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; otherwise, disasters will struck. 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. Substantial progresses have been made in understanding the machineries that perform DNA replication and chromosome segregation; yet new pathways are continuously being discovered that ensure these machineries perform their tasks with the precision needed for complex organisms like us. One such pathway is the SUMO pathway, which caught our interest 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. Together, these enzymes control reversible sumoylation of hundreds of proteins in cells. The importance of the SUMO pathways is evident by the fact that it is essential for cell viability and is well conserved from yeast to human. We are investigating a broad range of topics to understand how SUMO maintains genome integrity, as summarized below:

Enzymology of the SUMO pathways

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 mechanism for its exquisite substrate selectivity (Albuquerque, 2018). We have also 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 the other E3 ligases 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), whose formation requires the Rad52 homologous recombination pathway, the DNA damage checkpoint, the DNA helicase Rrm3 and DNA polymerase subunit Pol32. 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 in cell culture and its function in preventing chromosomal rearrangements often seen in cancer.

Novel SUMO signaling in the nucleolus

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. Our study has uncovered a novel signaling function of SUMO in this sub-nuclear compartment.

We have shown that loss of Ulp2 caused over 20-fold accumulation of sumoylated RENT complex and Tof2 in the nucleolus (Albuquerque, 2016). More recently, we found that mutations inactivating Ulp2-Csm1 binding caused a similar accumulation of nucleolar sumoylation; and interestingly, a drastic degradation of Tof2, a key protein required for rDNA silencing (Liang et al, 2017). However, unlike ubiquitin, SUMO has no known role in directly mediating protein degradation. Instead, we found that this SUMO dependent degradation of Tof2 requires the SUMO-targeted ubiquitin E3 ligase (STUbL) Slx5-Slx8, a family of evolutionarily conserved enzymes, suggesting SUMO and ubiquitin may work together to control Tof2 degradation via the antagonistic roles of Ulp2 and Slx5-Slx8. To explore it further, we are investigating sumoylation of the RENT complex in maintaining nucleolar integrity and regulating mitosis in yeast, as well as the corresponding pathway in human cells.

SUMO prevents aneuploidy

 Gascoigne et al. NCB 2012

Gascoigne et al. NCB 2012

The SMT3 gene encodes SUMO in Saccharomyces cerevisiae, which was originally identified as a high-copy suppressor of mif2, a mutation that impairs the essential kinetochore Mif2 protein, the ortholog of human CenpC. The Ulp2 protease is also known as SMT4, another high-copy suppressor of mif2. These earlier genetic findings provided the first clue to a key role of SUMO in preventing aneuploidy, a major cause of birth defect, Down syndrome and a hallmark of cancer. Kinetochore is the central macromolecular machine that powers chromosome segregation in all eukaryotes. Our recent finding that Ulp2 specifically desumoylates the inner kinetochore CCAN complex in yeast has triggered our investigation into the function of SUMO in preventing aneuploidy, an area with much to learn (Albuquerque, 2016).

Functional Proteomics: in vivo biochemistry on a proteome-wide scale

Cells are highly responsive to their environments. A widely used strategy is post-translational modification (PTM) of proteins that enables cells to detect, amplify and integrate signals into specific responses. We have developed quantitative phospho-proteomics technology to study the DNA damage response and quantitative SUMO proteomics technology 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 their defects may contribute to disease.