The MMTV LTR adopts a specific nucleoprotein organization when introduced into cells. This structure involves the positioning of six nucleosomes (A-F) over the 1300 base pair LTR. A specific chromatin transition is induced by the binding of steroid receptors to the B nucleosome. By comparing the activity of transient MMTV reporter constructs (which are not organized in specific chromatin structures) with identical sequence elements that have replicated (and manifest the phased nucleosome array), we have shown that the chromatin transition is mechanistically important in transactivation, and that steroid receptors function on replicated genes by relieving chromatin repression. Our previous work showed that each phased nucleosome corresponds to a family of octamer cores positioned in that region. Thus, the low resolution phasing pattern results from the frequency- biased occupancy of a subset of these frames. We developed a new assay, the """"""""linked enzyme assay,"""""""" that permitted us to examine the nature of the nucleoprotein transition. We showed that the chromatin alteration does not correspond to a single nucleosome event, but is more accurately modeled as a higher order structure transition in a wrapped nucleosome array. Thus it is likely that steroid receptors interact with, and induced transitions in, 30 nanometer structures, rather than simple polynucleosome arrays. The mechanism of chromatin antirepression by receptors must be explored in the context of these findings. We also demonstrated that activation of the MMTV promoter is significantly modified when organized in this highly reproducible chromatin structure. Transiently introduced progesterone receptor was found to be incapable of activating the replicated structure, although quite active on transient, disorganized templates. In contrast, transiently introduced glucocorticoid receptor was functional on both transient and stable templates. These studies confirm that the organized nucleoprotein structure found on the MMTV LTR is functionally important in gene regulation.
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