ABSTRACT
In vivo
, histone H1 plays an active role in establishing the transcriptionally
repressed chromatin state of the oocyte-type 5S RNA genes in the early stages of
Xenopus
development. By using fully defined
in vitro
system of chromatin assembly on plasmids with cloned oocyte- or somatic-type 5S gene repeats we found that the oocyte repeat which comprises
a 120 bp oocyte-type 5S RNA gene placed within the few hundred bp long native AT-rich flanks, but not the somatic repeat (a similar 120 bp somatic-type 5S RNA gene placed within native GC-rich flanks) enables histone H1 to realign the
nucleosomal core particles densely packed on plasmid DNA. The realignment
results in creation of the repeat unit of
~
240 bp and is achieved through complete removal of several core histone
complexes from plasmid template with the oocyte-type repeat. This effect of H1 is independent on the plasmid sequences and
seems to be solely due to the presence in the oocyte-repeat of the AT-rich flanks. The effects of H1 are completely suppressed by
distamycin A, a drug that specifically recognizes and binds oligo(dA)[middot]oligo(dT) runs in DNA. The binding of H1 results in increased protection
of DNA sites within the AT-rich oocyte-type 5S repeat. In an
in vitro
transcription assay performed with reconstituted chromatin templates containing
plasmids with the oocyte- or somatic-type repeats only the transcription of the oocyte-type 5S RNA gene was repressed in the presence of
physiological concentration of histone H1. These results support the view that
the AT-rich flanks of the oocyte-type 5S RNA gene are involved in histone H1-mediated chromatin reorganization that results in the
transcriptional repression observed
in vivo
.
The developmental regulation of 5S RNA gene transcription in
Xenopus laevis
has been a most thoroughly documented case of histone H1 involvement in modulation of transcription of a defined set of genes (reviewed in
1
). Two 5S RNA gene families are transcribed in early stages of embryonic development of
Xenopus
: the major oocyte-type, occurring in 20 000 copies per haploid genome, and the somatic-type, occurring in 400 copies per haploid genome. From late
gastrulation stage transcription of the oocyte-type 5S RNA genes becomes largely repressed whereas that of the somatic-type 5S RNA genes continues unaffected throughout consecutive
developmental stages and during the adult life of the frog. Studies on the
in vitro
transcription of
Xenopus
somatic cell chromatin were the first to establish that histone H1 is necessary
to maintain the repression of oocyte-type 5S RNA genes in somatic cells (
2
). This finding correlated well with the observation that normal somatic histone
H1 protein was absent in
Xenopus
oocytes (
3
). During early embryogenesis somatic H1 is gradually accumulated, its level in
chromatin correlating with the repression of the oocyte-type 5S RNA genes (
4
,
5
). A direct cause-and-effect relation between accumulation of somatic histone H1 and
repression of oocyte-type 5S RNA synthesis was documented by elimination of somatic H1 using
ribozyme strategies during early
Xenopus
embryogenesis. The elimination of somatic H1
in vivo
led to continued expression of oocyte-type 5S RNA genes by embryos that would normally have these genes switched-off (
6
,
7
).
In an effort to reveal the mechanism of differential effect of H1 on
transcription of
Xenopus
5S RNA genes, Chipev and Wolffe (
8
) found that
in vivo
the repressed oocyte-type 5S RNA gene was protected from nuclease digestion by incorporation
into nucleosome and that the entire oocyte-type 5S DNA repeats (genes with their flanks) were assembled into a
loosely positioned array of nucleosomes. In contrast, the potentially active
somatic-type 5S RNA gene was accessible to nuclease digestion and the majority of
somatic 5S RNA genes appeared not to be incorporated into positioned
nucleosomes. Chipev and Wolffe showed that histone H1 had an active role in
establishing these two different types of chromatin structure (
8
). However, the molecular basis of this gene-specific effect of H1 is not clear.
In our earlier work, based on the results of
in vitro
transcription studies, we suggested that the reason for the selective action of
H1
in vivo
may be the difference in base composition of the flanks accompanying the two
types of 5S RNA genes (
9
). In
Xenopus
the 5S DNA repeat consists of a 120 bp 5S RNA gene and two flanks of a total of a few hundred base pairs. The 360 bp 5'-flank and 56 bp 3'-flank of the oocyte 5S RNA gene comprise the
internally repetitous sequence most of which can be represented as follows:
C AA C G
CAA GTTTTCAA TTTGA TTTTCT
A GG A T
This sequence is 76% A + T, providing a strong H1-binding region in the direct vicinity of the oocyte 5S RNA gene. The
flanks of the somatic-type 5S RNA gene are G + C rich. We have shown
in vitro
that, due to the strong and selective binding of H1, the AT-rich oocyte-type flanks will confer the H1-dependent transcriptional repression to any of the two types
of 5S RNA genes. However, the simplicity of our
in vitro
system did not allow to probe the relation between the different affinities for
H1 of the flanks of the two types of 5S RNA genes and the differences on the
level of chromatin organization revealed by the later studies of Chipev and
Wolffe (
8
).
Here we address this problem using reconstituted minichromosomes with two types
of
Xenopus
5S DNA repeats. We show that the AT-rich flanks of the oocyte-type 5S RNA gene act as a strong local signal for H1-mediated reorganization of chromatin structure resulting in the increase of the spacing of nucleosomes to
>200 bp, increased protection of the 5S RNA gene repeat and the inhibition of the
transcription of 5S RNA gene in the
in vitro
transcription system.
Core histones were isolated from chicken red blood cell nuclei as described in (
10
). Histone H1 was derived from calf thymus by the method described in (
11
). The concentration of histones was determined using the Bradford procedure
with the BioRad Protein Assay (BioRad Laboratories, Inc.) and was done
according to the manufacturer's protocol. The concentration of histone H1 was
calculated from absorption at 280 nm using an extinction coefficient of 2.0 for
a 10 mg/ml solution of histone H1. Solutions of histones were stored in small
aliquots frozen at -80oC. DNA templates used for minichromosome reconstitution were
plasmids pBR327 and pUC19 with inserts consisting of
Xenopus laevis
somatic- or oocyte-type 5S RNA genes surrounded by their native (GC-rich or AT-rich, respectively) flanking sequences. The inserts were
excised from plasmids pXlsll and pXlo[Delta]3'+176 (described in ref.
9
) and subsequently cloned into the
Hin
dIII site of pBR327 or pUC19 plasmid. Plasmids were prepared by the alkaline
lysis method and purified twice on a CsCl/ethidium bromide gradient followed by
the repetitive extraction with water-saturated
n
-butanol, dialysis against two changes of TE buffer and precipitation with
ethanol (
12
). The concentration of DNA was measured spectrophotometrically at 260 nm using
an extinction coefficient of 1.0 for a 50 [mu]g/ml solution.
Minichromosomes were reconstituted by salt gradient dialysis (
13
). Defined amounts of supercoiled plasmid DNA and core histones were mixed in a
final volume of 50 [mu]l and the sodium chloride concentration was immediately adjusted to 2.0 M.
The sample was incubated at room temperature for 30 min and then for 1-2 h during which it was diluted by addition of five 20 [mu]l portions of TE buffer to obtain a final NaCl concentration of 1.0 M and a final OD
260nm
of 1.0 in a total volume of 100 [mu]l. The mixture was dialyzed for 5-6 h at room temperature or overnight at 4oC against 10 mM Tris-HCl pH 7.2, 1 mM EDTA with an NaCl concentration
decreasing linearly from 0.8 to 0 M. After dialysis sodium polyglutamate (average molecular weight 65 000) was added to a final concentration of 2.0 mg/ml (
10
). The sodium chloride concentration was then increased to 0.15 M and histone H1
(from a 10 mg/ml solution in water) was added to the desired H1:core histone
weight ratio. Finally, the sample was diluted with TE buffer (pH 7.2) to 200 [mu]l and the mixture incubated at 37oC for 10-12 h with occasional gentle stirring. After incubation, the
sample was dialyzed against 20 mM Tris-HCl (pH 7.2), 0.2 mM EDTA for 1.5 h at room temperature and centrifuged
for 1 min in a microfuge. The supernatant designated as a soluble
minichromosome fraction was stored at 4oC.
Before the addition of sodium polyglutamate to the minichromosome reconstitution mixture, distamycin (Distamycin A, Sigma) was added from a 1 mM
stock in 96% ethanol. The final distamycin concentration was 50 [mu]M and core histone-reconstituted DNA (~50 [mu]g/ml). Sodium chloride was added to 15 mM. The mixture was
incubated for 60 min at 37oC and subsequently subjected to H1 binding experiments with sodium
polyglutamate as described before.
The sample for digestion with micrococcal nuclease contained 2 [mu]g of histone-reconstituted DNA (in a soluble minichromosome fraction) diluted with
20 mM Tris-HCl (pH 7.2), 0.2 mM EDTA to a final volume of 50 [mu]l. Digestion was in the presence of 1 mM CaCl
2
. The mixture was preincubated for 1 min at 37oC prior to the addition of 10 U micrococcal nuclease (Eurogentec). The
incubation with the enzyme was for 4 min at 37oC. The reaction was stopped by the addition of an equal volume of stop
buffer containing 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 15 mM EDTA and 0.3% (w/v) SDS. Following
nuclease treatment the samples were deproteinized with proteinase K (Gibco BRL) (200 [mu]g/ml) and extracted with phenol-chloroform and chloroform before precipitation with ethanol. The extracted DNA was separated on 1.5% agarose in a 14 * 19 cm gel at a constant voltage of 70 V for at least 3 h. Gels were stained with ethidium bromide (0.5
[mu]g/ml) and photographed under UV light using the GDS camera (UltraViolet Products
Ltd).
Minichromosomes were incubated for 45 min at 37oC with 2.5 U topoisomerase I (Fermentas MBI) per microgram of DNA. The incubation was
stopped by the addition of SDS and EDTA to final concentration of 0.2% and 16
mM, respectively (
14
). The samples were subsequently incubated for at least 1 h at 37oC with proteinase K (1 mg/ml) and extracted once with phenol-chloroform before precipitation with ethanol. DNA was analyzed by two-dimensional electrophoresis in 1.2% agarose on 20 * 20 cm gels (
15
). The electrophoresis was carried out for 16-18 h in each dimension at constant voltage of 3 V/cm, in buffer
recirculating at a constant temperature of 5oC. Following the first dimension, the gel was soaked for 6 h at 5oC in the dark in TBE buffer containing 1.4 [mu]g/ml of chloroquine. Electrophoresis in the first dimension was in TBE buffer and in TBE buffer containing 1.4 [mu]g/ml of chloroquine in the second dimension. After electrophoresis the gel was soaked in deionized water and stained
with 0.5 [mu]g/ml ethidium bromide solution. The gel was photographed under UV light
using the GDS camera. Standard topoisomer mixtures of DNA templates were
prepared according to (
16
).
Transcription extracts from mouse Ehrlich ascites tumour cells were prepared by
the method of Dignam
et al.
(
17
). The reaction mixture (50 [mu]l) for a standard
in vitro
transcription experiment contained: 12.5 mM HEPES (pH 7.7), 10% glycerol, 70 mM
KCl, 0.5 mM dithiothreitol, 5 mM MgCl
2
, 600 [mu]M ATP, 600 [mu]M CTP, 600 [mu]M GTP, 60 [mu]M UTP, 20 [mu]M [[alpha]-
32
P]UTP (3000 Ci/mmol) (Amersham), 1 [mu]g DNA template and 25 [mu]l of a crude cell-free extract. The DNA template consisted of the oocyte- or somatic-type 5S RNA gene repeat from
X.laevis
cloned into the
Hin
dIII site of the pBR327 plasmid. DNA templates were used in a form of naked DNA
or reconstituted minichromosomes with or without histone H1. Before the
addition of the ribonucloside triphosphates, the mixtures were pre-incubated with the transcription extract for 30 min at 30oC. The transcription reaction was carried on for 60 min at 30oC and was terminated by addition of an equal volume of solution
containing 0.5% SDS, 0.1 M sodium acetate (pH 5.5) and 50 [mu]g carrier yeast RNA. The RNA was isolated from the reaction mixture
according to (
18
), redissolved in deionized formamide, heated at 95oC for 3 min and loaded on a 12.5% polyacrylamide (acrylamide:bis-acrylamide 16:1) gel containing 4 M urea. Electrophoresis was carried
out for 18 h at 4-5 mA. In the partially denaturing conditions of this electrophoresis system the somatic and oocyte 5S RNAs migrate at different distances
despite their identical length (
19
). The radioactive gels were exposed with Fuji RX films.
In order to compare the effects of the oocyte- and somatic-type
Xenopus
5S DNA repeats on chromatin assembly
in vitro
we choose, as a method of chromatin assembly, the reconstitution from pure DNA
and histones by dialysis from high salt. The DNA was that of pBR327 plasmid
into which was cloned (in the
Hin
dIII site of the polylinker) the full repeat (120 bp gene accompanied by native
5'- and 3'-flanks) of
Xenopus
5S RNA gene of the oocyte- or somatic-type (Fig.
1
A). The orientation of the inserts was confirmed by mapping the restriction
enzyme cutting sites in the insert and the nearby plasmid sequences. The final
plasmid constructions had 3943 and 4157 bp for plasmids containing the oocyte- and somatic-type repeats, respectively. For both constructs the final DNA length
was an integer multiple of the repeat length of 210 +- 3 bp (19 * 207.5 bp and 20 * 207.85 bp for the shorter and longer plasmid, respectively). Core
histones were purified from chicken erythrocytes and were homogeneous and fully
distinguishable from one another as determined by SDS-polyacrylamide electrophoresis (results not shown). Histone H1 was prepared from calf thymus and consisted of a set of native
variants as checked by SDS-polyacrylamide electrophoresis (results not shown). We determined the optimal core histone:DNA ratio performing reconstitutions with varying input histone:DNA ratio.
Chromatin samples with varying degrees of template saturation were digested
with micrococcal nuclease (MNase) to find the highest core histone:DNA ratio at
which the digestion pattern was still a 146 bp DNA ladder. The complexes formed
under these conditions were analyzed by topological assay. After the
reconstitution, complexes were incubated with excess of topoisomerase I to
remove any supercoils not constrained by nucleosomes (
20
). The only negative stress remaining in plasmids after deproteinization was
that constrained by histone octamers. Preliminary experiments were performed to
adjust the amount of topoisomerase I required to relax superhelical tension completely from the DNA template and assure that topoisomerase activity was not the limiting step in the assay. Distribution of DNA topoisomers was assessed by two-dimensional gel electrophoresis. The supercoiling assay generates a
Gaussion distribution of topoisomers about a mean and the linking number of the most intense spot
corresponds to the number of core particles in the minichromosome examined (
21
). The reconstituted complexes had on average 27 core particles, as shown in
Figure
1
B for plasmid with the oocyte-type insert (28 for plasmid with the somatic-type insert), indicating that at saturation the core particles on
both DNA templates were closely packed with spacing of ~150 bp. The estimation of protein and DNA content in reconstituted
minichromosomes showed that the ratio (w/w) of DNA to core histones was close
to 1 (results not shown).
In order to find out whether the formation of nucleoprotein complexes on the two
types of plasmids resulted in stable protection of specific sites on DNA we
digested the naked DNA, minichromosomes with and without histone H1 with
restriction enzymes cutting at sites located in various distance to the 5S RNA
gene (Fig.
2
A and C). Each sample containing an equal amount of naked or histone complexed
DNA was cut with 2-fold excess of restriction nuclease required for complete digestion of the
DNA as calculated for the naked template. After completion of digestion the DNA
was deproteinized and analyzed by electrophoresis in agarose gel (Fig.
2
B and D). Reconstitution of core particles resulted in partial protection of
restriction sites in both types of plasmid templates. An exception was the
Bam
HI site which was not protected in the plasmid containing the somatic-type 5S insert. This difference could reflect the increased stabilization of
core particles induced by the presence of the AT-rich flanks of the oocyte-type insert, a feature reflected also by the pattern of MNase
digestion seen in Figure
1
C and D (lanes 3). The addition of H1 had no effect on the protection of any of
the analyzed sites in the plasmid containing the somatic-type 5S insert. In contrast, in the plasmid containing the oocyte-type 5S insert the incorporation of H1 resulted in over 2-fold increase in protection of
Hin
dIII and
Stu
I sites compared with protection resulting from reconstitution of core
particles. The increased protection of these two sites which are located on the
boundaries and within the oocyte-type 5S DNA repeat, but not the
Bam
HI site located further away could result from strong binding of H1 to DNA in
this region and/or from H1-induced stabilization of positioned nucleosomes over the 5S DNA repeat.
In order to find the extent of H1-mediated reorganization of nucleosome alignment observed with templates
containing the oocyte-type insert we determined the linking number change related to
minichromosome reconstitution and thus the number of nucleosomes deposited on
the templates. Minichromosomes were reconstituted without H1 on plasmid
containing somatic- or oocyte-type 5S DNA insert. Half of the sample was treated with
topoisomerase I whereas the other half was used for reconstitution with histone
H1 at H1:DNA ratio of 1.0 and then treated with topoisomerase I. As shown in
Figure
3
, before the addition of H1 the average number of core particles in
minichromosomes was 26-27 (28 for the plasmid with the somatic-type insert). Thus, on both plasmids the core particles were
densely packed along the entire length of DNA. The reconstitution with H1 of minichromosomes containing plasmid with the somatic-type insert had no effect on the total number of nucleosomes. In contrast
to that, the reconstitution with H1 of minichromosomes containing plasmid with
the oocyte-type insert resulted in a decrease of the total number of nucleosomes to 16 (Fig.
3
A and B). This corresponds to the average nucleosomal spacing of 245 bp, a value similar to that estimated on the basis of MNase generated DNA ladder (Fig.
1
).
To check whether the effect of H1 observed with minichromosomes reconstituted on plasmids containing the oocyte-type insert was due to the preferential binding of H1 to AT-tracts present in DNA sequences flanking the oocyte 5S DNA and not
due to other differences between the two types of inserts (like slightly larger
size of the somatic insert or eight point differences between base sequences of the two types of 5S genes) we treated the minichromosomes
containing the oocyte-type insert with distamycin A before the reconstitution with H1. Distamycin A is known to recognize
and strongly bind the oligo(dA)[middot]oligo(dT) tracts in DNA and to inhibit the specific binding of H1 to
these sites (
22
). We have checked that the drug indeed prevents the selective binding of H1 to isolated oocyte-type DNA repeat (Fig.
4
A). Reconstitution of H1 on distamycin A-treated minichromosomes containing the oocyte-type insert did not result in the change of the total number of
nucleosomes (Fig.
4
B). Thus, the observed effect of H1 in minichromosomes depends on its specific
binding to the AT-rich tracts in DNA.
Plasmid pBR327 is known to possess a chromatin organizing region which
stimulates the nucleosome alignment reaction provided the plasmid DNA is an
integer multiple of 210 +- 3 bp and its total length is between 2400 and 3600 bp (
23
). While the constructs with the oocyte- and somatic-type inserts used by us had both the integer multiple of 210 +- 3 bp, the total length of DNA in these pBR327 derivatives
was >3900 bp. However, we could not exclude the possibility that the observed
effect of H1 is due to some specific features of pBR327 sequences that manifest
selectively, i.e. in the presence of the oocyte-type but not the somatic-type insert. To check this we prepared plasmid templates based on
pUC19 instead of pBR327 plasmid. The pUC19 templates containing somatic- or oocyte type inserts (the same as used in pBR327 constructs) were
reconstituted with core histones and H1 identically as described for pBR327
derivatives. The nucleoprotein complexes were analyzed by MNase digestion and
(for the minichromosome with the oocyte-type insert) by the topological assay, similarly as in the case of
minichromosomes containing the pBR327 constructs. The results of MNase
digestion revealed that upon the addition of histone H1 to the core particle-containing minichromosomes, the increase in the nucleosomal spacing (from 150 to >240 bp) occurred only for the template with the
oocyte-type insert (Fig.
5
A and B), similarly as for the minichromosomes based on pBR327 derivatives.
Interestingly, in the case of pUC19 templates the differences in digestion
patterns of minichromosomes with somatic- and oocyte-type inserts in the absence of H1 were much less pronounced compared
with those for pBR327 templates (Fig.
5
A and B, compare lanes 1 and 2). It is possible that the chromatin organizing
region of pBR327 and the AT-rich oocyte type insert produce some cumulative stabilizing effect on nucleosomal core particles. The topological assay showed that
the alignment of H1 on the pUC19 minichromosome with oocyte-type insert resulted in a drop of the total number of nucleosomes from 22
to 14 (Fig.
5
C). The addition to the minichromosome of the distamycin A before the addition
of H1 prevented the above effect of H1 (Fig.
5
C). Thus, we conclude that the H1-induced remodelling of minichromosome is independent on the plasmid sequence but is solely due to the presence of the AT-rich oocyte-type insert.
In order to check directly the effect on transcription of the histone H1-induced chromatin remodelling occurring in plasmid bearing the oocyte-type insert we used the cell-free transcription extract from Ehrlich ascites cells to
transcribe
in vitro
the 5S RNA genes. As templates for the
in vitro
transcription were used constructs in pBR327 plasmid containing the somatic- or oocyte-type inserts, in a form of naked DNA or in the form of reconstituted
minichromosomes with or without histone H1. Prior to the addition of nucleoside
triphosphates, the templates were preincubated for 30 min with the
transcription extract. The transcription reaction was then carried on for the
next 60 min. The isolated RNA products were separated on the semi-denaturing gel which allows distinction between the somatic and oocyte 5S
RNA. No noticeable differences were seen in the amount of somatic 5S RNA
transcribed from naked DNA, minichromosomes lacking H1 and minichromosomes
containing H1 (Fig.
6
, lanes 1-3). The amount of the oocyte 5S RNA transcribed from naked DNA was only
slightly smaller compared with that transcribed from minichromosomes lacking
H1. However, it dropped dramatically for minichromosomes with H1 (Fig.
6
, lanes 4-6). Thus, in the cell-free
in vitro
transcription system using reconstituted minichromosomes as templates, only the transcription of the oocyte-type gene surrounded by its native AT-rich flanks was sensitive to inhibition by histone H1.
Nucleosomes reconstituted from DNA and purified histones are irregularly spaced
and tend to pack closely together independent of whether linker histones are
present in the reconstitution system. The results of numerous experiments also
suggest that it is not possible to convert DNA molecules that are already
covered with closely packed nucleosomes into a chromatin-like arrangement characterized by nucleosomes that are spaced 50 or more base-pairs apart (
10
,
24
). Complete
in vitro
reconstitution of chromatin-like structures characterized by highly ordered and physiologically spaced
nucleosomes could only be generated on the synthetic polynucleotide poly[d(A-T)]-poly[d(A-T)], probably because of higher affinity of the poly[d(A-T)] duplex for H1 compared with average DNA (
25
), and on certain plasmid DNAs containing specific, chromatin organizing regions
(
13
). In the present work we have shown that a specific fragment of DNA comprising
the
Xenopus
oocyte-type 5S DNA repeat is capable of directing the histone H1-mediated major reorganization of already formed DNA:core histone
complexes on long stretches of DNA. The reorganization results in creation of
regular long gaps between packed core particles and is achieved through
complete removal of several core histone complexes from DNA. The DNA templates
that we initially used were the derivatives of the pBR327 plasmid which is
known to possess its own specific sequence able to induce the correct alignment
of H1, provided the DNA is an integer multiple of 210 +- 3 bp (
23
). We wanted to obtain physiologically spaced nucleosomes on plasmids with both
types of inserts in order to compare the differences in stability brought about
by the presence of AT-rich flanks of the oocyte-type gene. However, despite the presence in both constructs of an
integer multiple of 210 +- 3 bp, the correct alignment of H1 was only seen for the plasmid with
the oocyte- type insert. One reason for the lack of the effect of the pBR327
chromatin organizing sequences could be the total length of the DNA which, for
both plasmids used, exceeded the limit of the reported optimal size of 2400-3600 bp (
23
). This notwithstanding, and since the oocyte insert was smaller than the somatic insert, we could
not exclude that the observed effect of H1 was somehow linked to the difference
in length between the two pBR327 DNA templates. To check this we repeated the
reconstitutions with DNA derived from the pUC19, a plasmid lacking any
chromatin organizing regions (
23
). The identical results to those obtained with the pBR327 derived templates
strongly suggest that the observed correct alignment of H1 is independent of
the plasmid sequence but is solely due to the presence of the oocyte-type insert. The reason for this specific effect must be the highly AT-rich flanks of the oocyte-type 5S RNA gene. No such effect is observed for somatic-type 5S repeat which consists of a very similar 5S RNA
gene that is flanked by GC-rich DNA.
The binding of H1 results in a partial protection of DNA sites within the AT-rich oocyte-type but not within the GC-rich somatic-type repeat inserted into plasmid DNA. The above agrees
well with the results of our earlier studies (see also Fig.
4
A) showing a highly preferential interaction of H1 with isolated
Xenopus
oocyte-type 5S repeat (
9
). The major structural effect of H1 observed in minichromosomes containing the
oocyte-type repeat is completely suppressed by distamycin A, a drug shown to
block the accessibility of the AT tracts in DNA for H1.
The unusually long repeat length (~240 bp) observed in H1 containing templates with the oocyte-type repeat can be the result of the over-stoichiometric amount of H1. A similar effect of the increase in the repeat length (from 180 to 220 bp) as a result of increasing the amount of H1 was observed by Rodriguez-Campos
et al
. (
26
) and by Kamakaka
et al
. (
27
) upon reconstituting of chromatin with extracts from
Xenopus
and
Drosophila
embryonic cells.
In an
in vitro
transcription assay using as template the naked DNA, minichromosomes containing
H1 and minichromosomes lacking H1 the inhibitory effect of H1 manifested for
the transcription of the oocyte-type 5S RNA gene but not for the transcription of the somatic-type gene. Interestingly, there was not much difference in the
transcription efficiency of the somatic-type gene from the naked DNA and the DNA in a form of minichromosome with
or without H1. The same concerned transcription of the oocyte-type gene on the naked DNA and on the minichromosome lacking H1. Earlier,
O'Neill
et al
reported that the deposition of histone H1 onto core particles reconstituted on
tandem repeats of 5S rRNA gene inhibited both initiation and elongation of
transcripts by the T7 RNA polymerase (
28
). However, the transcription system we used, based on the crude cell-free extract, is completely different from the system based on the
purified T7 polymerase. It should also be noted that we have only measured the
net effect of the 60 min incubation with the transcription extract and did not
monitor the kinetics of the initial phase of the reaction. Nucleosome
movements, most likely their sliding, occurring even in the presence of histone
H1, were reported for plasmids reconstituted into chromatin and incubated in
the ATP enriched cell free system (
29
). Similar sliding probably occurred in the H1-lacking minichromosomes containing the 5S RNA gene inserts during the 60 min incubation in the cell free,
crude transcription system supplemented with ATP. The binding of H1 to the
minichromosome with the somatic insert did not induce the physiological spacing
and was also ineffective in preventing the sliding of nucleosomes (see Fig.
2
B) and thus in restricting the accessibility of the 5S RNA gene to the transcription apparatus.
In contrary to this, histone H1 binding to the plasmid containing the oocyte-type insert strongly inhibited the transcription of the 5S rRNA gene. The
AT-rich flanks of the oocyte-type gene could serve as a nucleation center for the correct binding
of H1 (presumably cooperatively) to its primary nucleosomal sites. Such
interpretation would account for the observed forceful removal of almost half
of the bound core histone complexes from template DNA. In addition, a strong
nucleation center of H1 binding located in the vicinity of a gene could form an
effective barrier for the sliding of local nucleosomes and thus block the access of transcription factors to their cognate promoter sequences. Chipev and Wolffe (
8
) showed that H1 had an active role in inducing the loosely positioned arrays of
nucleosomes over oocyte-type but not over somatic-type
Xenopus
5S RNA genes. The existence near the oocyte-type 5S RNA gene of the AT-rich DNA sequences which can act as the nucleation center enabling
the specific binding of H1 could be an important factor in the mechanism
underlying the
in vivo
effects of H1. Judging from the observed displacement by the H1 binding of
densely packed core histone complexes
in vitro
, a similar displacement could affect the transcriptional complex prebound to
the 5S RNA gene
in vivo
. It should be also noted that
in vivo
the oocyte-type 5S DNA repeats occur in tandem arrays, thus the effect of H1 binding
on reorganization and stabilization of nucleosome positions can be stronger than that observed by us
in vitro
with a template containing a single repeat.
We thank Beata Kilianczyk for excellent technical assistance. This work was
supported by Polish Committee of Scientific Research grants 662759203 (A.J) and
6P04A02208 (R.T.) and by Howard Hughes Medical Institute grant 79195-543403 (A.J.).
*To whom correspondence should be addressed at: Laboratory of Plant Molecular
Biology, Warsaw University, Pawinskiego 5A, 02-106 Warsaw, Poland. Tel: +48 22 659 6072; Fax: +48 22 391 21623; E-mail: andyj@ibbrain.ibb.waw.pl
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