Why study genome maintenance?

The genetic material is what defines all living organisms and thus cells have evolved sophisticated mechanisms to ensure that it is replicated and segregated faithfully during each cell division. This is especially true for complex organisms like us, considering an astronomical number of cell division must occur from one cell embryo to a grown adult, and each cell division requires accurate replication and segregation of over 3 billion base pairs of DNA! How do cells accomplish this amazing feat? How mistakes in doing so cause disease? The SUMO pathway caught our attention because of its profound role in genome maintenance, and its under-explored nature that provides a fertile ground for new discoveries.

Introduction

Small Ubiquitin-like Modifier (SUMO) is a member of the ubiquitin-like protein family. Like ubiquitin, the 10-kDa SUMO is covalently attached to lysine residues on target proteins via a cascade of an E1-activating enzyme (Aos1/Uba2 in yeast), an E2-conjugating enzyme Ubc9, and one of the several E3 ligases (Siz1, Siz2 and Mms21 in yeast). On the other hand, a family of SUMO isopeptidases (Ulp1 and Ulp2 in yeast) cleaves SUMO off its target proteins. Importantly, the SUMO pathway is essential for cell viability and is conserved from yeast to human. We are investigating how SUMO maintains genome integrity, regulates gene transcription, and how dysfunctions in these processes cause diseases.

Enzymatic Mechanism of the SUMO Pathway

A major interest of my team is to study the enzymology of the SUMO pathways. Using the model organism Saccharomyces cerevisiae, we found that SUMO E3 ligases have substantial overlapping and distinct substrates (Albuquerque et al, 2013). Most strikingly, we discovered that the SUMO protease Ulp2 specifically desumoylates three protein complexes at the centromeres, the ribosomal DNA (rDNA) and the origins of DNA replication, while Ulp1 suppresses the bulk of intracellular sumoylation (Albuquerque 2016). These breakthrough studies laid the foundation for our current investigations.

Ulp2-Csm1-structure.png

To understand Ulp2's exquisite substrate specificity, we collaborated with the Corbett lab to solve the crystal structure of the Ulp2-Csm1 complex (Liang et al, 2017), demonstrating that Csm1 directly recruits Ulp2 and thus facilitates its desumoylation activity in the nucleolus. Remarkably, once recruited to the nucleolus by Csm1, Ulp2 also utilizes its SUMO-binding to selectively target hyper-sumoylated substrates in the nucleolus, illustrating a novel dual substrate recognition and feedback control mechanism (Albuquerque, 2018). We have now implemented the CRISPR-Cas9 approach to investigate the enzymology of human SUMO pathways, an exciting and relatively unexplored area with much to learn.

SUMO Prevents Chromosomal Translocations

Human genetic studies have shown that mutation of NSMCE2, the human ortholog of yeast Mms21, causes genome instability syndrome and primordial dwarfism. Remarkably, inactivation of yeast Mms21 caused substantial chromosomal translocations (Albuquerque et al, 2013), while mutations of Siz1 and Siz2 had a much smaller effect. In collaboration with the Kolodner lab (Liang et al, 2018), we discovered that the primary function of Mms21 is to prevent the accumulation of spontaneous DNA breaks, most likely occurred during DNA replication, and in the absence of Mms21 SUMO ligase activity, these DNA breaks are processed to generate chromosomal translocations via break-induced replication (BIR). Combined with our finding that Mms21 specifically catalyzes sumoylation of the MCM complex in the DNA replisome, which is antagonized by the SUMO protease Ulp2 (Albuquerque et al, 2016), these findings led to the hypothesis that Mms21 dependent sumoylation of MCM prevents chromosomal translocations via protecting chromosomal DNA replication. We are studying its mechanism further.

An important reason of using yeast as a model organism is to rapidly derive new insights into fundamental process, which can then be used to understand how its dysfunction could cause disease. Therefore, we have begun to investigate human MCM sumoylation and its function in preventing chromosomal rearrangements, a hallmark of cancer genome.

SUMO and Gene Transcription

The nucleolus is the largest membrane-less organelle in the nucleus, where ribosome biogenesis takes place. Due to the repetitive nature of the ribosomal (rDNA) locus, the nucleolus is also transcriptionally silenced (darker region under EM) to prevent aberrant rDNA recombination and instability. Recently, we found that mutations disrupting the binding between Ulp2 and Csm1 caused an accumulation of sumoylated Tof2, leading to its degradation and a loss of rDNA silencing (Liang et al, 2017). Interestingly, this SUMO dependent degradation of Tof2 requires the SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5-Slx8, suggesting that SUMO and ubiquitin work together to control Tof2 degradation. Because both Ulp2 and Slx5 are conserved in humans, their human orthologs likely regulate gene transcription via a conserved mechanism, which we are investigating further.

SUMO Prevents Aneuploidy

 Gascoigne et al. NCB 2012

Gascoigne et al. NCB 2012

Aneuploidy, the presence of an abnormal number of chromosomes in cells, is a hallmark of cancer and a major cause of birth defects and mental illness. The kinetochore is the central molecular machine that controls chromosome segregation in all eukaryotes, whose dysfunction causes aneuploidy. The SMT3 gene, encoding the SUMO protein in S. cerevisiae, was originally identified as a high-copy suppressor of mif2, a mutation in the essential kinetochore Mif2 subunit, which is the ortholog of human CenpC. The Ulp2 protease, also known as SMT4, is the second high-copy suppressor of mif2 identified. These earlier genetic findings provided the first clue to a key role of Smt3 and Ulp2/Smt4 in regulating the kinetochore; however, the mechanism has been elusive for almost 20 years. Recently, we found that Ulp2 specifically desumoylates the inner kinetochore CCAN complex (Albuquerque, 2016), and this exciting discovery has opened the door to study the function of SUMO in preventing aneuploidy. Once again, S. cerevisiae provides us with powerful genetic and biochemical approaches to study this fundamental process, which is conserved from yeast to human.

Functional Proteomics: in vivo Biochemistry

Cells are highly responsive to their environments. A widely used strategy is post-translational modification of proteins that enables cells to detect, amplify and integrate signals into specific responses. These signals are transient and difficult to detect, we have developed quantitative phospho-proteomics to study phosphorylation in the DNA damage response; and more recently quantitative SUMO proteomics to study the SUMO pathways. With our recent acquisition of the latest Orbitrap Fusion-LUMOS mass spectrometer, we are excited to apply it to study these signaling pathways in human cells and examine how they are altered in disease such as cancer.