The general research interest of Liu lab is to understand the various mechanisms that cause genome instability in human cells, how cells counteract these threats, and why some regions in the genome are unusually susceptible to instability (e.g. common or rare fragile sites).
A) The characterization of DNA/RNA hybrids (R-loops) associated with common fragile sites
Common fragile sites (CFSs) are hot spots for deletions and chromosome rearrangements in cancer . It is well established that incomplete replication can cause a delay in chromatin condensation that leads to the ‘expression’ of CFSs (detected as breaks or gaps on metaphase chromosomes) [2, 3]. CFS regions have some intrinsic characteristics that predispose them to ‘fragility’ under replication stress conditions (RS). These include, 1) containing repeat sequences that could form secondary structures (e.g. hairpins or G-quadruplexes); 2) lack of replication origins; and 3) containing long genes or highly transcribed genes where replication forks are prone to collide with the messenger RNAs (e.g. regions where R-loops can form) [2, 4-8]. We have previously discovered that a process called mitotic DNA synthesis (MiDAS) that operates in mitosis is a strategy used by human cells to rescue the incomplete replication at those loci, particularly in cancers cells [9-12]. Our current focus is to identify the mechanisms employed by the cells to resolve stalled replication forks in the regions prone to form R-loops.
B) The analysis of folate deficiency induced genome instability
Folate deficiency is known to be associated with a diverse range of human disorders including fetal neural tube defects, age-associated dementia, infertility, and some type of cancers. Intriguingly, folate deficiency is known can cause the expression of group of rare fragile sites, all of which contain long stretch of CGG simple repeats. The most well studied locus of this kind is called FRAXA that is associated with Fragile X syndrome (FXS). We use FXS cells as a model to study how the FRAXA locus is affected by folate deprivation. Our data suggest that folate deprivation triggers extensive missegregation of this locus during cell division. Moreover, the entire chromosome X becomes unstable during a period of long-term folate deprivation . We are currently working on, 1) the characterization of the pathways employed by the cells to repair the collapsed replication folks caused by folate deficiency; and 2) the identification of genomic regions that are most vulnerable to folate deficiency.
C) Post-translational modification of proteins by sumoylation in response to DNA replication stress
In response to replication-associated DNA damage, human cells activate a highly conserved signalling network to prevent irreversible breakdown of DNA replication forks. One component of this DNA damage response is post-translational modification (i.e. phosphorylation, acetylation, mono- or poly-ubiquitylation and SUMOylation). While much is known about how phosphorylation affects protein function, little information is available on the SUMOylation of proteins following cellular stress. From an unbiased mass spectrometry based proteomic study, we have previously shown that, in response to RS (a condition that could initiate CFS expression), SUMO2 was conjugated to a panel of proteins in S phase in human cells [14, 15]. Interestingly, POLD3, a subunit of human polymerase delta, was found to be modified most significantly amongst these proteins . We are currently carrying out research to characterise how POLD3 SUMOylation by SUMO2 might play a role in the cellular response to RS.
D) Identification of genes that would predispose sensitivity to radiotherapy in cancer patients
Radiotherapy is an effective treatment for the majority of cancer patients. However, a small portion of cancer patients (5-10%) suffer significant toxicity from this treatment [16, 17]. To date, the cause for this ‘radiation-sensitivity’ is still unknown. We hypothesized that some of the reaction is caused by the DNA repair deficiency in these patients. In a normal environment, this deficiency would not be life-threatening for the patients. However, when the patients are exposed to radiotherapy that can cause damage specifically to DNA, the patients with those potential DNA repair deficiency would suffer more severely than those patients who have fully functional DNA repair systems. DNA samples of four cell lines derived from the lymphocytes of cancer patients who were sensitive to radiotherapy have been sequenced and analysed. Data analysis has been performed in a panel of genes that are known to be involved in DNA replication and repair. Functional analysis of the candidate genes will be carried out. The outcome of this project will facilitate the design of treatment for cancer patients.
- Richards RI. Fragile and unstable chromosomes in cancer: causes and consequences. Trends in genetics : TIG. 2001; 17:339-345.
- Helmrich A, Ballarino M, Tora L. Collisions between Replication and Transcription Complexes Cause Common Fragile Site Instability at the Longest Human Genes. Molecular Cell. 2011; 44:966-977.
- Letessier A, Millot GA, Koundrioukoff S, Lachages AM, Vogt N, Hansen RS, Malfoy B, Brison O, Debatisse M. Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site. Nature. 2011; 470:120-123.
- Barros AV, Wolski MA, Nogaroto V, Almeida MC, Moreira-Filho O, Vicari MR. Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role? Gene. 2017; 608:20-27.
- Hosseini SA, Horton S, Saldivar JC, Miuma S, Stampfer MR, Heerema NA, Huebner K. Common Chromosome Fragile Sites in Human and Murine Epithelial Cells and FHIT/FRA3B Loss-Induced Global Genome Instability. Gene Chromosome Canc. 2013; 52:1017-1029.
- McNees CJ, Tejera AM, Martinez P, Murga M, Mulero F, Fernandez-Capetillo O, Blasco MA. ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase. J Cell Biol. 2010; 188:639-652.
- Sfeir A, Kosiyatrakul ST, Hockemeyer D, MacRae SL, Karlseder J, Schildkraut CL, de Lange T. Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication. Cell. 2009; 138:90-103.
- Simonic I, Gericke GS. The enigma of common fragile sites. Human Genetics. 1996; 97:524-531.
- Bjerregaard VA, Ozer O, Hickson ID, Liu Y. The Detection and Analysis of Chromosome Fragile Sites. Methods Mol Biol. 2018; 1672:471-482.
- Garribba L, Wu W, Ozer O, Bhowmick R, Hickson ID, Liu Y. Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci. Method Enzymol. 2018; 601:45-58.
- Minocherhomji S, Ying S, Bjerregaard VA, Bursomanno S, Aleliunaite A, Wu W, Mankouri HW, Shen H, Liu Y, Hickson ID. Replication stress activates DNA repair synthesis in mitosis. Nature. 2015; 528:286-290.
- Ren L, Chen L, Wu W, Garribba L, Tian H, Liu Z, Vogel I, Li C, Hickson ID, Liu Y. Potential biomarkers of DNA replication stress in cancer. Oncotarget. 2017; 8:36996-37008.
- Bjerregaard VA, Garribba L, McMurray CT, Hickson ID, Liu Y. Folate deficiency drives mitotic missegregation of the human FRAXA locus. Proc Natl Acad Sci U S A. 2018;
- Bursomanno S, Beli P, Khan AM, Minocherhomji S, Wagner SA, Bekker-Jensen S, Mailand N, Choudhary C, Hickson ID, Liu Y. Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells. DNA Repair (Amst). 2015; 25:84-96.
- Bursomanno S, McGouran JF, Kessler BM, Hickson ID, Liu Y. Regulation of SUMO2 target proteins by the proteasome in human cells exposed to replication stress. J Proteome Res. 2015; 14:1687-1699.
- Barnett GC, West CM, Dunning AM, Elliott RM, Coles CE, Pharoah PD, Burnet NG. Normal tissue reactions to radiotherapy: towards tailoring treatment dose by genotype. Nat Rev Cancer. 2009; 9:134-142.
- Norman A, Kagan AR, Chan SL. The Importance of Genetics for the Optimization of Radiation-Therapy - a Hypothesis. Am J Clin Oncol-Canc. 1988; 11:84-88.
Dr. Cynthia T. McMurray, Berkeley National Laboratory, University of California, USA
Prof. Chunaram Choudhary, NNF Center for Protein Research, University of Copenhagen, Denmark
Prof. Liqun Ren, Chengde Medical University, China
Prof. Thanos Halazonetis, University of Geneva, Switzerland