Tag: Oral Session 3M

PTPN11 S502P and Tyrosine Kinase Non-Receptor-2 increase RAS/MAPK signaling in Acute Myeloid Leukemia

Presenter(s): Corinne Togiai − Biology

Faculty Mentor(s): Chelsea Jenkins, Dr. Bill Chang

Oral Session 3M

Research Area: Natural/Physical Science (Cancer Biology)

Funding: OHSU Knight Cancer Institute, Oregon Health and Science University, Howard Hughes Medical Institute, Druker Laboratory, Dr. Brian Druker, Dr. Bill Chang, Dr. Jeff Tyner and Dr. Chelsea Jenkins

PTPN11 is a gene which encodes the protein tyrosine phosphatase SHP2, an auto-inhibited protein that dephosphorylates targets in many of the proliferative pathways such as Ras/MAPK. This gene, PTPN11, is the driving force in 35% of Juvenile Myelomoncytic Leukemia (JMML) patients and 10% of Acute Myeloid Leukemia (AML) patients. Moreover, cells from a JMML patient were found to be sensitive to tyrosine kinase inhibitor dasatanib. This is thought due to interactions between PTPN11 and tyrosine non-kinase 2 (TNK2), which is a dasatanib target. Therefore, we hypothesized that HEK 293 T17 cells co-transfected with mutant PTPN11 S502P and TNK2 will display decreased phospho-TNK2 and increased phospho-ERK, as seen in the JMML mutant PTPN11 E76K. In my project, I worked with PTPN11 mutation identified in an AML patient sample (S502P) that has shown sensitivity to the drug Dasatanib, a kinase inhibitor that blocks the action of abnormal proteins that signal cells to proliferate, ultimately helping stop the spread of cancer cells. I performed multiple western blots consisting of: transfections, gel electrophoresis, and protein detection. Results show S502P mutant PTPN11 acts like E76K mutant in that it activates the RAS/MAPK pathway, and S502P mutant PTPN11 dephosphorylates TNK2. In conclusion, the patient sample S502P mutant has shown a dephosphorylating effect on TNK2 that has not been seen in any previous studies. Data suggests that this mutant also works with TNK2 to increase RAS/MAPK signaling. Through this interaction this mutation can be tested and targeted by Dasatinib to stop the proliferation of leukemic cells.

A Forward Genetic Screen for Genes Required in C. elegans Embryonic Morphogenesis

Presenter(s): Alexander Miller − Biology

Faculty Mentor(s): Bruce Bowerman, Molly Jud

Oral Session 3M

Research Area: Natural/Physical Sciences

Funding: NIH funding, Oregon Undergraduate Researchers in SPUR (OURS) summer scholarship

Our research in the Bowerman laboratory focuses on embryonic morphogenesis in the soil nematode, Caenorhabditis elegans. Morphogenesis is the coordinated movement and shape changes of cells that occur during development in all animals. Defects in this process can cause a variety of human disorders, including neural tube closure, vascular, and
limb developmental defects. My research involves a forward genetic screen for genes required in C. elegans embryonic morphogenesis, utilizing a collection of roughly 1,000 temperature-sensitive embryonic lethal (TS-EL) mutants previously created in the Bowerman laboratory. TS-EL mutants are first terminally phenotyped to identify mutants with penetrant morphogenetic defects, which are then genetically characterized to isolate recessive, loss-of-function, single-mutant alleles. We identify the mutant genes through a combination of SNP-mapping and whole-genome sequencing to find candidate genes, followed by complementation testing with null alleles when available. Mutant strains are sent to our collaborators, the Zhirong Bao laboratory at the Sloan Kettering Cancer Center, for single cell-fate lineaging. This allows us to more accurately distinguish between morphogenesis and cell fate patterning defective mutants. I have terminally-phenotyped nineteen mutants, fourteen of which have penetrant phenotypes (where 70% of embryos display a single phenotype), and genetically characterized six recessive, single mutants. Alleles or388ts and or1113ts have been genetically identified to be mutations in the gene let-19, which is a transcriptional coactivation subunit containing Mediator domains. Allele or542ts is a mutation in the gene chaf-1, a chromatin assembly factor likely regulating gene transcription. Allele or614ts is a mutation in the zwl-1, which codes for a kinetochore protein. While let-19 and chaf-1 fall within the scope of this project, our allele of zwl-1 is probably a weak cell division-defective instead of a morphogenesis-defective mutant, and thus is no longer being studied. Most of the genes we have identified are involved in gene expression regulation, suggesting there is a preexisting gene regulatory program for morphogenesis. Future work is to be done to expand our and the Bao laboratory’s roster of genes known to be required for morphogenesis. This research will help to create a deeper understanding of the basic genetic pathways and cell biological changes required for embryonic morphogenesis in C. elegans, thus improving our comprehension of human development.

Investigating the Role of Transposons in Temperature-Induced DNA Damage During Spermatogenesis

Presenter(s):  Colin Maxwell − Biology

Faculty Mentor(s): Diana Libuda, Nicole Kurhanewicz Codd

Oral Session 3M

Research Area: Biological Science

Meiosis is a specialized form of cell division that sexually reproducing organisms use to generate haploid sex cells. Developing sperm are particularly sensitive to temperature fluctuations, with some studies indicating that exposure to elevated temperature increases DNA damage in spermatocytes, but not oocytes. Although temperature-induced DNA damage has been observed, the underlying molecular mechanisms remain unknown. DNA transposons are mobile genetic elements that produce double-strand DNA breaks (DSBs) when excised from the genome. Additionally, transposons can excise from the genome under heat stress.

I hypothesize that heat stress causes transposon excision which may be observed as a linear relationship between transposon copy number and the quantity of DSBs in developing spermatocytes exposed to elevated temperature. To test this hypothesis, I conducted an immunofluorescence screen of wild type Caenorhabditis elegans strains with varying transposon copy numbers. Using deconvolution microscopy, DSBs were visualized via the recombinase RAD-51, a protein involved in the early stages of meiotic DSB repair. Quantification of RAD-51 foci was performed to determine the frequency of temperature-induced DSB formation. Preliminary results demonstrate that the CB4856 strain with ~15 copies of Tc1, a class of transposons active in C. elegans, exhibited half the amount of DSBs as the Bristol N2 strain with ~30 copies of Tc1 displayed upon heat shock. In contrast, comparisons of DSB quantities between additional strains with varying Tc1 copy numbers show no clear relationships. Taken together, these results indicate temperature-induced DNA damage in spermatocytes has multiple mechanisms, with excision of Tc1 transposons as one possible mechanism.

Investigating the Role Of Genomic Positioning in Directing Meiotic Double-Strand DNA Break Repair

Presenter(s): Rachel David − Biochemistry

Faculty Mentor(s): Diana Libuda, Erik Toraason

Oral Session 3M

Research Area: Biological Sciences

Funding: UO Alden Scholar Research Award, NICHD Grant

During meiosis, the specialized form of cell division that produces gametes, cells utilize recombination to maintain genomic integrity and promote proper chromosome segregation, ensuring fertility. Double-strand DNA breaks (DSBs), which serve as substrates for homologous recombination, are intentionally induced during meiosis. A fraction of DSBs must be repaired
as crossover recombination events with the homologous chromosome to forge a physical connection required to facilitate proper chromosome segregation. Although DSBs are induced across the genome, crossovers in C. elegans are preferentially formed along chromosome arms and not the center of chromosomes. What determines this crossover preference along chromosome arms is not well known. Intriguingly, previous studies in C. elegans have indicated that the crossover landscape is not determined by chromatin marks or specific sequence motifs. However, proximity of a DSB to the synaptonemal complex (SC), a meiosis-specific proteinaceous structure that connects homologous chromosomes together, has been suggested to influence DSB repair outcomes. To determine how genomic positioning of a DSB affects its repair outcome, I am exploiting genetic assays developed by the Libuda lab that enable controlled induction of a single DSB at a known genomic location and assess how that induced DSB was repaired. Utilizing CRISPR/Cas9 genome editing, I have targeted these assays to four unique loci that differ in position both along the chromosome length and in proximity to the SC. Together, our studies will elucidate how the position of a DSB within the genome influences how it is repaired to maintain genome integrity.

SMC-5/6 Facilitates Efficient DSB Repair During Meiosis in C. elegans

Presenter(s): Cordell Clark − Biology

Faculty Mentor(s): Diana Libuda, Erik Toraason

Poster 73

Oral Session 3M

Research Area: Biological Science

Funding: OURS Program Summer 2017, Dr. Diana Libuda’s laboratory is funded in part by an NICHD grant

Sexually reproducing organisms depend upon meiosis to form haploid sex cells necessary for reproduction. Despite the inherent risks of DNA damage to genome integrity, meiotic cells intentionally induce double strand DNA breaks (DSBs) throughout the genome. A specific and limited number of DSBs must be repaired as crossovers with the homologous chromosome to promote proper chromosome segregation. DSBs are induced in excess of the permitted number of crossovers. DSBs not repaired as crossovers must be repaired to maintain genomic integrity. In the Libuda lab, we have designed an assay to determine the repair outcome of a single induced DSB. We have demonstrated that in addition to the homologous chromosome, the sister chromatid is used as a repair template during DSB repair in C. elegans meiosis. My research aims to uncover the mechanisms that facilitate intersister repair, which are currently unknown. Experiments in multiple species have demonstrated that SUMOylation is required for crossover formation. Our preliminary immunofluorescence experiments in SUMO deficient mutants reveal SUMOylation is required for DSB repair in C. elegans oocytes. Prior research has hypothesized that the SMC5/6 complex, which contains a SUMO ligase subunit (NSE-2), facilitates intersister recombination. Using our intersister repair assay, I have demonstrated that the SMC5/6 complex is required for efficient intersister repair and intersister crossovers. My ongoing experiments are directly testing whether the SMC5/6 complex promotes intersister recombination via the NSE-2 SUMO ligase subunit. Overall, our studies are defining the mechanisms that facilitate intersister recombination to ensure genome integrity during sperm and egg development.