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Epigenetic Reprogramming at the Th2 Locus

Rao Venkata, Lakshmi Prakruthi

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2018, MS, University of Cincinnati, Medicine: Immunology.
Background and Aims: Atopic diseases are characterized by increased proliferation of T cells and an increase in levels of Th2 cytokines. Memory T cells are capable of producing cytokines much faster than naive T cells, upon exposure to antigen. This ability of memory T cells correlates to the presence of positive histone marks at the cytokine gene loci. This suggests that histone modifications play a major role in the poising of genes for transcriptional activity and hence, they can influence the expression levels of genes. We hypothesize that by targeting the histone modifications at the Th2 cytokine locus, we can regulate the expression of the cytokines that play a role in atopic disease progression. Methods: We have designed plasmids with TALE (transcription Activator-like Effector) DNA binding domains coupled with LSD1 and CRISPR/dCas9 domain with LSD1, G9a and SUV39h1. These plasmids are programmed to target the IE enhancer element in the IL4 locus. LSD1 is a demethylase that removes positive histone marks by demethylating H3K4me1/2. G9a and SUV39h1 are histone methyltransferases that deposit the repressive histone marks, H3K9me1/2/3. The plasmid constructs were delivered into D10G4.1 cells (a mouse Th2 cell line) using either electroporation or viral transduction and were then sorted based on GFP expression. Results: Upon successfully transfecting the TALE-LSD1 plasmid into D10G4.1 cells, we observe that the cells express the protein through western blot. Upon activation of the cells using PMA/Ionomycin, we expected to observe the decrease of IL4 expression. However, we did not see any significant decrease in IL4 expression. Hence, we considered cloning our enzymes into a CRISPR/dCas9 system and use a guide RNA to target the same IE enhancer at IL4 locus. We are currently in the process of optimizing the delivery of these plasmids into D10G4.1 cells. Conclusions: Our preliminary studies show that the TALE domain successfully fused to the enzyme, LSD1. We also showed that the protein is transcribed. However, the construct was not successful in modifying the expression of IL4 as we expected. We are further investigating possible alternatives to deliver LSD1 and other enzymes into cells to optimize efficiency and to see if there is a causal relationship between the presence of histone marks and gene expression.
Jonathan Katz, Ph.D. (Committee Chair)
Artem Barski, Ph.D. (Committee Member)
Ian Lewkowich, Ph.D. (Committee Member)
81 p.

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Citations

  • Rao Venkata, L. P. (2018). Epigenetic Reprogramming at the Th2 Locus [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543838686940608

    APA Style (7th edition)

  • Rao Venkata, Lakshmi Prakruthi. Epigenetic Reprogramming at the Th2 Locus. 2018. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543838686940608.

    MLA Style (8th edition)

  • Rao Venkata, Lakshmi Prakruthi. "Epigenetic Reprogramming at the Th2 Locus." Master's thesis, University of Cincinnati, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543838686940608

    Chicago Manual of Style (17th edition)