Potentiation, i.e., a region of chromatin adopting an open structure, serves as the cornerstone for the three tier mechanism of eucaryotic transcriptional regulation that consists of Potentiation-Initiation- Elongation. Only upon adopting a potentiated open chromatin state can the various members of the multigenic PRM1 yields PRM2 yields TNP2 gene cluster be transcribed. The underlying hypothesis of this research program is: transcriptional potentiation of a genic locus is mediated by the precise association of cell-specific factors with the region of locus control. To elucidate this mechanism, the PRM1 yields PRM2 yields TNP2 region of human chromosome 16 was characterized at the sequence, structural and biological levels. A transgenic animal model of the complete human locus was created. This is the first transgenic model of any multigenic haploid-expressed locus for any species. Sites of nuclear matrix attachment i.e., MARs, were identified within the chromatin domain. These sites often demarcate the ends of the genic domain and may act as regions of locus control. The first specific aim: Defining nuclear matrix association and the control of the PRM1 yields PRM2 yields TNP2 locus, will assess the role of MARs as part of the potentiative mechanism. This will be accomplished by creating a series of transgenic animals bearing various MAR deletions. Their effect on the expression of the various members of the locus will be determined. The second specific aim: Establishing the relationship of DNase I- hypersensitive sites and locus control will assess the role of DNase I- hypersensitive elements as regions of locus control. Their ability to act as potentiative elements will also be assessed by transgenic analysis. The third specific aim: Establishing the potentiative state of the PRM1 yields PRM2 yields TNP2 domain during differentiation and fine mapping the elements of locus control, will assess potentiative timing, thus establishing when cells of the spermatogenic lineage are committed to terminal differentiation. Regions of locus control will then be mapped in vivo during spermatogenesis. The corresponding cis- sequence elements and sites of transfactor occupancy will then be fine- mapped in vivo, using Linker Tag Selection Ligation Mediated PCR. Successful completion will directly test the hypothesis that spermatogenesis is controlled by a non-repressive selective potentiative mechanism while providing the foundation to isolate these potentiative factors.
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