modulated by histone modifications modifications are catalyzed by - - PowerPoint PPT Presentation

• „Nuclear inheritance which is not based on

differences in the DNA sequence“ (Holliday, 1994)

• chromatin structure influences gene expression
• modulated by histone modifications
• modifications are catalyzed by enzymes
• alterations influence cellular differentiation

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• chromatin structure influences gene expression
• modulated by histone modifications
• modifications are catalyzed by enzymes
• alterations influence cellular differentiation

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• chromatin structure influences gene expression
• modulated by histone modifications
• modifications are catalyzed by enzymes
• alterations influence cellular differentiation

• sequence specific recruitment of a histone

modifying enzyme to influence gene expression

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DNA- binding histone modifying

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• alters as a complex chromatin structure by

deacetylation  regulates gene expression

• could be artificially recruited in S. cerevisiae
• homologous to a human deacetylase HDAC1
• recuitment sequence specific in the cell
• hardly any off-target activity

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• transcription activator-like effector
• originally found to induce gene expression by

binding specific promotor regions ( AvrBs3 in Xanthomonas)

• two constant domains and one variable domain
• binds to specific target sequences
• „TALE code“ enabling creation of custom TALEs

(Boch et al., 2009)

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• created TALEs binding specific purposefully

targeted DNA sequences in the promoter

• recruitment of enzymes to specific sequences to

alter chromatin structure and thereby gene expression

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• same thing with demethylases and enhancers…

• SpyTag – SpyCatcher
• form a covalent bond
• avoiding fusion proteins

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Veggiani et al., Superglue from bacteria: unbreakable bridges for protein nanotechnology, Cell, 2014

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Advantages

• new approach beyond the possibilities of RNAi 

pre-transcriptional

• specific targeting
• modular system enables targeting of several genes

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• eYFP
• Rpd3
• TALE
• two TALES binding within promoter
• one binding an artificial sequence found via brute

force method confirmed by BLAST

CMV promoter eYFP cds Adh1 terminator Glühgü seq rpd3 cds SpyCatcher Adh1 terminator rpd3 promoter TALE cds SpyTag Adh1 terminator rpd3 promoter

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K699 (negative) YIYFP (positive)

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YIYFP with R YIYFP with T1 K699 (negative) YIYFP (positive)

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YIYFP with R YIYFP with T1 K699 (negative) YIYFP (positive) YIYFP with RT2 YIYFP with RT1 YIYFP with RT3

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n = 2

• CMV-promoter is active in S. cerevisiae (K699)
• SCATTER appears to induce gene knockdown

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Further testing

• protein detection
• state of acetylation
• change of chromatin state
• insert eYFP into different gene loci
• other chromatin-modifying enzymes
• test in cell culture and plants
• insert proteins directly

Application

• cancer: downregulation of oncogenes

• board game in collaboration

with iGEM Marburg

• investigation of

evolutionary processes and the construction of a cell

• cell components are gained

during the game

• mutations can be acquired
• compared to medieval

castle  age 10+

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• modular system of DNA-binding domain and

chromatin-modifying enzyme linked by SpyTag- SpyCatcher

• insertion of eYFP and regulatory constructs into yeast
• our aim: downregulation of target gene eYFP
• result: knockdown of eYFP transcription
• human practice: board game for children

• our instructors: Prof. Dr. Uwe Sonnewald, PhD

student Marlene Pröschel

• our advisor: Prof. Dr. Christian Koch
• division of Molecular Plant Physiology (clsm)
• host labs
• sponsors

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