From Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France
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ABSTRACT |
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The 5-untranslated region of the
Drosophila gypsy retrotransposon contains an
"insulator," which disrupts the interactions between enhancer and
promoter elements located apart. The insulator effect is dependent on
the suppressor of Hairy-wing (su(Hw)) protein, which binds to
reiterated sites within the 350 base pairs of the gypsy insulator,
whereby it additionally acts as a transcriptional activator of gypsy.
Here, we show that the 350-base pair su(Hw) binding site-containing
gypsy insulator behaves in addition as a matrix/scaffold attachment
region (MAR/SAR), involved in interactions with the nuclear matrix.
In vitro experiments using nuclear matrices from
Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with
topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in
culture demonstrate differential effects of the gypsy MAR sequence on
reporter genes, namely no effect under conditions of transient
transfection and a repressing effect in stable transformants, as
expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a
short "compacted" retroviral sequence three functional domains
(insulator, enhancer, and the presently unraveled MAR/SAR) dispersed
within more extended regions in other "boundary" domains, is
discussed in relation to previously proposed models for insulation.
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INTRODUCTION |
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Insulator elements play a fundamental role in genome organization. They isolate independent transcriptional units from cross-reaction with the neighboring regulatory elements (reviewed in Refs. 1-4). Within complex genetic loci, insulators participate in refined modulations of internal enhancer-promoter interactions (e.g. the bithorax complex in Drosophila; see Refs. 5 and 6).
One of the most extensively analyzed insulator element in
Drosophila is part of a retrotransposon, the gypsy
retrotransposon, where it occupies the 5-untranslated region
(5
-UTR)1 of this mobile
element. Actually, transposition of gypsy has been revealed by the
isolating activity of its insulator, which resulted for instance in the
disruption of the effect of enhancers sequences of the yellow
gene on the promoter of the gene (7-9). The gypsy insulator was
identified as entirely contained within a 350-bp sequence of the
5
-UTR; experiments performed in transgenic Drosophila with
this minimal sequence placed between a reporter gene and various
enhancer and/or silencers have shown that the gypsy insulator blocks
the effect of enhancers in a directional manner, since only those
located distally from the promoter with respect to the insulator
sequence are affected (10-14). Similar effects can be produced with
other well characterized insulator elements, including the
Drosophila scs (specialized chromatin structures) from the
87A7 heat shock locus (15-17), the human and chicken boundary elements
of the
-globin domain (18, 19), and the A element of the chicken
lysozyme gene (20, 21), all of which segregate DNA into distinct
functional domains.
However, the structural basis of this functional dissection is not so clear. Actually, boundary formation is thought to be connected with the establishment of specific chromatin structures, possibly associated with the formation of chromatin loops that would fold and assemble in such a way so as to increase the likelihood of interactions between regulatory elements within a domain, while decreasing these interactions between domains (reviewed in Refs. 2, 3, 22, and 23). Evidence for this type of model is still weak and stems from the observation of such loops by electron microscopy, and from the occurrence of specific DNA sequences, which would mediate the attachment of chromosomes to the nuclear matrix (see Refs. 24-27, and references therein). These sequences, also called MAR/SARs for matrix-associated or scaffold-attachment regions, have been operationally defined as DNA sequences that remain attached to the unsoluble nuclear matrix, and thereby are supposed to both define genomic structural domains and localize DNA segments close to the enzymatic machinery involved in DNA replication and transcription (22, 27-29). MAR/SARs are therefore likely structural elements to be involved in the segregation of the chromatin into functional domains and to participate in insulation.
Accordingly, we have devised experiments to determine whether the gypsy retrotransposon contains a MAR/SAR domain. The in vitro experiments presented in this report disclose such a sequence, which furthermore strictly co-localizes with the gypsy insulator. Experiments performed within cells in culture either transiently or stably transfected with reporter genes containing this sequence further demonstrate integration-dependent effects, consistent with a role in the organization and shaping of chromatin. The gypsy insulator, which contains previously identified binding sites for a protein, the suppressor of Hairy-wing protein (30-33), which regulates the extent of insulation (see above references and review in Ref. 4) and acts as a transcriptional activator (8, 34), therefore also interacts with components of the nuclear matrix. The complex in vivo regulatory effects of the gypsy element are discussed in relation with the characteristic features of this insulator.
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EXPERIMENTAL PROCEDURES |
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Plasmids and DNA--
The gypsy DNA was from plasmid cDm111
(35), re-cloned as BglII-EcoRI plus
EcoRI-BamHI fragments into the pSK (Stratagene) polylinker; a plasmid containing the gypsy LTR plus 5-UTR was obtained
by cloning the Klenow-treated HpaI-NcoI fragment
(positions 2-1201 in Ref. 36) from the above plasmid into the
EcoRV site of the pSK polylinker; plasmids with fragments I
to IV (see Fig. 1A) were obtained by inserting the
corresponding DNA fragments, obtained either by restriction from the
plasmids above or after polymerase chain reaction amplification, into
the pSK polylinker.
In Vitro MAR/SAR Binding Experiments-- High salt extraction of nuclei from Drosophila Schneider II, murine L, or human HeLa cells (2 M NaCl extraction after DNase I digestion) and in vitro MAR binding assays were performed as in Refs. 29 and 38. In vitro SAR binding assays with LIS-extracted nuclei (25 mM lithium diiodosalicylate) digested with a series of restriction nucleases (XbaI, BstXI, and XmnI) were performed as in Refs. 39 and 40. The gypsy fragments, radiolabeled either by Klenow treatment after restriction with appropriate enzymes or upon polymerase chain reaction amplification using 32P-labeled primers, were incubated 2 h with nuclear matrices prepared from 5 A260 units of purified nuclei, in the presence of competitor DNA (0.2 mg/ml sonicated salmon sperm DNA). Pellet (P) and supernatant (S) fractions were separated by centrifugation (1 min at 4 °C, 10,000 × g), treated with proteinase K and, after purification, were resolved on polyacrylamide or agarose gels (and transferred onto Hybond N+ membranes in the latter case); dried polyacrylamide gels or membranes were then autoradiographed. For competition experiments, the labeled fragment corresponding to the gypsy insulator (XmnI-Sau3A) was first preincubated with the antibiotics distamycin (Sigma) or chromomycin (Boehringer Mannheim) as in Refs. 41 and 42, and then assayed as above.
Precipitation and Cleavage Assay with Topoisomerase II--
For
the precipitation assay, radiolabeled fragments of the gypsy
retrotransposon were incubated 30 min at 30 °C with increasing amounts of highly purified yeast or human topoisomerase II (topo II;
gift from Dr. A. Larsen) in TEN buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.1% digitonin, 1 mM phenylmethylsulfonyl fluoride and 25 µg/ml bovine
serum albumin) in the presence of competitor DNA (sonicated salmon
sperm DNA) as in Refs. 41 and 42. Pellet and supernatant fractions,
separated by centrifugation (10 min at 4 °C, 12,000 × g) and treated with 0.5% SDS and 0.5 mg/ml proteinase K
(final concentrations), were analyzed on polyacrylamide gels, which
were dried and autoradiographed. For the topo II cleavage assay, a
fragment containing the 5 part of gypsy (positions 1-1201 in Ref. 36)
plus 1.5 kilobase pairs of the pSK vector was incubated with increasing
amounts of topo II in buffer B (20 mM Tris-HCl, pH 7.4, 20 mM KCl, 10 mM MgCl2, 70 mM NaCl, 0.05 mM spermine, 0.125 mM
spermidine, 0.1% digitonin, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml bovine serum albumin, 1 mM
dithiothreitol, and 1 mM ATP), supplemented with the
anti-tumor drug VM 26 (final concentration 50 µM; gift
from Dr. A. Larsen) as in Ref. 42. After electrophoresis and blotting,
the cleavage pattern was analyzed by hybridization with the indicated
probe (indirect end-labeling).
Precipitation and Protection Assay with Histone H1-- Precipitation assay and protection from digestion by DNase I (Sigma) were performed as in Refs. 43 and 44, with the gypsy insulator (fragment XmnI-Sau3AI) either as a single copy or as three directly repeated copies subcloned into the pSK plasmid. Plasmids were digested with restriction enzymes in the pSK polylinker, and fragments were labeled by Klenow treatment. DNA (2-5 ng of labeled fragments with 1 µg of sonicated salmon sperm DNA) was then incubated with histone H1 (Boehringer Mannheim) for 3 h at 37 °C. For the precipitation assay, pellet was recovered by centrifugation (15 min at 4 °C, 12,000 × g) and analyzed as above. For the DNase I protection assay, DNA with histone H1 was digested with DNase I (50 ng; Sigma) for 3 min at room temperature in the presence of 1 mM CaCl2, purified by phenol/chloroform extractions and analyzed by gel electrophoresis (44).
Cells, Transfections, and CAT Assays-- Drosophila SII cells were grown in Schneider medium (Life Technologies, Inc.) with 10% fetal calf serum (Life Technologies, Inc.), and murine L and human HeLa cells in Dulbecco's modified Eagle's medium with the same serum. Transfection of cells (about 5 × 106 cells) were by the standard calcium phosphate precipitation procedure, with 10 µg of DNA. Stable transformants were obtained upon co-transfection with the plasmid pSV2neo conferring Geneticin resistance (45), followed by selection with G-418 (0.7 mg/ml; Life Technologies, Inc.). For transient assays, cells were co-transfected with a plasmid containing the luciferase gene under control of Rous sarcoma virus LTR to normalize transfection efficiency. Protein extracts prepared as in Ref. 37 were assayed for CAT activity as in Ref. 46 using appropriate dilutions to obtain less than 50% conversion of [14C]chloramphenicol to acetylated forms. Reaction products were separated by thin-layer chromatography, and conversion of chloramphenicol was quantitated with a Bio-Imaging analyzer (BAS model 1000; Fuji, Tokyo, Japan). CAT activity was expressed as the percentage of [14C]chloramphenicol acetylated in 30 min/mg of protein at 37 °C. Reporter plasmid copy number in the stable transformants was measured by Southern blot analysis of the genomic DNAs restricted with HindIII and EcoRI, using a CAT gene fragment from the p6 plasmid as a probe. DNA isolation and Southern blot hybridization were as in Ref. 47.
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RESULTS |
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Sequence Analysis of the gypsy Insulator--
The gypsy insulator
is a short sequence (350 bp) with a complex organization (Fig.
1, A and B). It
contains reiterated binding sites for the suppressor of Hairy-wing
(su(Hw)) protein, which both regulates insulation efficiency and acts
as a transcriptional enhancer (see Introduction). These sites are part
of a sequence characterized by 12 copies of a core 12-bp sequence,
5-PyPuTTGCATACCPy-3
, with homology to the mammalian octamer motif
(see Ref. 30). These reiterated binding sites are interspersed among an
AT-rich domain (>70%), which is composed of short homopolymeric runs
of dA-dT base pairs and AT dimer repeats (Fig. 1B), and is
involved in the refined modulation of su(Hw) binding (30). Although
there is no definite consensus sequence for MAR/SARs, analysis of
previously characterized sequences revealed ATC regions with G residues
only on one strand of the DNA duplex, which were demonstrated to
contain binding sites for nuclear matrix specific proteins and which in a multimerized form actually bind to the nuclear matrix (48-50). Close
examination of the gypsy insulator actually discloses ATC regions,
with, for instance, four almost perfect (T/C)TTTTAATAAA(T/A)A(T/C)ATT repeats (Fig. 1B), organized as the ATC sequences in the MAR
of the mouse IgH gene, which harbors binding sites for the nuclear matrix-specific SATB1 and Bright proteins (ATC stretches with A-rich
core, flanked by AT-dimer repeats, see reference above). Finally,
MAR/SARs usually contain sequences related to the in vitro
topoisomerase II cleavage core consensus (A/T)A(C/T)ATT (51, 52);
such sequences can be found in the present sequence (see Fig.
1B). The possibility that the gypsy insulator co-localizes with a MAR/SAR was therefore tested, using classical assays for the identification of such domains.
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Binding of the gypsy Insulator to the Nuclear Matrix--
In
vitro binding assay of gypsy sequences with high salt-extracted
nuclei was performed as described in Refs. 29 and 38, using either
Drosophila (SII), murine (L), or human (HeLa) cells. Insoluble material (i.e. associated with the nuclear matrix)
was recovered as a pellet after centrifugation and treated, in parallel with the supernatant, with proteinase K, purified and analyzed on
agarose or polyacrylamide gels. As illustrated in Fig. 1C
using end-labeled fragments from the gypsy retrotransposon, a fragment is specifically recovered in the insoluble fraction, which coincides with the 5-UTR of gypsy; the other gypsy fragments are not associated with the nuclear matrix. A more refined analysis of the 5
-UTR region
using for the binding assay four labeled fragments (I-IV, see Fig.
1A) including the gypsy LTR, the most 5
part of the untranslated region (also AT-rich), the gypsy insulator (10, 12) and
the region corresponding to the N-terminal part of ORF1, further showed
that the MAR coincides with the insulator domain (Fig. 1C).
Identical results were obtained with nuclear scaffold (SAR assay, see
Fig. 1D) isolated after lithium diiodosalicylate (LIS)
extraction of nuclei from the SII cells as described in Refs. 39 and
40. In typical MAR/SAR assays, about 50% of total gypsy insulator DNA
was recovered in the pellet, as also observed (data not shown) with the
previously characterized histone MAR/SAR from Drosophila
(37). Finally, binding assays performed with nuclear matrices from
cells of other species revealed a conservation of the MAR/SAR activity
of the gypsy insulator (see Fig. 1C for murine and human
cells), as observed for other such domains (48, 53).
Interaction of Topoisomerase II with the gypsy Insulator--
All
MAR/SARs so far described have been shown to be functional targets of
topo II, one of the main protein components of the nuclear matrix (54,
55). Topo II is a key protein mediating interconversions between
different topological states of the DNA, through transient
double-strand breaks and rejoining. This enzyme is also required for
chromatin assembly and packaging, and it plays a significant role in
the establishment of chromatin loops (29, 56, 57). Assays for both the
aggregation of the gypsy MAR/SAR and for cleavage were therefore
performed, using highly purified yeast or human topoisomerase II. Topo
II-mediated aggregation was monitored as in Ref. 42 by centrifugation
using end-labeled gypsy fragments and analysis of the pellet as in the
MAR assay (Fig. 2A). As
illustrated in the figure using either a full-length gypsy element
restricted with appropriate enzymes or 5-UTR fragments including the
insulator and the AT-rich domain 5
to it (fragments III and II, see
Fig. 1A), topo II specifically precipitates the gypsy
insulator. In a second series of experiments (see Fig. 2B), topo II cleavage sites were assayed in vitro, using a
linearized fragment containing the gypsy 5
end and plasmidic DNA
incubated with increasing concentrations of topo II, in the appropriate cleavage buffer. After electrophoresis on an agarose gel and blotting, DNA was analyzed by an indirect end-labeling procedure using the probe
indicated in the figure. As illustrated in Fig. 2B, these experiments revealed the presence of a few strong cleavage sites within
the gypsy insulator, and no sites in the adjacent AT-rich untranslated
regions; sites in the LTR as well as in the flanking plasmidic DNA were
also detected, but possibly with lower cleavage efficiency, taking into
account the intensity of the bands and the position of the probe.
Altogether, these series of experiments reveal a specific interaction
of the gypsy insulator with topo II, as indeed observed for previously
characterized nuclear matrix associated regions (42, 58).
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The gypsy Insulator DNA Nucleates Histone H1-- MAR/SARs have been shown in vitro to bind histone H1 molecules in a highly cooperative manner. The tightly bound H1 facilitate in turn the further assembly -or nucleation- of "bulk" H1 onto flanking non-MAR/SAR DNA (41, 43, 44). Histone H1 binding has been shown to play a key role in the general repression of chromatin, whereas several drugs and cellular proteins (including the high mobility group proteins) induce chromatin "opening" as a result of a redistribution of histone H1 molecules preferentially bound to MAR/SAR-containing DNA to non-MAR/SAR DNA (44, 59). Possible interactions of the gypsy insulator with histone H1 was therefore assayed in vitro, using purified histone H1. In a first series of experiments, histone H1 cooperative binding was assayed by monitoring their ability to precipitate a gypsy insulator-containing DNA (Fig. 3A). A 1-kilobase pair DNA fragment containing three copies of the gypsy insulator together with the vector plasmid as a control were end-labeled and then incubated in vitro with increasing concentrations of histone H1. After centrifugation, DNA in the insoluble fraction recovered as a pellet was extracted and analyzed on agarose gels. As illustrated in Fig. 3A, histone H1 at a low concentration preferentially aggregates the insulator-containing DNA (10-fold ratio over control plasmid DNA), whereas higher histone H1 concentrations finally resulted in a general aggregation of both DNA fragments, as expected (43). Similar results were obtained using a single copy of the gypsy insulator (instead of 3-mer), but with a reduced specificity (data not shown). To analyze further the binding of histone H1, a second series of experiments was performed as in Ref. 43, to measure the possible protection of the insulator DNA domain from digestion by DNase I (Fig. 3B). The insulator DNA, together with the vector plasmid as a control, were incubated with purified histone H1 as above, then DNase I was added for 3 min and DNA was recovered and analyzed. As illustrated in Fig. 3B, the addition of histone H1 results in the protection of the insulator DNA, whereas complete degradation of the vector plasmid DNA takes place. This two series of experiments therefore strongly suggest that histone H1 actually binds in a specific manner to the gypsy insulator, resulting in a DNase I-resistant structure.
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Differential Effect of gypsy MAR/SAR-containing Reporter Genes in Transient and Stable Transfection Assays-- Common to the studies on MAR/SAR is the observation that MAR/SARs flanking a test gene have either a repressive or stimulatory effect on the level of its expression (Refs. 21 and 60-62; see the "chromatin switch model" in Refs. 22, 44, and 59). Moreover, in contrast to transcriptional enhancer elements, MAR/SARs exert this regulatory effect only if the reporter gene is stably integrated into the genome but have no effect in transient transfection assays (Refs. 20, 63, and 64; reviewed in Ref. 22). The gypsy MAR/SAR was therefore assayed as in Ref. 37 upon transfection of cells in culture under both transient and stable conditions. The SV-CAT reporter gene in Ref. 37 was inserted between the gypsy MAR/SAR, using as a negative control a vector without any bordering sequences and, as a positive control, a reporter with the previously characterized MAR/SAR from the hsp70 gene. These reporter plasmids were then introduced into mouse L cells by transfection and were either assayed 2 days after transfection or stably integrated upon further selection in G418 medium (the cells being co-transfected with a neo resistance-encoding plasmid). As illustrated in Fig. 4A, no effect of either the hsp or gypsy sequence can be detected in the transient assay, as expected for MAR/SAR sequences. However, both sequences had a significant effect in the stable transformants (Fig. 4B); the hsp MAR/SAR resulted, as reported previously for the same construction (37), in a 10-20-fold increase in CAT activity, whereas the gypsy sequence resulted in a 3-5-fold decrease. Identical results were similarly obtained using human HeLa cells (data not shown), with no effect in the transient assay and, again, a repressing effect (versus an increase for the hsp MAR/SAR) in stable transformants.
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DISCUSSION |
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The present investigation reveals an as yet unidentified
characteristic feature of the insulator domain of the gypsy
retrotransposon, i.e. a MAR/SAR activity. This 350-bp
domain, located in the 5-untranslated region of this
Drosophila retrotransposon, therefore combines three
functions, namely insulation, transcriptional enhancement, and nuclear
matrix attachment. This co-localization might be the consequence of the
pressure for compactness of retroviral genomes and could be responsible
for the complex "phenomenologic" behavior of the gypsy insulator
and the often divergent models that have been proposed to account for
the gypsy effects (12-14, 65).
A Multifunctional Domain within 350 bp of the gypsy Retrotransposon: an Enhancer, an Insulator, and a MAR/SAR-- The 350-bp gypsy sequence has been the subject of extensive analyses, which had already revealed two characteristic features of this domain. Actually, it had been demonstrated by several groups that this sequence acts as an insulator, therefore inhibiting in a directional manner the effect of enhancer sequences located distally from the promoter (10-13). Refined in vivo analyses have unambiguously demonstrated that this blockade is not due to a repression of the enhancer per se but to an inhibition of the effect of the enhancer on the insulated promoter, as promoters proximal to the enhancer remained fully regulated (12, 13). Most importantly, this insulating activity was shown to require the presence of the suppressor of Hairy-wing protein, which binds to the insulator sequence via well identified sites (reviewed in Refs. 2 and 4). Actually, a second protein is required for insulation, the mod(mdg4) protein, which most probably binds to the su(Hw) protein to form a complex active for insulation (14, 65).
A second function associated with the gypsy insulator is an enhancer activity which, although less extensively documented, also requires the su(Hw) protein (8, 34). Actually, in vivo analyses of the expression of a transgene containing the gypsy promoter plus 5A Tentative Model for gypsy Insulation-- A consequence of compaction is that the gypsy insulator and its associated components are most probably interacting, in vivo, with elements of the nuclear matrix. Accordingly, proteins of the nuclear matrix might play a role in the insulation process, and conversely the su(Hw) protein (which is essential for insulation) might interact with proteins of the matrix. Such interactions could actually account for the data on gypsy insulation and fit with previously proposed models for the gypsy effects. A first series of data strongly suggests that the gypsy insulator, as all previously characterized insulators, essentially prevents interactions between distal enhancer and promoter, without any direct repressing effect on the enhancer itself (12, 13). This directional effect can most easily be accounted for by the "looping model" involving generation of structural domains isolated one from the other by attachment of boundary sequences (MAR/SAR) to the nuclear matrix (see Ref. 22 and Introduction). Alternatively, a series of data on gypsy insulation (essentially in mod(mdg4) mutants) discloses bidirectional repressing effects (14, 65), which can be accounted for by a model involving heterochromatinization (65). The present data (showing that the gypsy insulator behaves as a MAR/SAR) are clearly in agreement with the structural looping model, but also support the heterochromatinization model. Indeed, the gypsy MAR/SAR DNA per se, in the absence of su(Hw) protein, is involved in histone H1 nucleation (as shown in this paper), and it has been demonstrated that histone H1 nucleation is associated with both DNA compaction and transcriptional silencing (reviewed in Ref. 67); in addition, Laemmli and co-workers have found that histone H1 could be removed from MAR/SAR domains by distamycin and distamycin-like proteins (D-like proteins, such as the high mobility group proteins), leading to the proposal that MAR/SARs could activate or repress transcription of adjacent genes depending on the nucleation/depletion of histone H1 ("switch model," see Refs. 22, 41, 44, and 59). The gypsy MAR/SAR could then be responsible for the repressing effect observed in the mod(mdg4) mutants (65), as well as in the present assay within heterologous cells (assuming further that appropriate D-like proteins are absent in those cells). Taking into account, in addition, that mutations in the mod(mdg4) or the su(Hw) genes modify position-effect variegation (65), it could be further hypothesized that the su(Hw)/mod(mdg4) complex acts as the D-like proteins and makes nucleation processes to switch from a repressing to an active state. Accordingly, a model in which the su(Hw) binding sites and the associated su(Hw)/mod(mdg4) complex modulate the effects of the MAR/SAR DNA sequence would rather simply account for the biological effects of the gypsy insulator in both the wild type and su(Hw)/mod(mdg4) mutants. The proposed model would then reconcile the two previous models for gypsy insulation, i.e. the heterochromatinization and the looping models.
In conclusion, we have shown that components of the nuclear matrix interact specifically with the gypsy insulator, and accordingly could be involved in establishing chromatin boundaries. The next important question, along these lines, concerns the role of the su(Hw) protein in this interaction. The su(Hw) protein could (i) be directly involved, as a possible component of the nuclear matrix itself, (ii) be involved as a "bridge" or intermediate protein, or even (iii) be dispensable (although necessary for insulation) if interaction with the nuclear matrix is mediated solely by the DNA sequences per se, possibly by the AT-rich interdomains within the insulator. Experiments to answer this question and delineate the role of the MAR/SAR for insulation in vivo are now in progress. ![]() |
ACKNOWLEDGEMENTS |
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We especially acknowledge Dr. Poljak for the gift of the p6 and p8 plasmids and Dr. A. Larsen for the gift of human and yeast topoisomerase II. We thank Dr. C. Lavialle for comments and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the CNRS (ACC-SV3), the Association pour la Recherche sur le Cancer (contract 6552), and Rhone-Poulenc Rorer (Bioavenir contract).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 33-1-42-11-52-76;
E-mail: heidmann{at}igr.fr.
1 The abbreviations used are: UTR, untranslated region; bp, base pair(s); MAR/SAR, matrix/scaffold attachment region; CAT, chloramphenicol acetyltransferase; LIS, lithium diiodosalicylate; LTR, long terminal repeat; topo, topoisomerase.
2 S. Nabirochkin, unpublished results.
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REFERENCES |
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