Regulation of T cell receptor α gene assembly by a complex hierarchy of germline Jα promoters

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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7930292 Regulation of T cell receptor α  gene assembly by acomplex hierarchy of germline J α  promoters  Article   in  Nature Immunology · June 2005 DOI: 10.1038/ni1189 · Source: PubMed CITATIONS 60 READS 31 3 authors , including:Abbas HawwariNational Guard Health Affairs 43   PUBLICATIONS   713   CITATIONS   SEE PROFILE All content following this page was uploaded by Abbas Hawwari on 05 December 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Regulation of Tcra   gene assembly by a complex hierarchy ofgermline J α  promoters Abbas Hawwari , Cheryl Bock , and Michael S. Krangel The Department of Immunology, Duke University Medical Center, Durham NC 27710  Abstract Tcra  gene assembly is characterized by an orderly progression of primary and secondary V α  to J α recombination events across the J α  array, but the targeting mechanisms responsible for thisprogression are largely unknown. Previous studies revealed that the TEA promoter plays an importantrole in targeting primary Tcra  rearrangements. We show that TEA and a novel promoter associatedwith J α 49 target primary recombination to discrete sets of C α -distal J α  segments and together directnearly all normal primary recombination events. Further, we show that TEA promoter deletionactivates previously suppressed downstream promoters and stimulates primary rearrangement tocentrally located J α  segments. Central promoter derepression also occurs following primaryrearrangement, thereby providing a mechanism to target secondary recombination events.The development of T lymphocytes bearing an α  T cell receptor (TCR) depends on the somaticassembly of variable (V), diversity (D) and joining (J) gene segments by the process of V(D)J recombination 1 . Tcrb  gene rearrangements occur first, in the CD4 - CD8 -  double negative (DN)subset of thymocytes. Production of a functional TCR   protein then promotes differentiationto the CD4 + CD8 +  double positive (DP) stage, during which Tcra  gene rearrangements occur.Following expression of a cell surface α  TCR, DP thymocytes are tested by positive selectionto identify those with useful TCRs 2 . Only a small fraction of DP thymocytes are ultimatelyselected for further maturation. Tcra  rearrangement has several unique features that are thought to increase the likelihood of positive selection 2,3 . First, V α  to J α  rearrangement occurs on both alleles without allelicexclusion. Second, initial, or primary rearrangements, are targeted to J α  segments at C α -distal,or 5 ′  end, of the 70 kb J α  array (polarity is denoted in reference to the sense strand throughout).If primary rearrangement fails to produce a selectable TCR, DP thymocytes may then undergomultiple additional rounds of secondary rearrangement that involve progressively more 5 ′ V α  gene segments and progressively more 3 ′  J α  gene segments. Moreover, the 5 ′  to 3 ′ progression along the J α  array is nearly synchronous on two Tcra  alleles. Tcra  rearrangementis ultimately terminated by positive selection, which silences recombinase expression, or bycell death resulting from a lack of positive selection. Thus, secondary V α  to J α  rearrangementprovides thymocytes multiple opportunities for positive selection, and is critical for theproduction of a robust and diverse TCR α  repertoire 4,5 .The mechanisms that direct primary Tcra  rearrangement are only partially understood. The Tcra  enhancer (E α ), situated 3 ′  of C α , controls the entire J α  array and is required for all V α  toJ α  recombination events 6 . The T early α  (TEA) promoter, located at the 5 ′  end of the J α  array,acts more locally, and controls usage of the most 5' J α  segments (J α 61-J α 53) 7 . However, TEA-deficient mice have quantitatively normal J α  usage downstream of this region, suggesting the Correspondence to: Michael S. Krangel.Correspondence should be addressed to M.S.K. (krang001@mc.duke.edu). NIH Public Access Author Manuscript  Nat Immunol . Author manuscript; available in PMC 2008 June 5. Published in final edited form as:  Nat Immunol . 2005 May ; 6(5): 481489. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    presence of one or more additional cis -acting elements with the potential to target primaryrearrangements. We previously analyzed the distribution of J α  usage in the short-lived DPthymocytes of mice lacking transcription factor ROR  (  Rorc -/- ) 5 . J α  usage was restricted to the5 ′  portion of the J α  array, as would be expected if thymocytes were limited to primaryrearrangements. Moreover, the detected rearrangements resolved into two discrete clusters,one just downstream of TEA and one spanning J α 50 to J α 45. The former is presumed to reflectprimary rearrangements targeted by the TEA promoter. The latter appears to identify a site of primary targeting that could account for the residual rearrangements in TEA-deficientthymocytes. However, to date, no known regulatory elements have been mapped to this region.Even less is understood about the targeting of secondary Tcra  rearrangements. An orderly 5 ′ to 3 ′  progression of rearrangements across the J α  array is implied by allelic synchrony in J α usage. Two types of models have been proposed, both relying on the ability of promoters toprovide local access to the recombinase 5,7,8 . The developmental windows model envisions aset of embedded germline J α  promoters that are successively activated according to an intrinsicdevelopmental program during DP thymocyte development. An alternative model envisions aV α  promoter-driven chain reaction, in which, following primary rearrangement, successivelyintroduced V α  promoters would target subsequent rounds of secondary rearrangement toprogressively more 3 ′  sets of J α  segments.To better understand the molecular basis for the ordered propagation of rearrangement eventsacross the J α  array, we searched this portion of the Tcra  locus for novel promoter elements.We identified a strong promoter element situated just upstream of J α 49, and tested its functionalrelevance by deleting it, either alone or in combination with the TEA promoter, usinghomologous recombination strategies. Our results indicate that the two promoters targetdiscrete sets of 5 ′  J α  segments for primary rearrangement, and together account for almost allnormal primary J α  recombination events. In addition, we found that 5 ′  promoter deletion leadsto the activation of previously suppressed downstream promoters, which can efficiently targetrearrangements into the central portion of the J α  array. Central promoter derepression alsooccurs following primary rearrangement. This provides a previously unappreciated mechanismfor the targeting of secondary V α  to J α  recombination events. RESULTS Identification of a germline promoter upstream of J α 49 We searched for the presence of promoter elements that could account for the recovery of J α rearrangements downstream of J α 53 in TEA-deficient mice 7 . As one approach, DP thymocytesfrom  Rag2 -/-  mice carrying a functional Tcrb  transgene (Rx  ) were analyzed for novel DNaseI hypersensitive sites within the J α  array ( Fig. 1a,b ). These mice provide a pure source of DPthymocytes that retain both a germline J α  array and all associated promoter elements. Basedon Southern hybridization using a J α 50 probe, DNase I digestion reduced a 12 kb Spe I fragmentto a 5.5 kb species. This mapped a strong hypersensitive site just upstream of the J α 49 genesegment. In the second approach, a C α  primer was used in 5' rapid amplification of cDNA ends(RACE) to amplify full-length germline transcripts that span the J α  array ( Fig. 1a,c ). Suchtranscripts are typically spliced from the most 5 ′  splice donor to C α . Two major species of 1.3kb and 0.6 kb were amplified from cDNA of Rx   DP thymocytes ( Fig. 1c, inset ). These specieswere cloned and sequenced. The 1.3 kb products were found to reflect initiation at the TEApromoter at the extreme 5 ′  end of the J α  array, with the TEA exon spliced to C α  (data notshown). The 0.6 kb products reflected initiation at sites associated with J α  segments (J α 58-J α 44), with splicing from J α  to C α . The predominant germline J α  transcripts included J α 49 andinitiated at several sites clustered within the J α 49 recombination signal sequence ( Fig. 1c,d ).To test for J α 49 promoter function in vitro , a 900 bp fragment spanning the J α 49 transcriptionstart site was cloned upstream of the luciferase gene in a reporter plasmid that contained or Hawwari et al.Page 2  Nat Immunol . Author manuscript; available in PMC 2008 June 5. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    lacked E α , and was tested for activity following transfection into Jurkat T cells ( Fig. 1e ). Whenintroduced into LUC-E α , the promoter fragment stimulated luciferase activity by about 200-fold. However, promoter activity was minimal in reporter substrates lacking E α . All activityof the 900 bp fragment was contained within a 5 ′  truncated fragment of 425 bp (data not shown).The position of the J α 49 promoter corresponds to the region of the J α  array in which J α  usageincreases to wild-type frequency in TEA-deficient mice 7  and to the cluster of primary J α recombination events observed in the short-lived DP thymocytes of  Rorc -/-  mice 5 . In contrast,promoter activity associated with the J α 58, J α 57, and J α 56 gene segments ( Fig. 1c ) localizedwithin the TEA-dependent zone. Therefore, we focused on the J α 49 promoter as a putativetargeting element for primary V α  to J α  recombination. T cell development in promoter-deleted mice The J α 49 and TEA promoters were individually deleted from embryonic stem (ES) cells derivedfrom 129:C57BL/6 (B6) heterozygous mice using a Cre- lox P homologous recombinationstrategy ( Fig. 2a,b ). Chimeric mice carrying a lox P-flanked neo r  cassette in place of TEA werebred to transgenic mice mice expressing Cre recombinase to delete the cassette, creating aTEA-deficient allele ( Δ TEA). The lox P-flanked neo r  cassette was removed from J α 49promoter-targeted ES cells by transient expression of Cre recombinase in vitro  to create aJ α 49-deficient allele ( Δ J49). These cells were then retargeted to delete the TEA promoter aswell. The use of heterozygous ES cells insured that the two homologous recombination eventswould occur on the same (129) allele. Cre-mediated recombination then yielded two differentalleles, one with simple deletions of both promoters ( Δ TEA Δ J49) and a second with a 16 kbdeletion encompassing the entire region between TEA and J α 49 ( Δ 5 ′ ) ( Fig. 2c ). The behaviorof all four targeted alleles was subsequently analyzed in homozygous mice ( Δ TEA, Δ J49, Δ TEA Δ J49, and Δ 5 ′ ).To test for developmental defects in the various strains, CD4, CD8 and TCR   expression wasanalyzed in thymocytes and splenocytes of 2-5 week old mice by flow cytometry. Nodifferences were detected in the absolute numbers and percentages of DN, DP, and singlepositive thymocytes and splenic T lymphocytes as compared to age matched littermate controls(data not shown). Similarly TCR   expression on thymocytes and splenocytes wasindistinguishable from littermate controls. Thus, there were no gross deficiencies in α  TCRexpression or α  T cell development in promoter-deleted mice. The J α 49 promoter controls primary rearrangement To study the effect of promoter deletions on primary targeting to the J α  locus, Δ J49 and Δ TEA Δ J49 alleles were bred onto the  Rorc -/-  background. V α 8 and C α  primers were used toamplify cDNA prepared from  Rorc -/- , Δ J49  Rorc -/- , and Δ TEA Δ J49  Rorc -/-  thymocytes, andpolymerase chain reaction (PCR) products were cloned and sequenced to identify thedistribution of J α  segments used ( Fig. 3 ). Because the V α 8 primer detects multiple V α 8 familymembers that are dispersed across the V α  region, V α 8 rearrangements are relatively unbiasedwith respect to J α  usage 5 . Consistent with previous results 5 , two clusters of J α  usage weredetected in  Rorc -/-  thymocytes, one encompassing J α 58 and J α 57, and a second encompassingJ α 50 to J α 45. The second cluster was absent in Δ J49  Rorc -/-  mice, indicating that the J α 49promoter is required for primary rearrangements to the region spanning from J α 50 to J α 45.Both clusters were absent in Δ TEA Δ J49  Rorc -/-  mice, indicating that the TEA promoter isrequired for primary rearrangements to the most 5 ′  J α  segments, and that the two promoterstogether are required for almost all normal primary targeting events into the J α  array. Withoutthese promoters, J α  usage in short-lived DP thymocytes was much more broadly distributed,although nearly 60% of rearrangements involved J α 42 to J α 26. Hawwari et al.Page 3  Nat Immunol . Author manuscript; available in PMC 2008 June 5. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Altered T cell repertoire in promoter-deleted mice To analyze the effects of altered primary rearrangement on the J α  repertoire, cDNA wasprepared from wild-type, Δ TEA, Δ J49, Δ TEA Δ J49 and Δ 5' thymocytes, and was amplifiedusing V α 8 and C α  primers. PCR products were then fractionated on agarose gels and transferredto nylon filters, and J α  usage was evaluated by hybridization with J α -specific oligonucleotideprobes ( Fig. 4 ). In comparison to wild-type thymocytes, Δ J49 thymocytes displayed anoverrepresentation of J α  segments from J α 58 to J α 50, with normal to slight underrepresentationof J α  usage further downstream. We interpret elevated usage of the most 5 ′  J α  segments toreflect an increase in primary targeting to the TEA-dependent zone in the absence of the J α 49promoter. The relatively normal usage of J α  segments across the J α 49 promoter-dependent zonesuggests that in the absence of primary targeting by the J α 49 promoter, these J α  segments canstill be used efficiently as a consequence of secondary rearrangements that follow primaryrearrangements targeted by the TEA promoter. Δ TEA mice revealed a profile of J α  usage similar to that reported previously 7 . By comparison, Δ TEA Δ J49 mice were more substantially impaired, with J α  usage further depressed by anaverage of 25% between J α 50 and J α 38. Δ 5 ′  mice revealed a nearly identical profile, exceptthat J α 48 usage was suppressed as compared to Δ TEA Δ J49 mice. That J α 49 promoter deletionimpairs usage of J α 50 to J α 38 on a Δ TEA but not a TEA +  background must reflect theelimination of secondary rearrangements that follow primary targeting by TEA. Thus, althoughthe TEA and J α 49 promoters target primary rearrangements in nonredundant fashion, they havepartially redundant influences on J α  usage as a consequence of secondary rearrangements.Because J α 52 and J α 50 are still used inefficiently on Δ TEA Δ J49 alleles, there may be weak targeting elements in the TEA to J α 49 interval, other than the TEA and J α 49 promoters. Theseelements must also account for low frequency J α 48 usage on Δ TEA Δ J49 alleles, given thatJ α 48 usage is minimal on Δ 5 ′  alleles. Downstream of J α 48, there was a gradual increase in J α usage in both Δ TEA Δ J49 and Δ 5 ′  mice, first to physiologic and then to superphysiologicamounts. This result indicates the presence of additional cis -acting elements that can targetprimary rearrangements to this region, consistent with the modified profile of primaryrearrangements in Δ TEA Δ J49  Rorc -/-  mice ( Fig 3 ).The preceding experiments monitored J α  usage relative to C α  usage in each mouse strain, butdid not measure the absolute magnitude of rearrangement across the central portion of the J α array. For this purpose, we quantitatively analyzed restriction digests of thymocyte genomicDNA by Southern hybridization. We generated heterozygous mice carrying a Δ TEA Δ J49 129allele and a wild-type B6 allele, and exploited restriction fragment length polymorphisms tosimultaneously examine rearrangement of the two alleles in individual DNA samples ( Fig5a ). The extent of recombination events involving a set of J α  gene segments was evaluated bymeasuring loss of the germline (unrearranged) restriction fragment on which they are located.Previous studies of wild-type alleles showed rearrangement to be extensive at the 5 ′  end of theJ α  array, and to diminish gradually from 5 ′  to 3 ′ 7,9,10 . Consistent with this, we observed thaton the B6 allele, retention of the germline J α 61-J α 56 fragment (Region 1) was only 29%,whereas retention of the germline J α 50-J α 43 fragment (Region 2) was 44% and retention of the germline J α 42-J α 37 fragment was 58% ( Fig. 5a,b ). Previous analysis of TEA-deficientalleles revealed almost complete retention of the germline signal for J α 61-J α 53, but only20-25% retention immediately downstream (J α 53-J α 39) 7 . This indicates that recombinationevents involving 5 ′  J α  segments are rare, whereas J α  segments immediately 3 ′  rearrange at highfrequency. Germline signals for the 5 ′  J α  segments persist despite extensive rearrangement of more 3 ′  J α  segments because the unrearranged 5 ′  J α  segments are excised ontoextrachromosomal circles that are stably maintained in DP thymocytes. Consistent withrepertoire analysis ( Fig. 4 ), rearrangement on the Δ TEA Δ J49 allele was more substantiallyimpaired than on a Δ TEA allele, since the retention of germline signal was 95% for J α 61-J α 56 Hawwari et al.Page 4  Nat Immunol . Author manuscript; available in PMC 2008 June 5. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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