evolution. The positions of the
bend sites were conserved even between the human b- and mouse bmaj-globin
genes (Wada-Kiyama and Kiyama, 1996b).
Mapping of over 90 bend sites in
the locus revealed that the periodicity of the bend sites exists throughout the
locus with an average interval of 680 bp (Wada-Kiyama and Kiyama, 1994, 1995,
1996b; unpublished results). However, we observed disturbance of the
periodicity at several locations. Interestingly, all of the locations of the
disturbed periodicity located upstream of the e-globin genes
that caused the distances of the adjacent bend sites to be longer than average
were found in or close to the DNase I-hypersensitive sites, which constitute
the locus control region (b-LCR). The b-LCR is composed of four or five developmentally-regulated DNase
I-hypersensitive sites (Crossley and Orkin, 1993; Evans et al., 1990; Felsenfeld, 1993). These sites are designated
as open chromatin regions and act as the sites of interaction of transcription
factors and the enhancer-binding protein NF-E2. This region interacts with the
promoter region of each member of the b-like globin gene
family and controls their expression during development. One of the DNase
I-hypersensitive sites, HS2 located 11 kb upstream of the cap site of the e-globin gene, was located in the center of two adjacent bend sites
separated by a distance of 860 bp, which is longer than average (unpublished
results). It seemed as if HS2 was placed far from the bend sites to minimize
the influence of the sites. This is discussed again below.
IV. The human c-myc and the immunoglobulin heavy chain m loci
The human c-myc gene has three exons and occupies a region of
approximately 5.5 kb on chromosome 8. As observed in the b-globin locus, this locus contained periodic bent DNA. DNA bend sites
were mapped at an average interval of 694.2 bp and were present in the 5'- and
3'- non-coding regions, introns and the non-coding exon (exon 1), but not
present in the coding region (Ohki et al., 1997). Interestingly, one of the bend sites corresponded to the
location of TATA box of the P2 promoter, suggesting that prebending of the
promoter region can facilitate transcriptional enhancement.
The c-myc gene is involved in the progression of Burkitt's
lymphoma by translocation of the locus into one of the immunoglobulin genes
located on chromosomes 2, 14 or 22. These translocation events often result in
reshuffling the location of regulatory elements. Deregulation of the expression
by juxtaposition of the m enhancer to the c-myc promoter is one of the mechanisms of tumor
progression caused by this oncogene. The mechanism of these translocation
events has not been well documented except that immunoglobulin-specific
recombination is somehow involved (Specer and Groudine, 1991). Translocation
junctions were formed at various locations in the locus yet no specific
sequences were commonly found in their immediate proximity. However, when the
periodic bent DNA was mapped in the c-myc and the Igm loci, at least three stable cell lines containing the
translocation junctions within these regions showed conservation of the
periodicity before and after the rearrangements (Figure 3). This would be readily explained if we assume that
the periodic bent DNA is a key element for chromatin structure. It would be
necessary for a stable cell line to maintain a similar chromatin structure as
to that before the rearrangement. Otherwise, a secondary rearrangement could
alter the sequence until a stable structure is eventually formed.
V. Other loci in eukaryotic
genomes
We have already determined
that periodic bent DNA is present in the human erythropoietin receptor and the
human estrogen receptor loci (Kuwabara et al., 1997; unpublished results). The intervals of the
sites in these loci were 651.2 or 688.1 bp, respectively, which are close to
the values for other loci (Table 1).
In both cases, its periodicity was disturbed by exons. For the estrogen
receptor gene, the alternative cap site located approximately 2 kb upstream of
the canonical site caused a shift of the nearby sites. For the erythropoietin
receptor gene, the 700 bp periodicity of bend sites was conserved even within
the long introns (1st and 6th introns) although the sites were shifted when the
length of introns was not sufficient to accommodate two sites. One of the sites
in the estrogen receptor gene contained motifs of the estrogen response
element, the binding site for the hormone-responsive trans-activating factors,
and had the affinity to the nuclear scaffold. This site might play a role in
determining the nuclear localization of this gene as well as a role in
transcriptional regulation.
For other loci of eukaryotes,
we examined the potential bend sites by a computer search (Wada-Kiyama, and
Kiyama, 1996a). Based on the observation that physically mapped bend sites
often contain A+T-rich sequences including An
or Tn tracts at intervals of ten or
multiples of ten nucleotides, we searched A2N8A2N8A2
and the complementary T2N8T2N8T2
for a periodicity. There was a statistically significant sequence periodicity
at an interval of roughly 700 bp in eukaryotic genomic DNA. This tendency was
absent in prokaryotes and in eukaryotic cDNA, suggesting that the periodicity
is universal among eukaryotic genomes, especially in intergenic regions.
VI. Biological significance of the bend sites
The observations that periodic
bent DNA is conserved during molecular evolution and its intervals are
maintained precisely in otherwise unstable intergenic regions suggested that
these sites are biologically relevant. Despite the systematic and organized
patterns of chromatin folding, no specific signals have been determined as key
elements for the folding mechanism. A computer search further revealed the
non-random distribution of nucleotide sequences on the genomic DNA, while it
failed to deduce any specific sequences in common, suggesting the presence of
unidentified codes which are not apparent from sequence information alone.
Therefore, judging from the periodicity and the length of their intervals,
periodic bent DNA may be closely associated with chromatin structure,
presumably with the formation of nucleosomes. We reported that some of the
sites were indeed involved in the formation of nucleosomes by having high
affinity to histone core particles (Wada-Kiyama and Kiyama, 1996b). Chromatin
structure seems to be extensively stabilized when the overall periodicity is
maintained before and after the rearrangement. Meanwhile, open chromatin
regions, revealed by DNase I-hypersensitivity (Gross and Garrard, 1988), could
be at least partly due to disturbance of the periodicity. While the nucleosome
phasing activity of these sites might be effective when the distances of the
bend sites are less than or equal to the length of four nucleosomes, open
chromatin regions would be more efficiently formed when their distances are
more than the length of four nucleosomes. Some of the sites seem to be used as
multiple sites for chromatin organization, as observed in the estrogen receptor
gene. We are currently investigating chromatin structure at the replication
origin based on the alignment of bend sites to examine the relationship of
these sites with DNA replication. Our results indicated that periodic bent DNA
is a key element of chromatin structure and plays a role in various biological
reactions.
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