The long-term research interest of my laboratory is the study of cellular mechanisms that govern chromosome inheritance and integrity. To do this, we use 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 removal of cohesin (Figure 1). 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 loaded after S phase at DSBs (termed postreplicative cohesin loading) 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. All three processes are regulated by the underlying chromatin structure, either locally or globally. We aim to understand the interplay and coordination of these processes. Uneven distribution of sister chromatids in mitosis or inefficient repair of 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.
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 inhibit 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. Our understanding of the spindle checkpoint in human cells, however, is far from complete. Our current research on the spindle checkpoint focuses on the two following areas.
Systematic identification of spindle checkpoint components. We are performing a genome-wide RNA interference (RNAi) screen to systematically identify spindle checkpoint genes in human cells. We will examine subcellular localization of candidate proteins identified in the screen and analyze binding among the candidate and known checkpoint proteins. These simple assays will confirm the validity of the hits from the screen and divide them into functional networks that include known checkpoint proteins. Selected networks will be studied in-depth, including characterization of cellular phenotypes upon their inactivation, monitoring of the steady state and dynamics of their localization, elucidation of the determinants and affinities of their interactions, and high-resolution structural studies.
Kinase signaling cascades in the spindle checkpoint. 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, Bub1, and BubR1 (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.
Sister-Chromatid Cohesion and DNA Repair
We are interested in the function of chromatin-directed sumoylation in postreplicative 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. 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 SMC5, SMC6, and two cohesin subunits in human cells. We will use purified recombinant proteins to reconstitute MMS21-dependent sumoylation of SMC5, SMC6, SCC1, and SA2, and we will test whether sumoylation of SMC5 and SMC6 affects their ATPase activities in vitro. We will map the sumoylation sites on these proteins through mutagenesis and mass spectrometry and test whether mutations of these sites will affect the loading of SMC5/6 and cohesin to DSBs in vivo. We will determine the structures of ATPase head domains of SMC5/6 that confer SUMO regulation. These studies may reveal the mechanism by which sumoylation regulates postreplicative cohesin loading and sister-chromatid recombination.
Histone Modification in Postreplicative Cohesin Loading
Loading of cohesin and the SMC5/6 complex at DNA DSBs requires ATM/ATR-dependent phosphorylation of histone H2AX (Figure 2). A systematic survey of histone modifications at DSBs in human cells is lacking, however. We will use the 293/A658 cell line with a single copy of the SceGFP gene stably integrated into its genome; SceGFP contains an 18–base pair I-SceI endonuclease recognition site that is absent in the human genome. Expression of I-SceI in 293/A658 introduces a single DSB in these cells. We will determine which known histone modifications are enriched at I-SceI–induced DSBs in the SceGFP-expressing 293/A658 cells using chromatin immunoprecipitation (ChIP). This will create a profile of histone modifications at DNA damage sites. Comparisons of this profile with known histone modification profiles of euchromatin, heterochromatin, and centromere chromatin will reveal the nature of the DSB-associated chromatin domain. We will then determine which histone modifications are required for postreplicative cohesin loading and sister-chromatid recombination by altering the levels of enzymes that control these modifications. We will then elucidate the mutual dependency of these modifications at DSBs. These studies will establish new links among epigenetic marks, chromatin structure, sister-chromatid cohesion, and DNA repair.
In summary, we will investigate the interdependency and coordination of multiple cellular processes that collaborate to maintain genomic stability, including epigenetic modifications, 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.
