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Cellular Mechanisms Governing Chromosome Inheritance and Integrity

Research Summary

Hongtao Yu studies the cellular mechanisms that govern chromosome inheritance and integrity, focusing on understanding the spindle checkpoint—a cellular system that ensures all chromosomes are properly aligned and segregated during mitosis.

The long-term research interest of my laboratory is to study cellular mechanisms that govern chromosome inheritance and integrity, with a combination of cell biological, biochemical, and biophysical methods. Genomic DNA is packaged into highly compacted chromatin. The nucleosome core particle, the basic building block of chromatin, consists of 147 base pairs of DNA and a histone octamer. Histone modifications regulate chromatin structure and dynamics, which in turn affect all processes that need to access genomic DNA, including DNA replication, sister-chromatid cohesion and segregation, and DNA repair.

During the cell cycle, cells duplicate their chromosomes in S phase, physically tether the replicated chromosomes through cohesin to establish sister-chromatid cohesion, and then partition the sister chromatids evenly into the two daughter cells in mitosis. Chromosome segregation is triggered by the stepwise removal of cohesin (Figure 1). During early mitosis, the mitotic kinases phosphorylate cohesin and its regulators, removing cohesin from chromosome arms. Cohesin at centromeres is protected from mitotic kinases by the complex of shugoshin and protein phosphatase 2A (Sgo1–PP2A). At the metaphase-anaphase transition, the anaphase-promoting complex or cyclosome (APC/C) mediates the ubiquitination and degradation of securin, an inhibitor of separase. Cleavage of cohesin by separase then allows sister-chromatid separation. A cell cycle surveillance system called the spindle checkpoint prevents premature sister-chromatid separation in response to misaligned chromatids that are not properly captured by spindle microtubules.

Sister-chromatid cohesion is also required for the efficient repair of DNA double-strand breaks (DSBs) within the genome through homologous recombination (HR) between sister chromatids (Figure 2). Cohesin is locally loaded at DSBs and facilitates HR by physically holding the two sister chromatids in close proximity.

Sister-chromatid cohesion, segregation, and recombination are interdependent processes and are temporally coordinated during the cell cycle. We aim to understand the interplay and coordination of these processes. Uneven distribution of sister chromatids in mitosis or failure to repair DSBs results in aneuploidy or chromosome translocations, which are two prevalent forms of genomic instability in cancer cells. Our studies will provide a better molecular understanding of chromosome instability in human cancers and may lead to new strategies to treat them.

The Spindle Checkpoint and Chromosome Segregation
Studies from my laboratory have helped to establish a general framework of how the spindle checkpoint operates. In this framework, the kinetochores of misaligned chromatids in mitosis recruit and activate the mitotic arrest deficiency (Mad)1–3 and budding uninhibited by benomyl (Bub)1–3 checkpoint proteins, which then diffuse away from these kinetochores to block Cdc20-dependent activation of APC/C in multiple ways (Figure 1). Inhibition of APC/C stabilizes securin and prevents cohesin cleavage, thereby delaying anaphase onset until all kinetochores achieve proper attachment to spindle microtubules.

A single unattached kinetochore is sufficient to activate the spindle checkpoint. This exquisite sensitivity of the spindle checkpoint suggests that certain steps of checkpoint signaling must be catalytic. Multiple spindle checkpoint proteins are kinases, including Aurora B, Plk1, Mps1, and Bub1 (Figure 1). These kinases likely form kinase cascades to transduce and amplify checkpoint signals from the kinetochores. However, the hierarchy and mechanism of activation for most spindle checkpoint kinases are unknown. We are studying how checkpoint kinases are activated by unattached kinetochores and how they transduce and amplify the kinetochore signals to inhibit APC/C. In particular, we aim to use purified recombinant proteins to reconstitute kinetochore-dependent activation of checkpoint kinases in a test tube. We also plan to determine the biochemical and biophysical basis of their activation.

Centromeres and Sister-Chromatid Separation
Centromeres are special regions of chromosomes that are crucial for chromosome segregation in mitosis. Mammalian centromeres contain up to megabases of tandem satellite DNA repeats. Centromeric DNA is neither necessary nor sufficient for the formation of functional centromeres. Epigenetic mechanisms contribute significantly to centromere identity. These mechanisms include the incorporation of the histone H3-like protein CENP-A into centromeric chromatin and other characteristic histone modifications. One such modification is phosphorylation of histone H2A by the spindle checkpoint kinase Bub1. Bub1-mediated H2A phosphorylation is required for the centromeric localization of Sgo1. Sgo1–PP2A dephosphorylates cohesin and its regulators to counteract the cohesin inhibitor Wapl at centromeres. We are studying how H2A phosphorylation recruits shugoshin to centromeres. We are also investigating the mechanism of action and cell cycle regulation of shugoshin.

Sister-Chromatid Cohesion and DNA Repair
We are interested in the function of chromatin-directed sumoylation in DNA damage–induced cohesin loading and sister-chromatid recombination. Small ubiquitin-like modifier (SUMO) is a ubiquitin-like protein that can be covalently conjugated to target proteins. Sumoylation regulates the functions of target proteins by multiple, context-dependent mechanisms. We have shown that Mms21, a subunit of the Smc5/6 complex, functions as a SUMO ligase. The SUMO ligase activity of Mms21 is required for DNA repair. Mms21 stimulates the sumoylation of the cohesin subunit Scc1 in human cells. Scc1 sumoylation promotes sister-chromatid HR by antagonizing the cohesin inhibitor Wapl. We are elucidating the molecular details of Wapl-mediated cohesin release from chromatin. We will establish how sumoylation and other post-translational modifications of cohesin counteract Wapl to stabilize cohesin on chromatin. We are also interested in the upstream signals that recruit cohesin to DNA damage sites and the mechanisms by which cohesin promotes sister-chromatid HR.

In summary, using a multidisciplinary approach, we are investigating the interdependency and coordination of multiple cellular processes that collaborate to maintain genomic stability, including epigenetic regulation, sister-chromatid cohesion, sister-chromatid recombination, and chromosome segregation. The integration of cell biological, biochemical, and structural methods will enable us to understand the fundamental principles of these processes in chromosome biology at the cellular, molecular, and atomic levels.

This research is also supported by the Cancer Prevention and Research Institute of Texas and the Welch Foundation.

As of August 27, 2012

Scientist Profile

Investigator
The University of Texas Southwestern Medical Center
Cell Biology, Structural Biology