Transcriptional Activation by Reorganizing Chromatin

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DESCRIPTION (provided by applicant): Chromatin remodeling serves as a functional key in multiple cellular processes, one of them being the regulation of gene expression through promoting formation of the transcription complex and elongation of the transcription complex. There are several well-documented examples of chromatin remodeling complexes working in conjunction with gene-specific transcription factors to make the DNA accessible to the transcription machinery. In addition, the nucleosome structure is a severe deterrent to the rearrangement of genes required for the production of immunoglobulins. Chromatin remodeling is apparently a mechanism used to tightly regulate vertebrate immune systems and is probably the key to the molecular mechanism underlying the "accessibility hypothesis" proposed 15 years ago. Chromatin remodeling is also involved in cell cycle control and interacts with the tumor suppressor protein Rb or retinoblastoma protein. In understanding how SWI/SNF and ISW2 remodel the nucleosome, it is important to know that it does not work randomly on chromatin, but they are recruited or targeted to specific locations by gene-specific transcription factors or repressors. Evidence indicates that chromatin remodeling can be tightly coordinated with DNA modifications such as methylation of DNA and DNA replication. The list of diseases linked to chromatin remodeling continues to grow and includes such diseases as rhabdoid tumours, a very aggressive form of pediatric cancers, breast cancer, leukemia, mental retardation, Williams syndrome, and Rett syndrome. It is not known which subunits of SWI/SNF interact with the transcription activator or how its interaction with the nucleosome may be different when recruited versus indiscriminate binding to nucleosomes. Our research plan is to examine the structure and its relation to function of the SWI/SNF chromatin remodeling complex by a series of approaches that uses either modified DNA or modified histone octamers. We will obtain the 3-dimensional structure of SWI/SNF by electron tomography and determine which regions interact with DNA and histone octamer by linking data from site-directed photoaffinity labeling and proteolysis to the structure. Next, we will determine how SWI/SNF and ISW2 remodel chromatin when recruited to specific sites within nucleosomal arrays by their respective "targeting" proteins. Data on these two different chromatin remodeling complexes suggest that they modulate chromatin structure in significantly different ways both in vivo and in vitro.