Alterations in chromatin structure are involved in the regu-lation of DNA transactions such as transcription and site-specific recombination.. A low level of recombination is observed wi
Trang 1Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin
Micha Schwikardi and Peter Dro¨ge
Institute of Genetics, University of Cologne, Germany
DNA transactions in eukaryotes require that proteins gain
access to target sequences packaged in chromatin Further,
interactions between distinct nucleoprotein complexes are
often required to generate higher-order structures Here, we
employed two prokaryotic site-specific recombination
sys-tems to investigate how chromatin packaging affects the
assembly of nucleoprotein structures of different
complex-ities at more than 30 genomic loci The dynamic nature of
chromatin permitted protein – DNA and DNA – DNA
inter-actions for sites of at least 34 bp in length However, the
assembly of higher-order nucleoprotein structures on targets
spanning 114 bp was impaired This impediment was
maintained over at least 72 h and was not affected by the
transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature
of chromatin Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin
Keywords: chromatin; DNA reactivity; nucleoprotein com-plex; site-specific recombination; transcription
Alterations in chromatin structure are involved in the
regu-lation of DNA transactions such as transcription and
site-specific recombination Recently, chromatin remodeling and
histone acetylation/deacetylation were identified as
import-ant regulators of chromatin structure at specific loci
(reviewed in [1 – 4]) Fundamental questions in this context
concern the general reactivity of DNA sites packaged into
chromatin For example, does the assembly of complex
nucleoprotein structures require active chromatin
remodel-ing throughout the genome, or is remodelremodel-ing only required
at specific loci? Further, the transcription process itself
transiently alters the structure of chromatin (reviewed in [5])
Little is known, however, whether these dynamic alterations
render sequences in vivo more accessible for DNA-binding
proteins and, thus, contribute to the formation of complex
nucleoprotein structures
Site-specific recombination has been used as a powerful
method to investigate fundamental questions both in
pro-karyotic and eupro-karyotic cells [6 – 10] In our present study,
we sought to address the questions outlined above by
employing two site-specific recombination systems that differ
markedly in their complexity The less elaborate system is
represented by the Cre recombinase encoded by Escherichia
coli phage P1 This enzyme is a member of the integrase family of conservative site-specific recombinases and functions efficiently in eukaryotic cells (reviewed in [11]) Two Cre monomers bind cooperatively to a 34-bp recom-bination sequence termed loxP (Fig 1A) Collision of two loxP-bound dimers results in the formation of a recombino-genic complex that catalyzes two reciprocal single-strand-transfer exchange reactions This leads to deletion of intervening DNA if two loxP sites are positioned as direct repeats
The second system employed in this study is derived from the E coli gd transposon-encoded resolvase The resolvase system is more complex than the Cre system In the first step leading to recombination resolvase binds to a recombination sequence called res A single res is composed of three binding sites (I – III) for resolvase dimers which together occupy 114 bp (Fig 1B) Three dimers bind cooperatively
to res with comparable affinities towards sites I and II in order to generate a recombinogenic complex, termed resolvo-some [12] Two resolvoresolvo-somes then synapse by random collision [13] Two res must be present as direct repeats on the same negatively supercoiled DNA molecule Only this site orientation leads to the formation of a functional synaptic complex, termed synaptosome, which entraps three ( – )supercoils [14] Strand exchange is catalyzed by dimers bound at paired sites I, while those bound at sites II and III serve accessory roles in synaptosome formation and in the activation of strand cleavage therein (Fig 1B) This rather complex architecture imposes directionality on recombina-tion, i.e strand exchange always results in deletion of DNA between two res
Recently, we have transferred the gd system to higher eukaryotes [10] Two resolvases containing activating mutations (E124Q or E102Y/E124Q) and a SV40-derived nuclear localization signal (NLS) at their C-termini are recombination-proficient on episomal DNA Full res are still
Correspondence to P Dro¨ge, Institute of Genetics, University of
Cologne, Weyertal 121, D-50931 Cologne, Germany.
Fax: 1 49 221470 5170, Tel.: 1 49 221470 3407,
E-mail: p.droege@uni-koeln.de
(Received 10 July 2001, revised 3 October 2001, accepted 5 October
2001)
Abbreviations: GFP, green fluorescence protein; Cre, cause of
recombination in phage P1; b-Gal, b-galactosidase; PGK, phospho
glycerate kinase; DMEM, Dulbecco’s modified Eagle’s medium; TRE,
tetracyclin-responsive-element; RLHRLZ,
res-lox-hygromycin-res-lox-lacZ.
Trang 2required for efficient recombination indicating that, even
in the absence of substrate supercoiling, the reaction
proceeds through the normal synaptic complex [10] This
has been demonstrated directly with topologically relaxed
substrates in a recent in vitro study employing
correspond-ing mutants of the related Tn3 resolvase [15] We also
demonstrated that the gd resolvase double mutant E102Y/
E124Q (hereafter referred to as gd102NLS) and Cre
recom-bine episomal substrates in CHO cells with comparable
efficiencies [10]
A low level of recombination is observed with gd102NLS
on episomal substrates containing two isolated, directly
repeated copies of the 32-bp long site I of res [10] Due to the symmetry of the central two base pairs at site I, random collision of two site I-bound gd102NLS dimers results, in this case, in either deletion or inversion of the intervening DNA segment (M Schwikardi and P Dro¨ge, unpublished results; see Fig 1C) Hence, depending on the presence or
on the accessibility of accessory sites in res, gd102NLS employs two recombination pathways in eukaryotic cells that can be distinguished by the resulting products
In order to compare the activities of Cre and gd102NLS
on substrates packaged in chromatin, we have generated reporter cell lines that carry target sites for both recom-binases randomly integrated into the host genome By comparing the efficiencies of recombination on episomal and on genomic targets, we have shown that the dynamic nature of chromatin renders a site of at least 34 bp in general reactive for recombination However, the assembly of and/
or the interaction between more complex nucleoprotein structures on 114-bp targets was significantly impaired in chromatin This impediment was not affected by the tran-scriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases
E X P E R I M E N T A L P R O C E D U R E S Vectors
Expression vectors for Cre, wild-type gd resolvase, and the two resolvase mutants have been described previously [10] pTRE-res-lox-hygromycin-res-lox-lacZ (-RLHRLZ) was generated by PCR using the recombination cassette of pCH-RLNRLZ as template [10] The neomycin gene was substituted by the hygromycin gene of pTK-Hyg (Clontech) The entire cassette was introduced into the Bam HI site of pTRE2 (Clontech) The derivate pTRE-SLHSLZ was gener-ated by PCR using pTRE-RLHRLZ as template The corre-sponding recombined product vectors were generated through transformation into E coli strain DH5a or 294-Cre [16] They were subsequently characterized by restriction digestion and DNA sequencing Substrate vectors were isolated using endotoxin free affinity chromatography (Qiagen, Germany)
Cell culture, cell lines, transfection, and recombination assays
CHO-AA8 Tet-Off cells (Clontech) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, streptomycin (0.1 mg:mL21), penicillin (100 U:mL21), and neomycin (400 mg:mL21) Stable reporter cell lines were generated with SapI-linearized pTRE-RLHRLZ Three days post-transfection, cells that had stably integrated the vector into the genome were selected with 350 mg:mL21hygromycin Transfection was generally performed with 5 106cells Electroporation was in 800 mL RPMI medium without phenolred and glutamate (Life Technologies) at 960 mF and 280 V Transfection efficiencies were determined by FACS (FACS Calibur; Becton Dickinson) using the program CELL QUESTand GFP as a marker They were typically in the range of 40 – 70% Trichostatin A (ICN Biomedicals, Germany), dissolved in ethanol, was added to culture medium at 3 mM final concentration Butyrate (Sigma, Germany), dissolved in sterilized water, was tested at 0,5
Fig 1 Schematic representations of recombination pathways.
(A) Cre-loxP pathway LoxP sites (arrows with open head) are present
as direct repeats on a circular substrate Synapsis occurs by collision of
two loxP-bound dimers (filled circles) The two sites are aligned in an
antiparallel orientation Strand exchange will then lead to deletion (B)
Recombination on two full res by wild-type or mutant resolvase The
res are depicted as direct repeats on a circular substrate DNA
super-coiling, required for the reaction with wild-type resolvase, is omitted for
clarity After all three cognate sites within res (I, II, and III) are bound
by resolvase dimers (filled circles) a synaptosome is generated The
interaction between dimers bound at accessory sites II and III, and the
catalytically active ones at paired sites I that is required to trigger strand
exchange is indicated by arrows Recombination will lead to deletion of
DNA between two res (C) Recombination by mutant resolvases on
sites I of res After resolvase dimers (filled circles) are bound to sites I,
random collision leads to two functional synaptic complexes When
subsites I align in an antiparallel orientation (top), recombination leads
to inversion of DNA between sites I If both sites align in a parallel
orientation, recombination leads to deletion (bottom) The fact that both
types of alignment leads to productive recombination complexes is due
to the symmetric nature of the site I of res.
Trang 3and 7 mM final concentration Camptothecin (Sigma,
Germany) was dissolved in dimethylsulfoxide and applied
at 50 mM, and EMD 50689, dissolved in dimethylsulfoxide,
was tested at 100 mM In order to inactivate the TRE-CMV
promoter, doxycyclin (20 ng:mL21) was added 2 weeks
prior to electroporation
b-Galactosidase (b-gal) assays, Southern blotting and
PCR
b-Gal assays and Southern blotting were performed as
described previously [10,17] The 32P-labelled probe was
generated by PCR with oligonucleotides priming in the
N-terminal domain of the lacZ gene Genomic PCR was
performed as described previously using 0.5 mg of purified
genomic DNA as template [10]
R E S U L T S
Cre- and gd102NLS-mediated recombination on episomal
substrates
We employed in this study a different cell line and different
recombination substrates than in our previous study In order
to use episomal substrates as controls it was therefore
necessary to re-investigate how Cre and gd102NLS perform
under these conditions Further, it was necessary to analyze
recombination on linearized episomal substrates as they
better resemble the topology of targets placed in chromatin
than circular substrates used before
The recombination substrate, termed pTRE-RLHRLZ,
contains a tetracyclin-responsive-element (TRE)-CMV
pro-moter construct placed upstream of a recombination cassette
(Fig 2A) Transcription from this promoter is regulated in
CHO-AA8 Tet-Off cells by doxycyclin The promoter is
active in the absence of drug, while its presence leads to
rapid transcriptional inactivation [18] The cassette is
com-posed of two directly repeated copies of res and of loxP
sites They flank the coding region of the hygromycin gene
which serves as the resistance marker for the generation of
stable reporter cell lines (see below) We placed the coding
region of the lacZ gene downstream of the cassette
Transcription is initiated 127-bp upstream of the first
nucleotide defining site III of the promoter proximal res,
Fig 2 Recombination on episomal targets (A) Diagram of substrate
vector pTRE-RLHRLZ Relevant genetic elements are marked and
explained in the text Start of transcription within the TRE-CMV
promoter is at 1374 (B) Normalized b-Gal activities as reporter for
recombination on episomal pTRE-RLHRLZ The activity of the
reporter is expressed in (%) relative light units (RLU) and normalized to
the amount of protein in crude cell extracts The activity resulting from
the recombined product (pTRE-RLZ) cotransfected with an expression
vector for a phage l integrase mutant was set as 100% In each case,
data were collected from six separate transfection assays, each
employing two wells containing about 2 105cells (C) Normalized
b-Gal activities as reporter for recombination on pTRE-SLHSLZ This
substrate contains two isolated sites I of res replacing the full res in
pTRE-RLHRLZ The recombined product, termed pTRE-SLZ,
cotransfected with pPGKIntss was used again as control (100%) The
graph shows the mean values of two assays with standard deviations
indicated by vertical lines.
Trang 4and proceeds through the entire cassette and the downstream
lacZ gene (Fig 2A) b-Gal assays performed with crude
extracts prepared from CHO-AA8 Tet-Off cells transfected
with pTRE-RLHRLZ confirmed that the lacZ gene is not
expressed (data not shown) Recombination by either Cre
or resolvase leads to deletion of the resistance gene and
to expression of b-Gal Recombination by either Cre or
resolvase thus generates identical product vectors, termed
pTRE-RLZ (Fig 2A)
Linearized substrate and product vectors were
cointro-duced with an expression vector for either Cre (pPGKCrebpa)
or gd102NLS (pPGKgd102NLS) Substrate and product
vectors cotransfected with an expression vector for the
phage lambda integrase mutant Int-h (pPGKInthss) served
as controls It is important to emphasize here that
recom-binases are expressed from the same eukaryotic promoter
(PGK) in the same vector background, and that Cre and
gd102NLS contain identical NLS [10]
Normalized b-Gal activities were determined in cell
extracts prepared 72 h after transfection The results show
that Cre and gd102NLS efficiently recombine linearized
pTRE-RLHRLZ (Fig 2B) In contrast to our previous study,
however, we found that recombination by gd102NLS is
reduced in this cell line to a level of 60% of that observed
with Cre Identical results were obtained when ( –
)super-coiled instead of linearized substrates were cotransfected
with recombinase expression vectors (data not shown)
We also analyzed recombination on a linearized
deriva-tive of pTRE-RLHRLZ, termed pTRE-SLHSLZ This
sub-strate contains two isolated sites I of res as direct repeats,
instead of two full res gd102NLS is also proficient to
recombine sites I in the absence of accessory sites The
efficiency of this reaction is significantly reduced, however,
reaching 10% of that observed with Cre (Fig 2C)
Recombination on genomic substrates
Hygromycin-resistant cell lines were generated with
linear-ized pTRE-RLHRLZ Southern blot analysis, PCR, and
DNA sequencing revealed that they contain between one
and about 20 copies of the substrate vector at different
genomic locations Hence, the vector integrated probably
randomly into the host genome (data not shown) We first
analyzed recombination in cell line TRE2/3 which contains
a single copy of the vector The analysis was performed in
the absence of doxycyclin, i.e the TRE-CMV promoter is
active and transcription proceeds through the entire
recombination cassette
The expression vectors for Cre, wild-type resolvase
(gdNLS), and the resolvase single (gd124NLS) and double
mutant (gd102NLS) were introduced separately into TRE2/3
cells by electroporation In addition, an expression vector
for GFP was used to determine transfection efficiencies
Genomic DNA isolated 72 h after electroporation was
digested with Bam HI and analyzed for recombination by
Southern blotting using a probe specific for the N-terminal
region of the lacZ gene (compare Fig 2A) The results show
that Cre efficiently recombines the genomic substrate
(Fig 3) Considering the transfection efficiency
indepen-dently determined in each experiment, a quantitative
analysis by phosphorimager from four different experiments
revealed that recombination occurred, on average, in about
66% of cells transfected with pPGKCrebpa However,
neither gdNLS nor gd124NLS generated a detectable amount
of products Only gd102NLS produced a faint signal In this case, quantitation revealed that recombination occurred in about 2% of cells transfected with pPGKgd102NLS (see also Table 1) Hence, compared to the reactions on episomal targets, the efficiency of recombination by gd102NLS is severely reduced on genomic res
We then tested whether treatment of TRE2/3 cells with doxycyclin affects recombination The drug was added to the medium 2 weeks prior to transfection This treatment leads to the rapid inactivation of the TRE-CMV promoter and should provide time for a potential re-setting of the chromatin structure Control experiments using cell line TRE2/3R containing one copy of recombined pTRE-RLZ, which was subcloned from Cre-treated TRE2/3 cells, con-firmed that b-Gal activity was reduced 200- to 300-fold compared to a control lacking the drug Further, the residual activity detectable in doxycyclin-treated TRE2/3R cells was only threefold to fivefold higher than that in parental TRE2/3 cells, indicating that the TRE-CMV promoter was efficiently inactivated by the drug (data not shown) When we tested recombination in doxycyclin-treated TRE2/3 cells, however, quantitation of Southern blots revealed that Cre and gd102NLS remained unaffected by the transcriptional status
of the recombination cassette (Table 1)
The entire set of experiments exemplified above with TRE2/3 cells expressing either Cre or gd102NLS was performed with five different cell lines, thus investigating recombination on more than 30 genomic copies of pTRE-RLHRLZ While Cre reproducibly recombined between 40
Fig 3 Cre, but not resolvase, efficiently recombines genomic targets Genomic DNA was prepared from TRE2/3 cells 72 h after electroporation with recombinase expression vectors DNA was digested with Bam HI, separated on a 0.8% (w/v) agarose gel, trans-ferred to nitrocellulose membrane, and hybridized to a probe derived from the N-terminal region of lacZ Bam HI-digested pTRE-RLHRLZ and pTRE-RLZ were used as unrecombined and recombined controls, respectively.
Trang 5and 80% of targets, gd102NLS exhibited only a residual
activity (Table 1) We conclude that Cre, irrespective of the
transcriptional status of loxP sites in chromatin, efficiently
recombines pTRE-RLHRLZ at the majority of genomic
loci However, gd102NLS appears to be severely impaired
when targets are packaged into chromatin Further, this
impediment is maintained irrespective of the transcriptional
status of target sequences
gd102NLS Recombines at genomicres through random
collision of I sites
The residual recombination activity observed in the Southern
analyses with gd102NLS (Fig 3; Table 1) could be due to
recombination at genomic sites I without synaptosome
formation This would be similar to the reaction on episomal
site I substrates To investigate this possibility, we used
b-Gal activity and PCR as reporters for recombination The
results exemplified with cell line TRE2/3 show that a
residual b-Gal activity is indeed detectable when gd102NLS
is expressed (Fig 4A) This activity (about 5% compared to
Cre) is in the range of that observed with episomal site I
substrates (about 10%; Fig 2C) Further, genomic PCR
employing primer pair P1/P2 (see Fig 2A) confirmed that
Cre and gd102NLS catalyzed deletion in TRE2/3 cells
(Fig 4B) If the activity observed with gd102NLS results
from a simple synapse generated by random collision
between dimers bound solely at sites I, inversion of the DNA
segment located between res should also be detectable PCR
analysis with primer pair P3/P4 and DNA sequencing of
products revealed that gd102NLS also catalyzed inversion
on genomic res (Fig 4C) Faint PCR signals indicative of
inversion and deletion were also observed in some
experiments with gd124NLS
Recombination is not affected by inhibitors of histone
deacetylases or topoisomerases
Histone modifications such as acetylation and
phosphoryl-ation play important roles in the regulphosphoryl-ation of chromatin
structure In particular acetylation of the N-terminal tails
of histones are thought to render chromatin more accessible
for DNA-binding proteins In fact, DNA transactions such
as V(D)J recombination and transcription are enhanced
when histone deacetylases are inhibited [19,20] Further,
eukaryotic topoisomerases appear to be involved in
chromatin organization, perhaps through direct interaction
with histone deacetylases [21] We decided therefore to test
whether inhibitors of histone deacetylases and eukaryotic
topoisomerases might render genomic res more accessible for gd102NLS, which could then lead to a significant increase in recombination activity
Two reporter cell lines were incubated with histone deacetylase inhibitors butyrate or trichostatin A (reviewed in [22]) at 24 h after transfection of recombinase expression vectors Cells were treated for 24 h, after which drugs were removed and cells were incubated for additional 24 h Untreated cells served as controls Further, controls with b-Gal-expressing TRE2/3R cells treated in the same way with trichostatin A showed that b-Gal activity increased up
to fourfold This might indicate that the TRE-CMV pro-moter becomes more accessible for the transcriptional machinery However, Southern analysis and b-Gal assays revealed that the efficiencies of recombination by Cre and gd102NLS remain unaffected by these inhibitors Following the same protocol, treatment of TRE2/3 cells with topo-isomerase type I inhibitor camptothecin and with the flavonoid EMD50689, the latter inhibits both type I and type
II topoisomerases [23], also showed no effect on recom-bination efficiencies (data not shown)
D I S C U S S I O N
We have investigated the reactivity of chromosomal DNA for two prokaryotic site-specific recombinases Episomal substrates were used first as controls because previous studies indicated that transfected, nonreplicating plasmid DNA is either not packaged into chromatin [10] or exhibits
an atypical chromatin structure even at 4 days after trans-fection [24] Our comparison between episomal and genomic substrates revealed that chromatin packaging of loxP sites did not significantly affect the activity of Cre at more than 30 different genomic loci Likewise, the rather inefficient gd102NLS recombination pathway employing a synapse consisting solely of two site I-bound dimers appeared to be functional to about the same extent on episomal and on genomic substrates The latter result is particularly important It implies that the steady-state intra-cellular concentration of gd102NLS at genomic and at episomal targets must be in the same range
The centers of the loxP site and of the site I of res located next to each other in the recombination cassette are separ-ated by 45 bp Hence, the two sites together occupy 78 bp The fact that both sites remained reactive when packaged in chromatin implies that the nucleosomal structure of chromatin must be rather dynamic, at least over the time frame of our experiments Assuming that the nucleosomal positioning is not determined by the sequence of our
Table 1 Recombination on genomic targets The values represent mean values of (%) recombination determined from two to four experiments.
An asterisk indicates data from one experiment ND, not determined.
Cell line
Trang 6substrate, the loxP site and the site I of res may be located either in the nucleosomal core or in the linker DNA; the length of the latter can vary significantly in vivo In addition, productive encounters readily occurred between sites I of res and between loxP sites separated by 1.2 kb, thus further strengthening the view of a rather flexible chromatin struc-ture [2,9] Importantly, our results have shown that these basic properties of chromatin are not significantly altered by the transcriptional status of DNA
In contrast to loxP sites and sites I of res, the reactivity of episomal and genomic full length res differed markedly in our analysis Genomic res were about 30-fold less reactive for recombination than their epsiomal counterparts We consider two explanations for this result Firstly, the ordered nucleosomal organization of chromatin prevents the co-operative binding of gd102NLS to all three sub-binding sites Even the repeated passage of the transcriptional machinery does not affect this accessibility limit Further-more, as a significant amount of gd102NLS is present in CHO cells throughout the time course of our experiments [10], neither changes in chromatin structure occurring during the cell cycle nor the passage of a replication fork render two 114-bp spanning res simultaneously accessible for the recombinase
Secondly, if we assume that two resolvosomes form simultaneously on genomic substrates, their interaction to generate a functional synaptosome may be prevented by unknown structural features of chromatin It is possible, for example, that the toroidal wrapping of DNA in eukaryotic chromatin somehow precludes the plectonemic intertwining
of res sites, which is required to assemble a functional synaptosome [14] Nevertheless, sites I of res aligned in two different orientations, leading to deletion or inversion
of the intervening DNA (Fig 4) This implies that the 1.2-kb long DNA segment connecting two res sites must be rather flexible despite its nucleosomal organization
Our analysis employing inhibitors of histone deacetylase and topoisomerases revealed that the reactivity of res remained unaffected, even though the transcriptional activity
of the TRE-CMV promoter was enhanced several-fold by both types of inhibitors Apparently an opening of the chromatin structure through hyperacetylation of histones and possible changes in DNA and/or chromatin topology resulting from topoisomerase inhibition were not sufficient
to render genomic full length res accessible for resolvase binding It will nevertheless be interesting to test in the future a wider range of substances for their potential to activate resolvase-mediated recombination on genomic res Our results thus suggest that there is a general requirement for substantial active chromatin remodeling in order to assemble multicomponent nucleoprotein complexes This requirement significantly increases the stringency with which DNA transactions are regulated in higher eukaryotes
A C K N O W L E D G E M E N T S
We thank members of our and of K Rajewsky’s laboratory for critical comments on the manuscript The flavonoid EMD50689 was a kind gift
of Dr J Ko¨hrle, Wu¨rzburg, Germany Special thanks go to K Rajewsky for support with cell culture facilities This work was financed through SFB 274 and Deutsche Forschungsgemeinschaft grant Dr187/8– 2 (PD).
Fig 4 Resolvase recombines at genomic res via random collision
of site I-bound dimers (A) Normalized b-Gal activities as reporter
for recombination in pTRE2/3 cells The graph shows the mean values
from five transfection experiments The 100% reference was obtained
with crude extracts prepared from pTRE2/3R cells transfected with
pPGKCrebpa Note that the RLU are plotted in a logarithmic scale.
(B) PCR to analyze deletion in TRE2/3 cells Genomic DNA was
prepared 72 h after transfection The PCR product indicative of deletion
is marked (del.) The product resulting from unrecombined genomic
pTRE-RLHRLZ is also indicated (unrec.) (C) PCR to analyze inversion
in TRE2/3 cells Only one PCR product (inv.) is generated Products
were analyzed on 0.8% agarose gels and visualized by UV after
ethidium bromide staining.
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