Chromosomes, the carriers of genetic information, regulate their own replication and segregation by acting as a signaling platform controlling the assembly of complex macromolecules such as mitotic spindles, nuclear envelopes, kinetochores, and heterochromatin. Chromosomes themselves also undergo major architectural changes during cell cycle progression and cellular differentiation. The dynamic nature of these macromolecules allows targeted chemotherapy, making its understanding highly important for medicine. The long-term goal of this project is to provide a mechanistic understanding of chromosome-associated macromolecular architectures. We have previously unveiled the Aurora B-mediated pathway that helps restrict assembly of the spindle and the nuclear envelope spatially to chromosomes and temporally to specific cell cycle stages. Here, we aim to achieve two distinct conceptual and technical breakthroughs toward understanding the regulation of chromosome-associated macromolecular architectures. First, we aim to develop a novel strategy to obtain a systems level understanding of how chromosomal compositions are controlled by specific histone modifications, cell cycle status, and major regulators of chromosome functions. We have established a method to directly manipulate nucleosomal compositions and histone modifications in frog egg extracts. Combining this method with quantitative mass spectrometry, we aim to identify proteins that are regulated by the mitosis-specific, enigmatic histone phosphorylation at Ser10 of histone H3 (H3S10) and heterochromatin associated trimethylation at Lys9 of histone H3 (H3K9me3). Applying our refined clustering strategy to this large-scale proteomic data we aim to identify novel functional protein networks on nucleosomes. Our preliminary data suggest that this strategy can work at an unprecedented level of efficiency and resolution, and can identify novel potential interactions between proteins associated with chromatin. We will further validate the functional significance of these novel interactions. Second, our preliminary data using super-resolution microscopy indicate that kinetochore architecture is spatially and functionally segmented into a stable core and a polymeric expandable module whose assembly on chromatin is induced in the absence of microtubule attachment by mitotic kinases, Aurora B and Mps1. Unlike the conventional kinetochore assembly model where the kinetochore assembles in a unidirectional order from the inner to the outer kinetochore components, we hypothesize that the expandable module copolymerizes in a manner dependent on both inner and outer kinetochore components. We will determine the mechanistic basis behind the expandable module assembly and its functional significance. This finding helps answer the long-standing enigma of how distinct kinetochore functions are adapted in response to microtubule attachment status; overall, our studies will generate new concepts of chromosome functions and regulation, broadly impacting life sciences and medicine.
Proteins that control dynamic assembly and disassembly of macromolecular architectures associated with chromosomes are great candidates for therapeutic targets of a number of diseases, including cancers. We propose to develop a novel proteomic strategy that can effectively identify proteins and protein networks that functionally associate with chromosomes in response to specific local and temporal cues. We also aim to provide a novel mechanistic framework for assembly and disassembly of the kinetochore, which is the macromolecular machine, associated with chromosomes and mediates chromosome segregation during cell divisions.
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