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V(D)J recombination targeted in CIS by transcription induced DNA supercoiling
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Acronym: Idei 2-2012
Contracting Authority: Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI)
Number / Date of the contract: 2/2012 / 2012-06-01
Idei - Proiecte Complexe
Project Manager: Mihai Ciubotaru
Partners: Institute of Biochemistry of the Romanian Academy
Starting date / finishing date: 2012-07-01 / 2015-06-30
Project value: 5788200 RON
Abstract: Somatic recombination assembles and diversifies the antigen receptor genes, of B and T cells. RAG recombinase binds two specific DNA sites (RSSs) to initiate recombination, a process called paired complex (PC) or synapsis formation. Synapsis is followed by cleavage and then by repair of the double stranded breaks which physiologically occur only between gene segments located intrachromosomally. Pairing preserves the integrity of the genome, preventing aberrant interchromosomal joinings (translocations) implicated in human B and T cell lymphomas. Despite its significance the mechanism that physiologically directs somatic recombination for sites located in cis with respect to DNA is entirely unknown. Prior to recombination, dechromatinization, and germline transcription of the variable gene segments of the immunoglobulin (Ig) or T cell receptor (TCR) loci lead to a substantial dynamic change in the torsional tension and supercoiling of the adjacent DNA (2-7). This work studies the configuration of RSSs in the PC, the role of transcription and its induced DNA supercoiling in RSS site orientation as active factors in controlling RAG intrachromosomal (in cis) sites pairing. To investigate the configuration of RSSs in the PC we developed a FRET based assay with which we show that both 12 and 23RSSs are arched in the PC in a U shape configuration which similarly positions in both RSS types the extreme bends at the junctions of RSS consensus heptamer/nonamer with the spacers. Using the same FRET assay we prove considerable 12RSS conformation differences to occur in the PC versus its signal complex with RAG, configurations which may account for 12/23 rule and its implications in hairpin formation. To supercoil the DNA subjected to recombination and to test both in vitro in the test tube as well as intracellularly the targeting mechanism we used a T7 phage heterologous controllable transcription unit juxtaposed in the proximity of two reporter 12RSSs with respect to a constitutive 23RSS. Although regardless of their deletional or inversional orientation intracellularly transcription enhances recombination from both reporter 12RSSs, the effects are substantially more pronounced for the deletional orientation and for the distal 12RSS(with respect to its 23RSS partner). We first studied the effects of site orientation on RAG catalysis by in vitro coupled cleavage on planar short constrained DNA circles and show that spatial phase RSS orientation is important displaying a sinusoidal activity correlation. Using longer DNA circles containing RSS sites of defined topology and orientation, we show that the rate and efficiency of RAG synapsis is affected both by their configuration and type of supercoiling two factors that we suggest favor in vivo RAG preferential in cis recombination. Moreover, we bring strong evidence in support that local transcription directly facilitates recombination, a novel and unexpected finding which should neither be associated with its subsidiary role in enhancing RAG site accessibility nor with its locally induced supercoiling. To study and contrast RAG in trans effects we developed a LacI DNA tethering artificial system which facilitates RAG induced targeting in trans with respect to DNA and with it we reinvestigated in this context the effects of transcription. Lac I presence enhances dramatically in trans RAG coupled cleavage but unlike the in cis situation local transcription has only a moderate to weak enhancing catalytic effect. The knowledge of RAG preferred RSS configuration, orientation and topology allowed us to develop this LacI in trans DNA tethering system an unprecedented experimental setup with which we can induce intracellularly RAG targeted chromosomal translocations. -The paragraphs denoting the objectives, the methods, the experiments addressing them and their interpretation keep the same initial labels with those used in the original project proposal.

Objectives: At the end of our project we succeeded to accomplish all our proposed goals fulfilling at each objective the following remarkable deliverables: Objective 1- D3A. Identify the mechanism by which DNA supercoiling activates RAG catalysis. a) Routinely express and purify RAG1, RAG2 and HMGB1/2 of various sources, active proteins that constitute valuable reagents for all our in vitro studies. b) We have identified based upon IS264 RAG facilitated ligation assays, that RAG induces rapidly (<5min) substantial accumulation of supercoiled circles. Their production was initially postulated then proved by FRET experiments to be due to a mechanism by which RAG synapsis wraps plectonemically bent 12&23 RSS actively inducing DNA supercoiling. This effect facilitates dramatically in cis synapsis, which is evidenced by monomeric circles accumulating rapidly and efficiently, versus multimers which barely detectable in the assay. c) We have investigated the mechanism by which RAG cleavage is activated by the degree of supercoiling of its substrate. We performed RAG coupled cleavage on 10 distinctly supercoiled topoisomers of various helical phase internal positionings of the 12/23RSSs. The cleavage is substantially increased in supercoiled topoisomers and respects the helical orientations of RSSs which synapse optimally. This suggests that RSS supercoiling simply facilitates a lower activation energy for the formation of the synaptic complex, fostering its configuration. d) Using FRET methodology to circumvent an unproductive design in our original proposal(IHF mediated DNA bending) we have determined the 12&23RSS configuration in the RAG synaptic complex, and we further exploited these findings building a model of the two RSSs in the presence of the RAG complex. The configuration of 23-RSS in the synaptic complex was published in 2013 Ciubotaru, M. Trexler A. J., Spiridon L., Surleac M. D., Rhoades E, ., Petrescu A. J., Schatz D.G. "RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes. Nucleic Acids Research, 2013,vol 41, 2437-2454 e) We have shown both by in vivo and in vitro experiments that transcription driven supercoiling can activate dramatically RAG activity in the promoter adjacent RSSs.

Accomplished Results

The present report (Engl_ScientificReportMihaiCiubotaru_July_2012_15Oct2016_PN-II-ID-PCCE-2011-2.pdf) summarizes the main results obtained throughout the entire project duration from the 1st July 2012 to the final stage 15th October 2016 for the project PN-II-ID-PCCE-2011-2-0024, entitled: "V(D)J recombination targeted in cis by transcription induced DNA supercoiling." funded from PNII Romanian National Research funds via UEFISCDI and lead by Project Director Dr. Mihai Ciubotaru.

FINAL CONCLUSIONS
At the end of our project we succeeded to accomplish all our proposed goals fulfilling at each of the 4 objectives the following valuable fundamental findings and remarkable deliverables:
Objective 1- D3A. Identify the mechanism by which DNA supercoiling activates RAG catalysis. a) Routinely express and purify RAG1, RAG2 and HMGB1/2 of various sources, active proteins that constitute valuable reagents for all our in vitro studies.

b) We have identified based upon IS264 RAG facilitated ligation assays, that RAG induces rapidly (<5min) substantial accumulation of supercoiled circles. Their production was initially postulated then proved by FRET experiments to be due to a mechanism by which RAG synapsis wraps plectonemically bent 12&23 RSS actively inducing DNA supercoiling. This effect facilitates dramatically in cis synapsis, which is evidenced by monomeric circles accumulating rapidly and efficiently, versus multimers which barely detectable in the assay.

c) We have investigated the mechanism by which RAG cleavage is activated by the degree of supercoiling of its substrate. We performed RAG coupled cleavage on 10 distinctly supercoiled topoisomers of various helical phase internal positionings of the 12/23RSSs. The cleavage is substantially increased in supercoiled topoisomers and respects the helical orientations of RSSs which synapse optimally. This suggests that RSS supercoiling simply facilitates a lower activation energy for the formation of the synaptic complex, fostering its configuration.

d) Using FRET methodology as an alternative to circumvent an unproductive design in our original proposal(IHF mediated DNA bending pg27) we have determined the 12&23RSS configuration in the RAG synaptic complex, and we further exploited these findings building a model of the two RSSs in the presence of the RAG complex. The configuration of 23-RSS in the synaptic complex was published in 2013

Ciubotaru, M. Trexler A. J., Spiridon L., Surleac M. D., Rhoades E, ., Petrescu A. J., Schatz D.G. "RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes. Nucleic Acids Research, 2013,vol 41, 2437-2454
We have identified the configuration of 12-SC and that of 12RSS in the synaptic complex published in 2015
Ciubotaru M. , Surleac M. D., Metskas L. A., Koo P., Rhoades E., Petrescu A. J., Schatz D.G." The architecture of the 12RSS in V(D)J recombination signal and synaptic complexes" , Nucleic Acids Research, 2015, vol43, 917-931
Objective 2-D3B3 Test how DNA supercoiling affects RAG deleterious synapsis in trans.
e) We assembled 3 very important constructs in which an octamer of LacI poly operator sites is located in the vicinity of reporter 12 or 23RSS and with it we showed beyond any doubt the remarkable effect LacI has in mediating the in trans RAG cleavage at the reporter 23RSS via docking of the two poly8x operators. Within a range of concentrations between 0.1-1?M of LacI the single cleavage at 23RSS r increases almost five folds, increase which occurs only when the appropriate in trans 12RSSr partner is present in the reaction(Fig.24 of the report).
f) Our results(Fig.25) suggest that in the context of LacI mediating in trans docking of a pair of reporter 12 and 23RSSs located on distinct DNAs, transcription of one site does not influence their RAG synapsing levels beyond the normal levels observed when the reaction is not assisted by transcription. Thus, it seems that T7 transcription requires a local effect to enhance RAG synapsis on transcribed RSSs located in cis as shown in the experiments described in Fig.16 and Fig.19. at Obj. 3
These results and those from Obj 3 were presented as an oral presentation: Ciubotaru M,."V(D)J recombination targeted in cis by transcription induced DNA supercoiling", Invited talk presented at 14th edition of the Site-Specific Recombination, Transposition and DNA dynamics workshop, DNA transactions, Sept. 7-12, 2014 Isle d'Oleron, France. Objective3 -D3C. Prove transcription driven DNA supercoiling to be the selective factor in favoring PC formation in cis.
g) We have shown both by in vivo and in vitro experiments that transcription driven supercoiling can activate dramatically RAG activity in the promoter adjacent RSSs. We have shown from our intracellular assays(Fig.15 and 16) that T7 polymerase transcribing not only that it does not sterically hinder in any way the access of RAG to its RSSs(located within 75bp proximity from the transcription initiation site) but enhances its catalysis to them. This notable effect is a direct facilitation of RAG activity induced by transcription, and must not be confused with the described accessibility phenomenon by which the local chromatin modifications are fostered by the endogenous RNA polymerase II action.
h) In our in vitro assays we show high RAG enhancement near a 12RSS reporter in deletional under the T7 transcription influence in underwound topoisomer UnwC2(Fig.19).This finding can be explained by our original hypothesis that the negative supercoiling created behind a RNA marching polymerase in the deletional orientation helps bending and reconfigures the 12RSS located behind the promoter, making it more amenable for synapsis with its 23 partner(Fig.19). The findings described at d), e) and h) were presented first as a review paper on theoretical grounds in 2014 Ciubotaru M., Surleac M.D., Musat G. M., Rusu A. M., Ionita E., Albu C. C. Paul " DNA bending in the synaptic complex in V(D)J recombination: turning an ancestral transpososome upside down", Discoveries, 2(1):e13:1-15, 2359-7232(2014). and later in an International conference :Ciubotaru M. "V(D)J recombination targeted in cis by transcription induced DNA supercoiling", Invited talk presented at Albany 2015 19th Conversation, June 9th-13, 2015, University of Albany, NY, USA.
j)The data presented in Obj. 2 and Obj. 3 are currently incorporated in a manuscript :"Transcription direct effects that influence V(D)J recombination " Andreea Maria Rusu, Elena Ionita, Cristina Jiglaru, Ioana Popa, Mihaela G. Musat, David G. Schatz , ?tefan E. Szedlacsek and Mihai Ciubotaru, which should be submitted for publication by the end of 2016.
Objective 4-D3D.Use DNA supercoiling to induce in vivo RAG translocations on a synthetic chromosomal mililocus. i)We succeeded to assemble and make a 3T3 stable cell line 3T3 21xLacIOp carrying endogenously in a chromosome a 21x LacI Operator minilocus and we proved its presence and integrity by PCR and FISH technique(Fig.26 and 27).
k) Using the stable cell line 3T3 21xLacIOp we have developed an unprecedented intracellular in trans RAG assay with which we can test various endogenous DNA insertions in the chromosome of the host cell. This is a valuable reagent with which we intend to study the translocations involved in lymphoagenesis.
l) The data presented in Obj. 4 are currently incorporated in a manuscript :"Intracellular Induced in trans V(D)J recombination " Mihaela G. Musat, and Mihai Ciubotaru, which should be submitted for publication by the end of 2016.

THE STAGES OF THE PROJECT AND DELIVERY DATES
1. Single phase for year 2012 (2012-12-15)
2. Single phase for year 2013 (2013-12-15)
3. Single phase for year 2014 (2014-12-15)
4. Single phase for year 2015 (2015-06-30)
RESULTS
PUBLISHED ARTICLES
RESEARCH TEAM

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