Reorganization of the local chromatin environment is a fundamental component of gene regulation. While considerable progress has been achieved in the identification and characterization of activities that are involved in chromatin modification, the mechanisms by which the many modifications and transitions are orchestrated into the final effects of gene activation, repression, and epigenetic inheritance are poorly understand. We have studied nuclear receptors as important models in understanding chromatin modification and restructuring. Not only are these proteins critically important in many disease processes, but the ease with which their activities can be manipulated by ligand stimulation make them experimentally attractive model systems.1) We established a new approach for the analysis of native, in vivo chromatin structures. Using the """"""""multi-gel"""""""" system established by Hansen and colleagues, we characterized the """"""""native,"""""""" higher-order structure of the MMTV promoter, and showed that the promoter exists in three states in vivo. We demonstrated that the promoter nucleosome array is capable of folding into higher order structures, and characterized several activities associated with these structures. This work represents one of the first physical characterizations of an undenatured, in vivo nucleosome array, and provides the first evidence for the organization of active promoters into tertiary chromatin structures.2) We developed an in vitro chromatin remodeling system that is receptor dependent and faithfully duplicates, in vitro, the well described in vivo MMTV chromatin transition. Using this system, we showed that the promoter transition encompasses all of the B nucleosome, but only the 3' half of the C nucleosome. This finding is difficult to reconcile with the prevailing model of nucleosome sliding, and points to the need to explore these transitions in more detail.3) Using our in vitro chromatin remodeling system, we developed new technology to study protein/DNA interactions in vitro in real time. We established a laser-based, UV crosslinking system that allows us to efficiently detect receptor/DNA and remodeling complex/DNA interactions with short, 5 nanosecond UV pulses. Using this system we showed that receptor template interactions during chromatin remodeling are transient and periodic, and that the receptor is actively ejected from the template during the remodeling reaction. This unexpected finding has led to a paradigm shift in the field, and provides a new mechanistic basis for the transient interactions of receptors with promoters we first reported in living cells.4) The nuclear receptor coactivator CARM1 methylates histone H3 and synergizes with p160-type coactivators and the p300/CBP coregulators to enhance gene activation by steroid and nuclear hormone receptors. We showed that CARM1 cooperates with GRIP1 to enhance steroid hormone-dependent activation of stably integrated MMTV promoters, and that this coactivator function required the methyltransferase activity of CARM1. We showed by Chromatin IP (ChIP) assays and immunofluorescence studies that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.
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