Potentiation, i.e., a region of chromatin adopting an open structure, serves as the cornerstone for the four tier mechanism of eukaryotic transcriptional regulation that consists of POTENTIATION->ACTIVATION/ INITIATION->ELONGATION. Only upon adopting a potentiated open chromatin state can the various members of the multigenic PRM1->PRM2->TNP2 gene cluster be transcribed. As we and others have shown, a likely key player in this mechanism of transcriptional regulation is the nuclear matrix attachment region. Accordingly the primary hypothesis of this research program is: the initial step coordinating potentiation among co-expressed genes is the association of their chromatin domains with the nuclear matrix. This project will continue to build on the foundation of our previous studies using the human PRM1->PRM2->TNP2 domain as a model. We will expand this model to include both the single copy human transgenic domain model that we have created and the corresponding endogenous mouse domain. A five-year research program directly testing the nuclear matrix hypothesis is outlined in three specific aims. The first: To define the organization of the mouse Prm1, Prm2, Tnp2 domain, will establish the level of concordance of the endogenous mouse domain. The potentiative timing of the human locus will be determined using the single copy transgenic animal model. This will critically assess the usefulness of this comparative transgenic model. The second: To establish the relationship of nuclear matrix attachment/association and potentiation during differentiation, will establish whether nuclear matrix association precedes, is coincident or follows the opening of this chromatin domain and whether sites of attachment appear and/or change in a differentiation dependent manner for this suite of haploid expressed genes. The third: To establish the role of other epigenetic modifiers in the potentiative mechanism, will provide a high resolution view of the various modifications and sites of trans-factor contact during differentiation. Comparative DNase I hypersensitive site mapping during differentiation will be undertaken to identify potential sites of trans-factor interaction. This will be correlated with potentiation to identify sites of trans-factor contact modulating large scale changes in chromatin structure. In parallel, using the ChIP assay and the newly created genomic array that we have developed, we will assess Heterochromatin Protein 1, histone acetylation and methylation status of the transgenic human domain during differentiation. Successful completion of this project will, for the first time, reveal the order of events leading to the formation of a potentiated open chromatin domain. This milestone provides the foundation to isolate and functionally characterize the associated agents that open chromatin domains.
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