Chromosome movements that regulate tissue-specific gene expression

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During FY16 we accomplished the following: 1. Developed reagents and cell lines to carry out live cell imaging of RAG1/2 recruitment to antigen receptor loci in pro-B cells. These included tagging RAG1 and RAG2, and mutant RAG2 proteins, with a HaloTag or a SunTag. These proteins were expressed in Abelson virus-transformed pro-B cell line 6312. 2. Detection of SunTag requires co-expression of a single-chain Fv protein that binds to the SunTag to amplify the fluorescence signal that increases the probability of detecting single molecules in vivo. We generated stable cell lines that express this single chain Fv protein. 3. To test recruitment of enzymatically inactive Cas9-SunTag (dCas9-SunTag) to specific sites in the genome, we generated pro-B cells that express guide RNAs (gRNAs) targeted to telomere repeats. 4. We carried out preliminary visualization studies of RAG1-Halo and RAG2-Halo in live pro-B cells at Janelia Farms. Discrete puncta of each fluorescent derivative were observed. The next step is to determine the stoichiometry of RAG complexes generated by these fluorescent proteins. 5. We developed vectors to express gRNAs that target the variable (VH) domain of the IgH locus. If telomeric repeats are readily visualized in the experiments described above, we will recruit dCas9-SunTag to the VH domain to study dynamics of the IgH locus.