Combining cell biological, biochemical, and biophysical methods, my research program aims to understand cellular mechanisms governing genomic stability at the molecular and atomic levels. Genome maintenance relies on high-fidelity DNA replication, sister-chromatid cohesion, and chromosome segregation. Defects in these processes cause genomic instability and aneuploidy, which can promote tumorigenesis depending on context. Successful execution of these processes requires their interactions with underlying chromatin and proper coordination during the cell cycle, particularly in S phase and mitosis (Figure 1).
In S phase, DNA replication is coupled to the establishment of sister-chromatid cohesion. Cohesion establishment requires coordinated interactions among the ring-shaped Cohesin and its regulators, including Pds5, Sororin, and Wapl. Sororin and Wapl are positive and negative regulators of cohesin, respectively. Pds5 has dual roles, and binds to Sororin and Wapl mutually exclusively. Binding of the Pds5-Sororin heterodimer to Cohesin with its Smc3 subunit acetylated establishes cohesion, whereas binding of Pds5-Wapl releases Cohesin from chromatin and inhibits cohesion. Antagonistic binding of Sororin and Wapl to Pds5 thus dictates the status of sister-chromatid cohesion.
During early mitosis, the kinases Plk1 and Cdk1 phosphorylate Cohesin and Sororin, triggering their Wapl-dependent removal from chromosome arms. A complex of shugoshin (Sgo1) and PP2A protects cohesin from these kinases at centromeres. At metaphase, centromeric cohesion enables sister kinetochores to attach to microtubules emanating from opposite poles, a state termed bi-orientation. The balance of cohesion and spindle-pulling force creates tension across kinetochores. After all kinetochores are bi-oriented, the protease Separase cleaves centromeric cohesin to initiate anaphase. Through attachment to microtubules anchored at opposing poles, the separated chromatids are evenly partitioned into daughter cells. Kinetochores not bi-oriented activate the spindle checkpoint. This checkpoint inhibits the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase, to block separase activation, anaphase onset, and mitotic exit.
My research program focuses on, and has contributed to, two major interconnected areas: (1) the spindle checkpoint and (2) sister-chromatid cohesion and chromosome segregation. Our research highlights an emerging principle in cell biology: exquisite spatiotemporal coordination of opposing activities or functionalities underlies transitions between cellular states.
The Spindle Checkpoint
The spindle checkpoint is an intracellular signaling network during mitosis that senses and responds to kinetochores not under bi-orientation and delays anaphase (Figure 1). In the past two decades, our research, along with contributions from many others, has established that unattached kinetochores recruit and activate checkpoint proteins. The activated checkpoint proteins collaborate to inhibit Cdc20, the mitotic activator of APC/C, thereby delaying anaphase. A critical APC/C inhibitor is the mitotic checkpoint complex (MCC) comprising BubR1-Bub3-Mad2-Cdc20. Microtubule binding and tension generation at kinetochores displace and inactivate checkpoint proteins, and silence the checkpoint. Checkpoint signaling is thus controlled by the spatiotemporally regulated antagonism between checkpoint proteins and microtubules at kinetochores.
A highlight of our work is the demonstration that Mad2 is a prion-like, multi-state protein. The conformational transition of Mad2 is critical for MCC assembly and checkpoint signaling, and is regulated by positive (Mad1) and negative (p31comet) regulators and by phosphorylation. More recently, we have established the KMN network of kinetochore proteins (consisting of Knl1-Zwint, the Ndc80 complex, and the Mis12 complex) as a major signaling platform of the spindle checkpoint in human cells. We have characterized the mechanisms by which KMN is anchored to centromeric chromatin during mitosis. Furthermore, we have shown that competition between the checkpoint kinase Mps1 and microtubules for binding to the Ndc80 complex constitutes a direct mechanism for detection of unattached kinetochores (Figure 2). Finally, we have shown that a heterodimeric kinase complex of Bub1 and Plk1 phosphorylates Cdc20 and inhibits APC/C in a mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.
Our ongoing research focuses on the in vitro reconstitution of KMN-dependent spindle checkpoint signaling and microtubule-mediated inactivation of this process in the test tube. These experiments will define the minimal checkpoint sensor, and establish the activation and inactivation mechanisms of the checkpoint. Another ongoing effort is to explore the physiological functions of spindle checkpoint components using mouse genetics. Finally, we are interested in studying the plasticity of mitotic programs and consequences of chromosome missegregation in different cell types using the human embryonic stem cells and induced pluripotent stem cells as models.
Sister-Chromatid Cohesion and Chromosome Segregation
Human sister chromatids at metaphase are primarily linked by centromeric cohesion, forming the iconic X shape. Premature loss of centromeric cohesion disrupts orderly mitotic progression. Sgo1–PP2A) localizes to centromeres in mitosis, binds to Cohesin in a reaction requiring Cdk-dependent phosphorylation of Sgo1, dephosphorylates Cohesin-bound Sororin, and protects Cohesin at inner centromeres. The kinetochore kinase Bub1 phosphorylates histone H2A at T120 (H2A-pT120) and recruits Sgo1 to kinetochores, 0.5 µm from inner centromeres. Recently, we have shown that Sgo1 is a direct reader of the H2A-pT120 mark. Bub1 also recruits RNA polymerase II (Pol II) to unattached kinetochores and promotes active transcription at mitotic kinetochores. Pol II-dependent transcription enables kinetochore-bound Sgo1 initially recruited by H2A-pT120 to reach Cohesin embedded in centromeric chromatin (Figure 3). Our study demonstrates the importance of spatially constrained tug-of-war between opposing enzymatic activities in cell biology, and implicates mitotic transcription in targeting regulatory factors to highly compacted mitotic chromatin.
The ring-shaped Cohesin complex regulates transcription, DNA repair, and chromosome segregation by dynamically entrapping chromosomes to promote chromosome compaction and sister-chromatid cohesion. The Cohesin ring needs to open and close to allow its loading to and release from chromosomes. Recently, we have determined the crystal structures of the protease domain of Separase alone or in complex with an Scc1-derived peptide inhibitor. The structures reveal the structural basis of phosphorylation-enhanced Cohesin cleavage by Separase. In addition, we have determined the crystal structures of Wapl, Pds5B, and SA2/Scc3 bound to an Scc1 fragment. Our biochemical analyses further suggest that Pds5–Wapl stabilizes a transient, open state of cohesin to promote its release from chromosomes. Sororin inhibits a functional interaction between PDS5 and WAPL, thus stabilizing Cohesin on chromosomes.
The current available data collectively support the following model for Cohesin loading and release (Figure 4). Scc2–Scc4, Cohesin, and DNA form a transient complex, in which Scc2–Scc4 strengthens the engagement and ATP binding of the Smc1–Smc3 ATPase domains. ATP hydrolysis disengages the ATPase domains and opens the Cohesin ring to entrap DNA. ATP rebinding closes the inner gate and encircles DNA in the top Smc1–Smc3 closure. The entrapped DNA promotes ATP hydrolysis and inner-gate opening, allowing DNA to escape from the top closure. Wapl–Pds5 binds to the nucleotide-free Cohesin and, upon ATP binding, disrupts the Smc3–Scc1N interface to release DNA. Smc3 acetylation prevents the entrapped DNA from stimulating the ATPase activity of Cohesin, blocking inner-gate opening and Wapl–Pds5-dependent outer-gate opening.
Our ongoing research aims to determine the high-resolution structures of Cohesin in different nucleotide states, alone or bound to its regulators and DNA. In addition, we are investigating the mechanism by which cohesion establishment is coupled to DNA replication. These structural and functional studies will unveil the mystery behind the magic acts of DNA entrapment and release by the Cohesin ring.
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, 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, the Clayton Foundation for Research, and the Welch Foundation.
As of March 11, 2016