Identification of Snail1 as a repressor of the humantelomerase reverse transcriptase (hTERT) gene in response to transforming growth factorβ(TGF-β)

다국어 초록 (Multilingual Abstract)

Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is the primary determinant for telomerase enzyme activity, which has been associated cellular immortality. The expression of hTERT gene has been reported to regulate by various extracellular(external) stimuli and is aberrantly up-regulated in more than 90% of cancers. Here we show that hTERT gene expression was repressed in response to transforming growth factor-β(TGF-β) by a mechanism dependent on transcription repressor Snail. TGF-βdown-regulated the level of c-Myc and activated the expression of Snail gene. In addition, ectopic expression of Snail strongly inhibited the hTERT promoter activity; however, co-expression of c-Myc abrogated Snail-mediated down-regulation of hTERT promoter activity. Chromatin immunoprecipitation (ChIP) analysis revealed that TGF-βdecreased occupancy of c-Myc whereas dramatically increased the recruitment of Snail onto E-box motifs of hTERT promoter, thereby repressing the expression of hTERT. Our findings suggest that a dynamic alteration in hTERT promoter occupancy by c-Myc and Snail is a mechanistic basis for TGF-β-mediated regulation of hTERT.

다국어 초록 (Multilingual Abstract)

Protein kinase Calpha(PKCalpha) phosphorylates the Ser33/37/Thr41 residues of β-catenin, which lacks a typical PKCalpha canonical sequence, but little is known about its underlying mechanism. Here we showed thatSer33/Ser37/Thr41 of β-catenin fragments encompassing the armadillo repeats 1-5 (β-catenin1-781, β-catenin1-682, and β-catenin1-422) are phosphorylated by PKCalpha whereas β-catenin1-138 lacking these repeats is not phosphorylated. Binding-site analysis revealed that PKCalpha directly interacts withβ-catenin through the sites on the armadillo repeats 1-5. In addition, axin fragments (365-500), which interacts with β-catenin through armadillo repeats 3-5, disrupted PKCalpha/β-catenin association and inhibitedβ-catenin phosphorylation by PKCalpha. In HEK293 cells, the levels of β-catenin1-781 and β-catenin1-422 were decreased whereas the amount of β-catenin1-138 was unchanged by pharmacological stimulation of PKCalpha. Our results suggest that the association of PKCalpha with the armadillo repeats of β-catenin placed the Ser33/37/Thr41 residues of β-catenin in close proximity to PKCalpha, thereby facilitating PKCalpha-mediatedβ-catenin phosphory lation.

목차 (Table of Contents)

• Chapter 1
• List of Figure
• Abbreviations
• Abstract
• 1.Introduction
• 1. 1. Human telomerase reverse transcriptase(hTERT)
• 1. 2. Transforming growth factor-β(TGF-β)
• 2. Meterials and Methods
• 2. 1. Cell Culture and reagents
• 2. 2. Plasmids, transfection and luciferase assay
• 2. 3. Western blot analysis
• 2. 4. RNA extraction and semi-quantitative RT-PCR
• 2. 5. Chromatin immunoprecipitation (ChIP) assays
• 3. Results
• 3. 1. TGF-βrepresses hTERT gene expression in immortalized human keratinocytes
• 3. 2. TGF-β down-regulates c-Myc and up-regulation Snail in immortalized human Keratinocyte
• 3. 3. Snail represses hTERT promoter activity through binding to E-box sequences
• 3. 4. A dynamic alteration in hTERT promoter occupancy by c-Myc and Snail in responseto TGF-β
• 4. Discussion
• 5. Reference
• 6. 국문요약
• Chapter 2
• List of Figure
• Abbreviations
• Abstract
• 1. Introduction
• 1. 1. Protein kinaseCa(PKCa)
• 1. 2. β-Catenin
• 2. Meterials and Methods
• 2. 1. Cell culture, transfection, and chemical
• 2. 2. Plasmid and recombinant proteins
• 2. 3. In vitro kinase assay
• 2. 4.GST pull-down assay
• 2. 5. Western blot analysis
• 2. 6. Statistical analysis
• 3. Results
• 3. 1. Armadillo repeats are required for PKCa-mediatedβ-catenin Phosphorylation
• 3. 2. PKCa interacts with armadillo repeats of β-catenin
• 3. 3. Axin disrupts PKCa/β-catenin interaction
• 3. 4. Armadillo repeats are required for PKCa-mediated β-catenin degradation
• 4. Discussion
• 5. Reference
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