Splicing Factor SF3B1: Putative Chronic Lymphocytic Leukemia Driver Gene

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dc.contributor.advisor Woodruff, Rachel en_US
dc.contributor.advisor Reed, Robin en_US
dc.contributor.author Huang, Jingyuan
dc.date.accessioned 2018-06-22T18:25:45Z
dc.date.available 2018-06-22T18:25:45Z
dc.date.issued 2018 en_US
dc.identifier.uri https://hdl.handle.net/10192/35797
dc.description.abstract Background The protein SF3B1 is an essential component of the SF3B complex, which is a constituent of the general splicing factor U2 snRNP. Recent studies found that mutation of SF3B1 is associated with numerous hematological cancers such as chronic lymphocytic leukemia (CLL), but the mechanism for its pathogenesis remains unclear. Methods Done by the core facility at Harvard Medical School (HMS), CRISPR was used to construct human embryonic stem (ES) cell lines bearing the SF3B1K700E point mutation, which is the most common mutation in hematological cancers. My lab had DNA sequencing performed to identify the cell line carrying the K700E mutation, and RNA sequencing (RNA-Seq) was performed by Dr. Tan. I prepared nuclear extracts of K700E mutant MT and WT cells to analyze 17S/12S U2 snRNP in the K700E mutation, and I used Western blots to determine whether MT SF3B1 has any effect on U2 snRNP and the spliceosome. Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR) were conducted to validate that MT SF3B1 affects gene expression. Results RNA-Seq showed that there was extensive mis-splicing in SF3B1K700E ES cells compared to WT ES cells. In addition, expression of ~1000 genes were upregulated in the SF3B1K700E cells. In my project, I, together with a postdoc and my superivor Dr. Reed, used a combination of literature searches and Gene Cards to identify the functions of all of the upregulated genes as well as their disease associations. Remarkably, we found that numerous master regulators of hematopoiesis were upregulated. For example, the TAL1 gene, which is required for hematopoiesis, was 140-fold upregulated, and PAX5, which is required for the B cell lineage, was 20-fold upregulated. In addition, we found that most of these transcription factors are associated with hematological cancers, which is the type of cancer associated with the SF3B1 mutation. Moreover, we found that 22% of the upregulated genes are involved in downstream events in hematopoiesis. In additional studies, we validated the upregulation of the blood transcription factors by RT-PCR and qPCR. We also found that the K700E mutation caused lability of the U2 snRNP, which may explain why mis-splicing occurs. I then carried out western blots to determine whether the K700E mutation affected the expression level of U2 snRNP components and other splicing factors in the spliceosome, but did not see any apparent effect. Further studies are needed to determine how the K700E mutation causes U2 snRNP lability. Conclusions Our study reveals that SF3B1K700E causes extensive mis-splicing in ES cells, similar to what has been reported in blood cancers carrying this mutation. Moreover, our data are the first to show upregulation of important hematopoietic transcription factors and downstream blood genes, which may explain the blood cell type specificity of a general splicing factor in cancer. en_US
dc.format.mimetype application/pdf en_US
dc.language English en_US
dc.language.iso eng en_US
dc.publisher Brandeis University en_US
dc.rights Copyright by Jingyuan Huang 2018. en_US
dc.title Splicing Factor SF3B1: Putative Chronic Lymphocytic Leukemia Driver Gene en_US
dc.type Thesis en_US
dc.contributor.department Undergraduate Program in Biology en_US
dc.degree.name BS en_US
dc.degree.level Bachelors en_US
dc.degree.discipline Biology en_US
dc.degree.grantor Brandeis University, College of Arts and Sciences en_US


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