Use 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 remodeling 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 eukaryotic 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 resolvosomes 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. Eur. J. Biochem. 268, 6256–6262 (2001) q FEBS 2001 required 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. EXPERIMENTAL PROCEDURES 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 m ML-glutamine, streptomycin (0.1 mg : mL 21 ), penicillin (100 U : mL 21 ), and neomycin (400 mg : mL 21 ). 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 : mL 21 hygromycin. Transfection was generally performed with 5 Â 10 6 cells. 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 QUEST and 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 m M 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. q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6257 and 7 mM final concentration. Camptothecin (Sigma, Germany) was dissolved in dimethylsulfoxide and applied at 50 m M, and EMD 50689, dissolved in dimethylsulfoxide, was tested at 100 m M. In order to inactivate the TRE-CMV promoter, doxycyclin (20 ng : mL 21 ) 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 32 P-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]. RESULTS 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 Â 10 5 cells. (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. 6258 M. Schwikardi and P. Dro ¨ ge (Eur. J. Biochem. 268) q FEBS 2001 and 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. q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6259 and 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 genomic res 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 regulation 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). DISCUSSION 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 Cre gd102NLS (–)Doxy (1)Doxy (–)Doxy (1)Doxy TRE2/3 (single copy) 66.8 74.2 2.0 2.5 TRE3/2 (single copy) 42.3 39.1 2.2 1.3 TRE3/5 (single copy) 75.0 ND ND ND TRE2/2 (. 20 copies) 50.2 50.2 2.5* ND TREII/3 (. 10 copies) 84.0 76.2 2.2 2.3 6260 M. Schwikardi and P. Dro ¨ ge (Eur. J. Biochem. 268) q FEBS 2001 substrate, 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. ACKNOWLEDGEMENTS 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. q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6261 REFERENCES 1. Kadonaga, J.T. (1998) Eukaryotic transcription: An interlaced network of transcription factors and chromatin-modifying machines. Cell 92, 307–313. 2. Kingston, R.E. & Narlikar, G.J. (1999) ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev. 13, 2339–2352. 3. Oettinger, M.A. (1999) V(D)J recombination: on the cutting edge. Curr. Opin. Cell Biol. 11, 325 –329. 4. Wolffe, A.P. & Guschin, D. (2000) Chromatin structural features and targets that regulate transcription. J. Struct. Biol. 129, 102–122. 5. Orphanides, G. & Reinberg, D. (2000) RNA polymerase II elon- gation through chromatin. Nature 407, 471– 475. 6. Bliska, J.B. & Cozzarelli, N.R. (1986) Use of site-specific recom- bination as a probe of DNA structure and metabolism in vivo. J. Mol. Biol. 194, 205 –218. 7. Dunaway, M. & Dro ¨ ge, P. (1989) Transactivation of the Xenopus rRNA promoter by its enhancer. Nature 341, 657–659. 8. Higgins, N.P., Yang, X., Fu, Q. & Roth, J.R. (1996) Surveying a supercoil domain by using the gamma delta resolvase system in Salmonella typhimurium. J. Bacteriol. 178, 2825–2835. 9. Ringrose, L., Chabanis, S., Angrand, P.O., Woodroofe, C. & Stewart, A.F. (1999) Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. EMBO J. 18, 6630–6641. 10. Schwikardi, M. & Dro ¨ ge, P. (2000) Site-specific recombination in mammalian cells catalyzed by gd resolvase mutants: implications for the topology of episomal DNA. FEBS Lett. 471, 147–150. 11. Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109. 12. Salvo, J.J. & Grindley, N.D.F. (1988) The gd resolvase bends the res site into a recombinogenic complex. EMBO J. 7, 3609 –3616. 13. Sessions, R.B., Oram, M., Szczelkun, M.D. & Halford, S.E. (1997) Random walk models for DNA synapsis by resolvase. J. Mol. Biol. 270, 413–425. 14. Dro ¨ ge, P. & Cozzarelli, N.R. (1989) Recombination on knotted substrates by Tn3 resolvase. Proc. Natl Acad. Sci. USA 86, 6062–6066. 15. Arnold, P.H., Blake, D.G., Grindley, N.D., Boocock, M.R. & Stark, W.M. (1999) Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity. EMBO J. 18, 1407–1414. 16. Buchholz, F., Angrand, P.O. & Stewart, A.F. (1996) A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs. Nucleic Acids Res. 24, 3118–3119. 17. Lorbach, E., Christ, N., Schwikardi, M. & Dro ¨ ge, P. (2000) Site- specific recombination in human cells catalyzed by phage l integrase mutants. J. Mol. Biol. 296, 1175–1181. 18. Gossen, M. & Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracyclin responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551. 19. Kwon, J., Morshead, K.B., Guyon, J.R., Kingston, R.E. & Oettinger, M.A. (2000) Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA. Mol. Cell 6, 1037 –1048. 20. Strahl, B.D. & Allis, C.D. (2000) The language of covalent histone modifications. Nature 403, 41–45. 21. Tsai, S C., Valkov, N., Yang, W M., Gump, J., Sullivan, D. & Seto, E. (2000) Histone deacetylase interacts directly with DNA topoisomerase II. Nat. Genet. 26, 349– 353. 22. Yoshida, M., Horinouchi, S. & Beppu, T. (1995) Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function. Bioessays 17, 423–429. 23. Boege, F., Straub, T., Kehr, A., Boesenberg, C., Christiansen, K., Andersen, A., Jakob, F. & Ko ¨ hrle, J. (1996) Selected novel flavones inhibit the DNA binding or the DNA religation step of eukaryotic topoisomerase I. J. Biol. Chem. 271, 2262–2270. 24. Jeong, S. & Stein, A. (1994) Micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous DNA lengths for chromatin assembled on non-replicating plasmids in transfected cells. Nucleic Acids Res. 22, 370–375. 6262 M. Schwikardi and P. Dro ¨ ge (Eur. J. Biochem. 268) q FEBS 2001 . Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin Micha Schwikardi and Peter Dro¨ge Institute of Genetics,. 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