Cell Biology of Mammalian Nuclei

Project Summary/Abstract I have a long-standing interest in chromosome biology, and in recent years this has expanded to cover multiple aspects of mammalian nuclear cell biology. We are pursuing investigations in two areas, both involving dramatic protein relocalizations related to human health. One project is focused on tumor marker protein Ki-67, and how it protects chromosomes. Ki-67 organizes heterochromatin at the nucleolus during interphase, relocalizes to coat mitotic chromosomes, and is important for tumor growth and metastasis. We discovered that acute depletion of Ki-67 causes DNA damage during mitosis, and that defects are more severe when Ki-67 and p53 are simultaneously depleted. We also discovered that the C-terminal chromatin-binding domain of Ki-67 (termed “LR”, based on its leucine-arginine dipeptide motifs) is necessary and sufficient to protect cells from damage when endogenous Ki-67 is depleted. Therefore, we will analyze how Ki-67 protects DNA, and map where in the genome this is most important. We will determine which LR residues affect its ability to anchor Ki-67 to chromosomes and protect them from mitotic damage, focused on sites of mitotic phosphorylation and blocks of charged residues. Data from these cellular function experiments will guide biochemical studies of nucleic acid and nucleosome binding. Concurrently, we are working collaboratively to solve the structure of the LR-nucleosome complexes. We have obtained cryoEM data indicating where LR binds the nucleosome surface, and binding studies with mutant nucleosomes confirm these findings. In addition to biochemical and biophysical investigations, we will determine whether specific genomic loci are damaged when Ki-67 is lost. To test this, we will take advantage of our finding that the 3’OH groups at DNA strand breaks caused by Ki-67 depletion can be enzymatically labeled. We will use this property to generate genome-scale libraries to map the damage at the nucleotide level and we will determine whether the patterns are cell-type specific. Macrophages are a critically important cell type in our innate immune system, which must mount rapid responses to the presence of infectious agents. The speed and importance of these responses make macrophages an ideal system for studying dynamic events in the mammalian nucleus. We have established a research project based on our discovery that macrophages relocalize proteins and nuclear bodies related to RNA splicing in response to interferon-gamma and bacterial lipopolysaccharides, which together promote pro- inflammatory “M1” differentiation. These rearrangements require specific signaling events, because they are blocked by inhibition of p38 MAPK kinases and do not occur during anti-inflammatory “M2” differentiation in response to cytokine IL-4. We are therefore investigating which RNA processing events and protein partners are the key regulatory molecules related to these nuclear body dynamics during macrophage differentiation.