Chromatin Assembly Factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes. CAF-1 is a nucleosome assembly factor important for DNA replication, DNA repair, and heterochromatin formation. CAF-1 protein levels correlate with cell proliferation and cancer prognosis, making these studies a high priority for the medically important processes of genome stability and the maintenance of epigenetic states. Via mass spectrometry, we discovered multiple nucleolar proteins associated with the human CAF-1-p150 subunit. Microscopy detects a subset of cellular p150 associated with nucleoli, the sites of ribosomal RNA (rRNA) synthesis and ribosome assembly. Notably, RNAi-mediated depletion of p150 causes a dramatic loss of nascent rRNA transcripts and spatial redistribution of some nucleolar proteins. Therefore, we have discovered that p150 has a previously unrecognized role in the structure and function of the nucleolus. rRNA synthesis is regulated by energy supply, differentiation, cell cycle progression, tumor suppressors and oncoproteins. Nucleolar alterations are also important for cancer diagnoses, and the rRNA synthesis machinery is increasingly viewed as a therapeutic cancer target. Therefore, these studies are crucial for understanding clinically relevant interactions between DNA replication, gene expression, and growth control. We plan to explore three Aims to explore how p150 functions to regulate rRNA synthesis, and to extend these findings via genome-scale studies:
Aim 1. The mechanism of regulation of rDNA transcription by CAF-1 p150. We will test several hypotheses raised by our observations: (a) p150 could be acting as part of a transcriptional activation complex at the rDNA promoter, perhaps distinct from its role as a CAF-1 subunit, (b) p150 could regulate the percentage of transcriptionally accessible rDNA repeats, (c) p150 could promote transcriptional elongation, or (d) p150 might be critical for maintaining the epigenetic modification state of the rDNA repeats. Alternatively, (e) p150 might prevent cryptic transcription events. We note that these possibilities are not mutually exclusive.
Aim 2. Molecular analyses of p150 recruitment to repetitive DNAs. We will determine whether specific p150 protein domains are required for association with rDNA, and also assess the role of the new nucleolar interaction proteins in p150 recruitment. We will also determine the extent of cell cycle regulation of these associations.
Aim 3. Genome-wide analysis of p150 localization, transcriptional targets, and effects on rDNA conformation. We will test our hypothesis that p150 is a master regulator of three-dimensional interactions of rDNA repeats. We will compare these data to genome-scale analyses of p150's transcriptional targets and genomic localization. Together, these studies will provide candidate direct targets of p150 regulation, and lead us to test dependency relationships for these observations.
Ribosomes, the cellular machines that synthesize proteins, contain large ribonucleic acid (RNA) molecules. We are pursuing our recent discovery that a protein previously thought to be dedicated to chromosome duplication is also an important regulator of ribosomal RNA production. Notably, ribosomal RNA synthesis also is regulated by cell growth and energy supply, as well as by tumor suppressors and oncoproteins, and is increasingly viewed as a therapeutic cancer target. Therefore, these studies will help us understand clinically relevant interactions that link DNA replication, gene expression, and growth control in human cells.
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