Enhancer Induced Changes In Chromatin Structure And Function
Bennett, Jonathon D.
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Abstract Gene regulation in mammals is a cornerstone issue in developmental biology. One example of gene regulatory elements are enhancers, which have been shown in transfection assays to be cis-regulatory sequences that activate transcription regardless of location and orientation to their gene promoters. However, no studies have examined in vivo whether the distance of a mammalian enhancer is critical for its function, and whether enhancers affect other genes beyond proximal regulatory domains. One significant mammalian enhancer is located within the immunoglobulin heavy chain (IgH) gene locus that spans a distance of 3 Mb on mouse chromosome 12. The IgH locus contains variable (VH), diversity (DH), and joining (JH) gene segments, and constant region (CH¬) exons. These IgH gene segments recombine early in bone marrow B-lymphocyte differentiation to yield functional antigen receptor genes. Coupled to this, it has been demonstrated that the Eµ intronic enhancer, located between the J¬H-Cμ intron, has a significant role in gene regulation, including V(D)J recombination. The first project is to test whether the location of the Eµ enhancer is required for its function with respect to chromatin, transcription, and recombination. The second project is to determine whether the Eµ enhancer affects other genes in cis within the IgH locus, or elsewhere in the murine genome. For the first project, BAC transgenic mice were developed that have the Eµ enhancer in different locations and experiments assayed for changes in chromatin, transcription, and recombination as a result of the relocated Eµ enhancer. Our results demonstrate that regions of heterochromatin are changed to euchromatin based on the location of the Eμ enhancer, and that when the location of the Eμ enhancer is changed, unique transcripts and gene rearrangements are induced. For the second project, genome-wide DNase Hypersensitivity sequencing was performed on samples with the Eμ enhancer present and absent. Several novel peaks associated with DNase-I sites within the IgH locus were identified, and some peaks were lost when the Eμ enhancer was absent. Therefore, the Eμ enhancer has other unique gene regulatory features in its proximal domain, and each of the unique DHS peaks may serve a gene regulatory purpose for the IgH locus that needs further exploration.