Chromosome movements that regulate tissue-specific gene expression

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During fiscal year 2010 we accomplished the following: 1) Used 7 pairs of 10 kb FISH probes to measure spatial proximity of different parts of the IgH locus in primary bone marrow pro-B cells that contain wild-type or Eμ-deleted IgH alleles. We found Eμ-dependent and Eμ-independent forms of locus contraction. These observations corroborated the biochemical assays described in the Summary of project Z1A AG000383. 2) Developed conditions to visualize chromatin loops in the IgH locus by barcode experiments. Specifically, we used 6 short FISH probes simultaneously to detect IgH alleles in primary pro-B cells; 3 of these were labeled with red fluorophore and the other 3 with green fluorophore. We marked IgH alleles with a bacterial artificial chromosome probe labeled in blue. We recorded 20-40 fluorescent images per nucleus by taking 0.2μM Z sections and traced the path of single IgH alleles in 3 dimensions. We found that WT IgH alleles form closed loops between Eμ and the 3'RR; the chromosome conformation in the loop varies between nuclei indicating a high degree of diversity and conformational flexibility.