INHIBITION OF GENE TRANSCRIPTION USING XANTHOMONAS DERIVED TALE PROTEINS
Şeyda Şaziye Temiz
Biological Sciences and Bioengineering, M.Sc. Thesis, 2013
Assoc. Prof. Batu Erman (Thesis Supervisor), Prof. Dr. Selim Çetiner, Prof. Dr. Uğur Sezerman, Asst. Prof. Erdal Toprak, Prof. Dr. Canan Atılgan
Date &Time: July,30th, 2013 - 14.00
Place: FENS L067
Keywords: Transcription activator-like effector, TALEN, Golden Gate cloning, site directed mutagenesis, competitive binding, IL-7R alpha, NF-kappa B
Transcription activator like effector (TALE) proteins derived from bacterial plant pathogens of the Xanthomonas genus have recently emerged as powerful molecules for genome modification. Naturally occurring TALE proteins have a modular DNA binding domain and obey a simple DNA recognition code. This permits easy assembly of artificial TALE proteins of desired specificity and functionality. In the first part of this study, we aimed to mutate several transcription factor binding sites in the IL-7R alpha gene locus by generating TALE nucleases (TALEN) targeting these sites. We designed, constructed and expressed 3 pairs of TALENs targeting the NF-kappaB, Notch, and glucocorticoid receptor (GR) transcription factor binding sites in the IL7R gene enhancer. We generated cell lines with insertion and deletion (INDEL) mutations induced by these TALENs at these target sites and determined the effects of these mutations on IL-7R alpha gene expression. We assessed TALEN induced mutations in murine Neuro-2a and RLM11 cell lines by a modified restriction fragment length polymorphism (RFLP) assay and by DNA sequencing. We demonstrate that mutations induced by TALEN pairs targeting the IL7R enhancer NF-kappaB site reduce IL-7R alpha gene expression, while mutations in the Notch binding site did not change IL-7R expression. In the second part of this study, we aimed to inhibit the transcription activation function of the NF-kB protein by competitive binding of target sites with TALE proteins. We generated plasmids encoding TALE-dsRed fusion proteins that were designed to bind NF-kB binding sites in a reporter cell line. TNF-alpha treatment of this cell line results in NF-kB nuclear translocation and a resultant increase in GFP fluorescence. TALE-dsRed fusion proteins with increasing numbers of DNA binding repeats (12-17) competed for NF-kB binding to this reporter and resulted in reduced GFP expression upon TNF treatment. Our experiments demonstrate that TALENs and TALEs can efficiently inhibit gene transcription by mutating transcription factor binding sites or by competition with transcription factor binding.