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The chicken light chain locus: a model of homology-directed repair of SSBs?

The chicken light chain locus: a model of homology-directed repair of SSBs?.  V.  V.  V.  V. C. V. J.  V.  V.  V.  V. C. V. J. Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation.  V.  V.  V.  V. C. V. J.

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The chicken light chain locus: a model of homology-directed repair of SSBs?

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  1. The chicken light chain locus:a model of homology-directed repair of SSBs? V V V V C V J V V V V C V J Chicken pre-B cell line DT4O: carries out constitutive templated Ig hypermutation

  2. V V V V C V J • Regulators of homology-directed repair after the DNA lesion • What makes an attractive donor? • SSBs • What is the true recombination potential of nicks? • Are nicks blocks to aberrant recombination at the light chain locus?

  3. Regulators of homologous recombination donor repression donor activation point mutation homology-directed repair

  4. 90 80 AcH3 AcH4 70 60 Fold Enrichment 50 40 30 20 10 0 FlankV25 V24 V13 V5 V1 V1-V VR Pseudogenes have an accessible chromatin state in DT40 cells

  5. No IPTG 100 µM IPTG 15 µm The experimental system

  6. 10 GFP-LacI 9 LacI-HP1 8 7 6 Fold Enrichment 5 4 3 2 1 0 AcH3 AcH4 AcH3 AcH4 V17 pol ChIP analysis of LacI-HP1 effects on chromatin

  7. IgM loss: a measure of mutation time in culture GFP-LacI LacI-HP1 Homology-directed events 77% 3% Nontemplated 23% 97%

  8. VP16-LacI: Tethering an activator GFP-LacI VP16-LacI AcH4 IgG AcH4 IgG Ova V17 Enrichment: 4.5 12.4

  9. H3 variants and HIRA Hake and Allis, PNAS (2006) Polo and Almouzni (2004)

  10. Effect of tethering HIRA HIRA-LacI GFP-LacI

  11. DT40 PolyLacO clones expressinghistone H3 variants H3.3-FLAG H3.1-FLAG C 1 2 3 4 1 2 FLAG actin

  12. V V V V C V J • Regulators of homology-directed repair after the DNA lesion • What makes an attractive donor? • SSBs • What is the true recombination potential of nicks? • Are nicks blocks to aberrant recombination at the light chain locus?

  13. Recombination potential of SSBs versus DSBs I-SceI / AniI P T Puro eGFP eGFP • have stable clones in DT40, Ramos and Nalm6 cell lines • analysis of the DSB and nickase activities of wild-type and mutant AniI • constructing integrating retroviral vectors for delivery of endonucleases

  14. Avoiding aberrant recombination at light chain locus C V J V V V V V V C V J V V V + direct repeats or V C V J V V V inverse repeats V V V

  15. Aberrant recombination at light chain locus can occur by crossovers donor recipient SSBs rarely progress to Holliday intermediate (1) Holliday junctions are rarely resolved to crossovers (2)

  16. RuvC resolvase: a possible diagnostic agent gene conversion crossover

  17. Maizels lab

  18. Homology-directed repair(HDR) Rad51…

  19. Repair factors are limiting for gene conversion Ellen Ordinario Munehisa Yabuki

  20. Competing repair pathways HDR w/ exogenous donor Non-homologous end joining HDR w/ sister chromatid xx xx xx xx xx xx Smc5/6 … Ku80 …  x xx xx xx xx Sequence correction Loss of sequence Maintain sequence information

  21. Optimize HDR with an exogenous donor sequence • Express factors that are limiting for HDR (e.g. NBS1) • Knockdown non-homologous end joining factors (e.g. Ku80) • Knockdown factors (e.g. SMC5) that promote HDR with the sister chromatid

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