(Received for publication, August 17, 1995)
From the
In order to increase our knowledge about the mechanisms that
regulate expression of human -like globin genes, we have used a
novel technique to analyze the chromatin structure in living cells.
This approach allowed us to detect specific DNA regions in vivo where nucleosome folding or unconstrained DNA supercoiling in
erythroid cells differs from that in non-erythroid cells. In this
method, we use 4,5`,8-trimethylpsoralen (TMP) as a probe capable of
detecting altered chromatin conformations. Our results show that TMP
binds to DNA with a higher affinity over the regions in the locus that
are actively expressed, including both the promoter and the transcribed
region. This higher affinity detected when comparing erythroid cells
with non-erythroid cells does not extend to other regions inside the
-globin cluster. Our data suggest that the observed effect is
likely due to nucleosome displacement. Alternatively, it could result
from localized DNA supercoiling, but not from widespread torsional
stress across the entire
-like globin locus as hypothesized
previously.
Human -globin cluster is located in chromosome 11 and
contains six genes expressed only in erythroid cells at different
developmental stages. The pattern of expression of these genes during
development is precisely regulated:
-gene is expressed during the
early stages of embryo development, the
-genes (
and
) are expressed during the fetal stages, and the
product of the
-gene is the most prominent form in adults. A
sequence located 5 to 15 kb (
)upstream from the embryonic
-globin gene plays an essential role on both the tissue-specific
and the developmental specific expression of the
-globin cluster.
This sequence has been named locus control region (LCR) and contains
four DNase I-hypersensitive sites: 5`HS-1, 5`HS-2, 5`HS-3, and 5`HS-4.
Chromatin structure analysis on the human
-globin cluster has
revealed that the entire locus is preferentially sensitive to DNase I
in erythroid cells(1, 2, 3, 4) .
However, natural deletions that eliminate the LCR elements, such as
that occurring in Hispanics (
)
-thalassemia,
prevent tissue-specific changes in chromatin structure and render the
-globin locus resistant to DNase I(5) . On the other hand,
there are evidences indicating that some LCR elements are able to exert
their effect only when integrated into the genome or at least when they
are folded into chromatin(6, 7, 8) .
According to these results, there is reason to believe that the LCR
elements may regulate
-globin locus expression through alterations
on the chromatin structure within the locus.
In the present work we
use a novel technique to compare chromatin structure in vivo in the -globin locus in K562 cells and HeLa cells. Our
approach is based on the original technique described by Cook et
al.(9) , where 4,5`,8-trimethylpsoralen (TMP) is used as a
probe for altered chromatin conformations. Variations on nucleosome
folding or unconstrained DNA supercoiling affect the rate of TMP
binding to DNA. Our results show that TMP cross-links DNA more
efficiently over the regions in the
-globin locus that are
actively transcribed in K562 cells than over the same DNA fragments in
the HeLa cells. The higher cross-linking rates detected in erythroid
cells when compared with non-erythroid cells do not extend to other
regions inside the
-globin cluster.
In this work we have mapped different regions in the
-globin locus using TMP as a probe. Mapping was carried out by
quantitating the in vivo TMP-induced double-stranded DNA
cross-linking frequency in the region analyzed. The quantification is
based on the ratio between single-stranded DNA and double-stranded DNA
obtained after a denaturation and renaturation process (see
``Experimental Procedures''). In the present assays, living
cells were treated with TMP at 4 °C and subsequently irradiated
with 360 nm UV light for various periods of time. Increasing
cross-linking efficiencies were obtained as a function of the time of
irradiation. This effect is shown in the Southern blot in Fig. 1. It was observed that after irradiation of the cells for
increasing periods of time, the intensity of the double-stranded DNA
band was augmented while that of the single-stranded DNA band
diminished.
Figure 1:
TMP binding affinity to DNA in K562
cells and HeLa cells over the human -globin gene. A,
- and
-globin genes
restriction map. Arrows indicate directions of transcription. Bracket indicates the 2.6-kb EcoRI fragment analyzed.
The probe used is shown by the solid bar. B = BamHI; R = EcoRI. B, agarose
gel analysis of TMP binding to DNA over
-globin gene.
Cross-linking rates are estimated based on the ratio between
double-stranded DNA (ds) and single-stranded DNA (ss)
for each lane (see ``Experimental Procedures''). Living cells
were exposed to different 360 nm UV radiation times: 0 min, lanes 1 and 6; 2 min, lanes 2 and 7; 4 min, lanes 3 and 8; 6 min, lanes 4 and 9; and 8 min, lanes 5 and 10. Lanes 1-5 represent the results obtained for HeLa cells and lanes
6-10 the results obtained for K562 cells. C,
, cross-linking rates for K562 cells.
, cross-linking
rates for HeLa cells. Slope value in K562 cells is higher than in HeLa
cells, which indicates a stronger affinity between TMP and DNA over the
-globin gene in K562
cells.
Fig. 1shows the results obtained when we
analyzed the -globin gene region in the erythroid cell
line K562 and in the non-erythroid cell line HeLa. When the
cross-linking rates for the 2.6-kb EcoRI fragment that
contains most of the
-globin gene were represented as
a function of the time of irradiation with UV light, the slope of the
regression lines obtained for both cell lines varied significantly.
Cross-linking efficiencies for K562 cells were higher than those
obtained for HeLa cells as indicated by the slope values of the
regression lines. According to this result, DNA in the
-globin gene of K562 cells is more accessible to TMP
than in the same region in HeLa cells.
K562 cells have normal globin
genomic maps and synthesize - and
-globin chains. In contrast with adult erythropoietic
cells, they don't contain detectable levels of
or
transcripts(2, 12, 13) . However, because
variations in expression have been described, we have analyzed the
transcriptional pattern in the actual K562 cells used for the
cross-linking studies. The cells expressed
- and
-globin transcripts as indicated by the presence of a
73-nucleotide-long primer extension product from primer 2 (Fig. 2). Neither
-globin gene nor
-globin gene
expression was detected. Control experiments were performed to confirm
that the absence of the expected 56-nucleotide-long primer extension
product from primer 1 was due to a lack of expression of
- and
-globin genes in K562 cells. On the other hand, HeLa is a
non-erythroid cell line, and, consequently, those cells don't
express any of the genes contained into the
-globin cluster.
Comparison between the cross-linking rates in the same fragments in
both cell lines allowed us to avoid any variations in TMP affinity to
DNA due to differences in the DNA sequence.
Figure 2:
Expression analysis of -,
-,
-, and
-globin genes in K562 cells.
Endogenous
- and
-globin mRNAs gave
primer extension products 73 nucleotides long. The absence of a
56-nucleotide-long primer extension product from primer 1 is indicative
of the lack of expression of
- and
-globin genes in K562
cells. Lanes 1 and 3, primer extension from 10 µg
total RNA; lanes 2 and 4, primer extension from 30
µg of total RNA.
In order to prove that
the differences found between K562 and HeLa cell lines were not due to
particular cell line properties, TMP affinity was analyzed over a
4.5-kb BamHI fragment containing the keratin 16 human gene
using as a probe the 3`-untranslated region of the keratin 16 cDNA (Fig. 3). The slopes obtained for K562 and HeLa cell lines were
almost identical, indicating that TMP has the same affinity for the DNA
in the human keratin 16 gene in both cell lines. Since neither K562
cells nor HeLa cells express keratin genes, we can therefore assume
that the TMP cross-linking behavior is independent of cell line
properties. Thus, it is more likely that the differences found in the
-globin cluster between K562 and HeLa cells reflect the existence
of specific chromatin structures associated to the active region in the
K562 cells.
Figure 3:
TMP binding affinity to DNA in K562 cells
and HeLa cells on the human keratin 16 gene. A, keratin 16
gene restriction map. Arrow shows the direction of
transcription of the keratin 16 gene. Bracket indicates the
4.5-kb BamHI fragment analyzed. Solid bar indicates
the 0.7-kb PstI fragment used as probe. B = BamHI; P = PstI. B, agarose
gel analysis of TMP binding to DNA over keratin 16 gene. ds = double-stranded DNA; ss = single-stranded
DNA. Living cells were exposed to different 360 nm UV radiation times:
0 min, lanes 1 and 6; 2 min, lanes 2 and 7; 4 min, lanes 3 and 8; 6 min, lanes 4 and 9; and 8 min, lanes 5 and 10. Lanes 1-5 represent the results obtained for HeLa cells,
and lanes 6-10 the results obtained for K562 cells. C, , cross-linking rates for K562 cells;
,
cross-linking rates for HeLa cells. Slope values obtained for both cell
lines are almost identical, which indicates identical TMP affinity to
DNA in K562 cells and HeLa cells for the fragment
analyzed.
Several other fragments from the genomic region
containing - and
-globin genes were
analyzed in order to define in more detail the specific domains where
TMP binds more efficiently to DNA. The
-globin gene is
located 4 kb upstream from the
-globin gene, and it is
also actively transcribed in K562 cells. We measured the TMP
cross-linking rate over the 2.6-kb BamHI fragment containing
the 5` end of the
-globin gene and its promoter. The
slope values for both cell lines are shown in Fig. 4a.
It could be observed that the slope obtained in K562 cells for this
fragment was higher than that obtained in HeLa cells. The cross-linking
rates found over
- and
-globin genes
indicated that in vivo TMP binding to DNA detects chromatin
structure alterations associated to actively transcribed regions in the
-globin cluster.
Figure 4:
TMP binding affinity to DNA in K562 cells
and HeLa cells over the -globin locus. A,
-globin
locus restriction map. Brackets indicate the fragments
analyzed. DNase I hypersensitive site 2 is indicated as Hs-2.
Different
-like globin genes are indicated. H = HindIII; B = BamHI; D = DraI; R = EcoRI; Bg = BglI; P = PstI. B, comparison of the cross-linking rates between K562 cells
and HeLa cells. Fragments analyzed are indicated by lowercase
letters. Slope values obtained from experiments similar to those
shown in Fig. 1and Fig. 2are plotted on the vertical axis. Solid bars show the data obtained for K562
cells, and hatched bars show the data obtained for HeLa cells.
Standard deviation is shown by the error bars. The ratio
between the slope values obtained in both cell lines is indicated for
every fragment.
Fig. 4b presents the results
obtained when we analyzed the 2.9-kb DraI fragment containing
the intergenic region between - and
-genes. In this case, the slope values obtained for
both cell lines did not display any difference, suggesting that
chromatin alterations are restricted to the transcribing regions and do
not extend to the adjacent regions. We also analyzed the 2.7-kb BamHI + BglI fragment containing 1 kb of the
3`-noncoding region next to
-gene (Fig. 4e). Even though some variations in slope value
between K562 cells and HeLa cells could be observed in this fragment,
the difference in both values was smaller than that obtained for the
2.6-kb EcoRI fragment already described.
This result can be explained by a dilution effect on the total
cross-linking efficiency in K562 cells over the
2.7-kb BamHI + BglI DNA fragment as a
consequence of the presence of a large 3`-nontranscribing region.
The 1.6-kb EcoRI fragment containing the promoter and a
small portion of the coding region (Fig. 4c) showed the highest cross-linking rates in
K562 cells. Taken together, this result and the small cross-linking
rates observed in the 2.9-kb fragment containing the intergenic region (Fig. 4b) are consistent with our notion that the
promoter region binds TMP very efficiently. The high cross-linking
rates detected in the promoter region could be explained by nucleosome
displacement over the promoter in K562 cells(14, 15) ,
which would increase the effective DNA length able to bind TMP. This
possibility is discussed below.
DNase I sensitivity experiments had
previously shown that chromatin structure is altered over more than 100
kb in the -globin locus(3, 4) . In order to
determine whether the overall change in chromatin structure does also
affect TMP cross-linking efficiency, we extended the TMP cross-linking
analysis to other regions inside the
-globin locus. The data
obtained for the DNA fragments including the
-globin gene and the
-globin gene are shown in Fig. 4, f and g, respectively. Both genes are expressed neither in K562
cells nor in HeLa cells, but a 5` DNase I hypersensitive site has been
described for the
-globin gene in K562 cells(2) . Our
results indicated that the chromatin structure alterations already
described in these regions in K562 cells do not increase TMP
cross-linking efficiency. In fact, the slope values obtained for the
fragment containing the
-globin gene were smaller in K562 cells
than in HeLa cells. We next analyzed the DNase I hypersensitive site 2
in the LCR because of its well known property to act as a powerful
enhancer in transfection experiments. The results are shown in Fig. 4h. K562 cells bound TMP at a significantly lower
rate than HeLa cells over this region, which probably reflects a higher
level of DNA protection in K562 cells as a consequence of protein
binding over erythroid-specific elements present in this fragment.
However, the possibility cannot be excluded that the enhancer and
insulator effects associated to LCR activity alter chromatin structure
or DNA supercoiling and, therefore, limit TMP binding.
Even though TMP binding to DNA depends largely on chromatin
structure, the results obtained when using TMP as a probe to reveal
structural alterations differ from the data obtained in DNase I
sensitivity experiments. Our results demonstrate that most of the
-globin locus does not display higher cross-linking rates of the
intercalating drug TMP in the erythroid cell line K562 than in the
non-erythroid cell line HeLa. In fact, some regions such as the DNase I
hypersensitive site 2 in the locus control region (LCR) do bind TMP
less efficiently in K562 cells. These results indicate that in vivo TMP binding to DNA does not seem to be affected by high order
chromatin structures.
DNA supercoiling is one of the main chromatin
properties able to affect TMP binding to DNA(16, 17) .
It had previously been hypothesized that the active state of the
-globin locus in erythroid cells could be associated to widespread
supercoiling across the entire locus. Our findings, in contrast,
showing higher TMP cross-linking rates in K562 cells than in HeLa cells
only in the regions that are actively transcribed do not favor the
notion of a large supercoiled domain associated to the
-globin
locus in erythroid cells.
Nucleosome folding is also able to affect
TMP binding to DNA(18, 19) . Several DNase I
hypersensitive sites have been mapped over the flanking regions of the
-globin genes(2) . Those hypersensitive sites are probably
associated with nucleosome displacement, as it has been shown for the
-globin genes(14, 15) . However, the DNase I
hypersensitive site located 5` to the
-globin gene does not seem
to increase significantly TMP cross-linking rates into the EcoRI 2.3-kb fragment (Fig. 4f), indicating
that displacement of only one nucleosome is not sufficient to cause a
detectable increase in the TMP binding rate over the fragments
analyzed. We therefore conclude that the significant increase in DNA
cross-linking rates observed in both
-globin genes is not only due
to nucleosome displacement on the promoter but it does more probably
reflect the existence of some other major structural changes associated
with transcription. It must be pointed out that changes are strictly
associated with the transcribed domains and do not affect the flanking
regions. Thus, the higher TMP cross-linking rates observed over the
transcribing regions in K562 cells compared with those obtained over
the same DNA fragment in HeLa cells should be due to a generalized
nucleosome release over the transcribing regions or to specific DNA
supercoiling associated to the RNA polymerase movement. It must be
pointed out that the domains that show differences in TMP cross-linking
rates are so small that none of the in vivo nicking treatments
already described are able to affect the DNA inside the domains at a
significant level(20, 21) . As a consequence, at this
moment we are not able to distinguish whether nucleosome displacement
or DNA supercoiling causes the observed differences in cross-linking
rates.
It has been shown that the nucleosome core is displaced and reformed elsewhere as a result of transcription(22) , but, as it has been concluded(23) , the histone octamer seems to step around the transcribing polymerase over a very short distance without leaving the template. According to this, nucleosome displacement during transcription would not increase the amount of nucleosome-free DNA inside the transcribing region. In this case, the high rates of cross-linking detected over the transcribing regions should be due to specific changes in DNA structure. Moreover, nucleosome transfer from DNA ahead of the polymerase to DNA behind it induces the formation of a transient bridging complex in which the octamer contacts both donor and acceptor DNA(23) . Thus, the DNA fragments located immediately behind and ahead of the polymerase would form a loop whose ends are attached to the same nucleosome. In this structure, the DNA inside the loop would accumulate most of the unconstrained negative supercoiling generated by the transcription complex (24) and would, therefore, become highly susceptible to TMP binding.
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