Analysis of chromatin-RNA interactions during the cell cycle.

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DNA is packaged by histone proteins into chromatin in order to fit into the nucleus of a cell. Globally, chromatin can be separated into open, transcriptionally active and closed, transcriptionally silent compartments. Many different nonhistone, chromatin-binding proteins contribute to global chromatin structure. Chromatin remodeling enzymes, transcription factors, and transcription-associated RNAs are important for decondensing transcriptionally active portions of the genome. Heterochromatin proteins and associated noncoding RNAs are important for condensation of the transcriptionally inactive portions of the genome. Changes in chromatin organization and compaction are critical for cell state changes during development and chromosome segregation during mitosis. Chromosome structure changes dramatically at the beginning of mitosis as chromosomes compact and individualize in preparation for segregation. As cells enter into mitosis all of the structure present in the interphase nucleus is erased. Erasure of interphase nuclear structure is correlated with the stepwise removal of the Cohesin complex. Chromosome condensation and individualization are accomplished by the combined action of the Condensin complexes and Topoisomeriase IIα. Interestingly, dramatic changes in chromosome structure are temporally correlated with suppression of transcription. At the end of mitosis chromosomes cluster and decondense to reform a single interphase nucleus. Chromosome decondensation and clustering is accomplished by the rebinding of many different proteins that are removed from chromatin during mitosis and is temporally correlated with the resumption of nuclear transcription. While changes in chromatin structure during mitosis are correlated with changes in nuclear transcriptional activity, little is known about how the two processes are linked. We have recently discovered that prophase removal of the Cohesin complex form chromosomes is critical for silencing mitotic transcription and that removal of chromatin-bound RNAs from chromosomes during mitosis is mediated by phosphorylation of SAF-A. The identification of molecular pathways linking changes in chromosome structure to changes in transcriptional activity presents an opportunity to understand how these events are linked. In this proposal we will reconstitute the biochemistry of nucleic acid interactions mediated by SAF-A to provide a picture of how this abundant protein controls chromosome structure. We will then examine how removal of SAF-A from chromatin promotes chromosome condensation during prophase. We will then examine how rebinding of SAF- A to chromatin at the end of mitosis promotes nuclear reformation and transcriptional activation. Collectively, the experiments outlined in this proposal will provide new insight into changes in chromatin-RNA interactions that control chromatin structure and how these changes contribute to accurate chromosome segregation and transcriptional regulation.