Discipline: Biological Sciences
Subcategory: Cancer Research
Room: Marriott Balcony A
Besmira Alija - City University of New York- Hunter College
Co-Author(s): Aaron Viny MD, MS, Memorial Sloan Kettering Cancer Center, New York, New York; Ross Levine MD, Memorial Sloan Kettering Cancer Center, New York, New York
Genes coding the subunits of cohesin are often mutated in Acute Myeloid Leukemia (AML). Cohesin, a protein complex with multiple subunits including Stromal Antigen 1/2 (STAG1/STAG2), localizes at genomic regions called superenhancers, sites that regulate expression of genes required for cell differentiation. By stabilizing DNA loops, cohesin allows for the interaction between promoters and enhancers, which is crucial for the expression of genes required for differentiation. The expression of these genes also relies on the accessibility of DNA, which is provided by the activating histone mark, H3K27Ac. STAG2 mutations are present in 12% of myeloid neoplasms, but the role of STAG2 mutations in the pathogenesis of myeloid malignancies is incompletely understood. We hypothesize that Stag2 mutations may decrease the expression of the activating histone mark H3K27Ac, limiting accessibility of DNA, thereby interfering with expression of genes necessary for differentiation.
In order to test this hypothesis, Western blotting analysis was used to determine the effects of Stag2 loss of function mutations on activating histone marks in mouse models. We used wild type mice (Stag2+/+) as a control, mice with deletion of one copy of the Stag2 gene (Stag2+/-), and mice with deletion of both copies of the Stag2 gene (Stag2-/). Genotypes were assessed through polymerase chain reaction (PCR). Proteins from the spleens of these wild type mice (Stag2+/+), heterozygous knockout mice (Stag2+/-), and homozygous knockout mice (Stag2-/-) were extracted. Western blotting was used to assess expression of Stag2 protein and expression of the activating histone mark H3K27Ac. Through PCR, we were able to confirm the genotypes of the mice used. The results of Western blotting analysis demonstrated the presence of Stag2 protein in wild type mouse (Stag2+/+) as well as in heterozygous knockout (Stag2+/-). The lowest levels of Stag2 protein were found in the homozygous knockout (Stag2-/-). Western blotting also showed that one copy of the Stag2 gene (Stag2+/-) was sufficient to maintain a similar H3K27Ac expression level compared to the wild type mouse. Deletion of both copies of the Stag2 gene (Stag2-/-) showed a marked decrease in H3K27Ac. Our findings show that loss of both copies of the Stag2 gene (Stag2-/-) resulted in decreased H3K27Ac expression. Decreased expression of this activating histone mark may result in decreased DNA accessibility and hence decreased expression of genes involved in differentiation. In future work, we aim to determine if H3K27Ac loss occurs uniformly or at specific genes. These questions will be explored with Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) to elucidate the mechanism by which Stag2 mutations are associated with myeloid malignancies.
References: Lasada, A. 2014. Cohesin in Cancer: Chromosome Segregation and Beyond. Nature Reviews.14: 389-393.
Rao et al. 2017. Cohesin Loss Eliminates All Loop Domains. Cell.171, 305-320
Funder Acknowledgement(s): I thank Dr. Aaron Viny and Dr. Ross Levine at Memorial Sloan Kettering Cancer Center for their unconditional support throughout this project. I also thank the Office of Faculty Development at Memorial Sloan Kettering Cancer Center for their help, and for funding the research that was conducted.
Faculty Advisor: Aaron Viny and Ross Levine, firstname.lastname@example.org
Role: Polymerase Chain Reaction (PCR), the western blot protocol, and protein extraction was conducted by me.