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, 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 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). Considering the opposing activities of Mms21 and Ulp2 control sumoylation of MCM in the DNA replisome (Albuquerque et al, 2016), we are further studying how sumoylation of MCM may prevent chromosomal translocations via the control of chromosomal DNA replication. 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 role 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 over 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.

Proteomics: a powerful analytical tool for biology

Mass spectrometry (MS) is arguably the most powerful analytical tool for protein analysis. Numerous applications for MS have been described, which are based on the remarkable ability of MS to measure the mass/charge ratio of protein and/or peptide with increasing sensitivity, mass resolution, and accuracy; as well as its ability to break down peptide via collision-induced dissociation to produce a fine fingerprint of each peptide and thus reveal its amino acid sequence. The availability of protein sequence databases via genome sequencing projects has greatly advanced the use of MS to identify protein with sub-femtomole sensitivity and mass resolution better than 1 ppm, using our latest ORBITRAP LUMOS-FUSION mass spectrometer. Moreover, the use of stable isotope labeling via metabolic or chemical methods has made MS the most accurate analytical tool for protein quantification.

Over the past decade, we have worked extensively in applying MS for specific biological applications. First, we routinely use MS to identify protein-binding partners purified from cell extract via either immunoprecipitation or other protein affinity methods. Second, we apply MS to identify post-translational modifications of proteins, including phosphorylation and sumoylation. Third, we are developing chemical cross-link to map protein interaction interface and thus obtain structural information. Last but not least, we apply MS to measure protein expression to determine the effect of aneuploidy on the proteome. Importantly, these applications incorporate stable isotope labeling via Stable Isotope Labeling via Amino acid in Cell culture (SILAC) or TMT (Tandem Mass Tag), to obtain specific biological insights. Therefore, students can expect to acquire this unique expertise, besides receiving diverse trainings in genetics, molecular biology and biochemistry.