During fiscal year 2011, we accomplished the following: 1. We spent a great deal of effort discovering that the original experimental design (see Annual Report 2010) of using mono-nucleosomes assembled with recombinant histones would not work because such mono-nucleosomes were refractory to cleavage by full length or core RAG1 and RAG2. Therefore, we decided to use chromatin-assembled plasmids in coupled-cleavage (12/23-RSS-dependent) reactions to further investigate the role of H3K4 trimethylation (H3K4me3) in initiating VDJ recombination. 2. In an alternate route to the same question, we used recombinant wild-type and mutant RAG proteins with purified DNA substrates to test the hypothesis that H3K4me3 recognition by the PHD domain of RAG2 is an allosteric activator of recombinase activity. We used recombinant RAG1 and RAG2 proteins to introduce single-strand nicks at a 12- or 23-RSS in the presence, or absence, of a peptide containing the N-terminus of histone H3 that was trimethylated at lysine 4. We found that RSS-specific nicking by wild-type RAG proteins increased in the presence of H3K4me3 peptide. We inferred that this was due to recognition of the peptide by the RAG2 PHD domain because the effect was absent when recombinant core RAG2, or a point mutation in the PHD domain, was used instead of full-length RAG2. Moreover, RAG2 mutations that neutralized autoinhibition by the PHD domain were less affected by inclusion of the peptide. These observations are consistent with allosteric activation of RAG activity by H3K4me3 recognition.
| Yan, Zhijiang; Delannoy, Mathieu; Ling, Chen et al. (2010) A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability. Mol Cell 37:865-78 |
| Ishii, Haruhiko; Du, Hansen; Zhang, Zhaoqing et al. (2009) Mi2beta shows chromatin enzyme specificity by erasing a DNase I-hypersensitive site established by ACF. J Biol Chem 284:7533-41 |
| Du, Hansen; Ishii, Haruhiko; Pazin, Michael J et al. (2008) Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription. Mol Cell 31:641-9 |
