Matthews Lecture 2_final 140115

excellent tool for exploring mechanisms of action

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Unformatted text preview:   sequence whole genomes comparative/evolutionary analysis •  Personalized medicine/toxicology •  Template preparation •  Solid phase (illumina/solexa) 35 bp reads •  Clonally amplified by emulsion PCR (454) 100-200 bp 12 Illumina sequencing by synthesis approach Gene Knockdown siRNA RNAi (siRNA or shRNA) – mediated knockdown of protein of interest. -  Excellent tool for exploring mechanisms of action -  Verifying target genes 14 SiRNA-mediated Knockdown Nuclear Receptor siNR#1 siNR#2 NR However, this is knockdown not knockout usually 70-80% 15 Estrogen Receptors (ERs) •  ERs - mediators of the bioactivity of estrogens (E2) •  implicated in breast, ovarian and prostate cancers •  most important therapeutic target for breast cancer •  activated by many xenobiotic and dietary compounds •  17β-estradiol > estrone ≥ estriol C D A B •  Nuclear receptors, two subtypes; ERα (NR3A1) and ERβ (NR3A2) •  Two transactivation domains; AF-1 and AF-2 16 ERα Signalling Estradiol CH3 OH HO 17β- oestradiol (E2) cytosol Ligand binding nucleus CoR ep ER CBP Homodimerization & DNA Binding SRC p300 TIF2 TFF1 GREB1 ER ER Proteasomal ER degradation coac/vators ERE TBP RNA Pol-II 17 Many different isoforms of ERα and ERβ AF- 1 hERα hERβ1 A/B 20% DNA binding dimerisa?on C D 95% 30% Ligand binding dimerisa?on E 60% AF- 2 F 30% Different isoforms hERα - 46 hERβ1short (hERβcx) hERβ2 hERβ ∆ 5 hERβ4 hERβ5 18 Forma/on of homodimers and binds EREs (estrogen response elements) ERα/β ERα/β A G G T C A n n n T G A C C t IR- 3 or palindromic sequence with a 3 bp spacer 19 Direct DNA binding of ER homo- and heterodimers α α β β ERE ERE ERE ERα homodimers β α ERβ homodimers ERα/ERβ heterodimers β α ? α β β α ERα ERβ ERα/ERβ co- expression Tethering to other transcrip/on factors 20 NR- mediated mechanism of ac/on is complex A NR NR NRE B C NR NR NRE Increase in gene expression TATA Transcrip/onal repression TATA Tethering or Crosstalk with other receptor pathways NR or ac/va/on NR or ac/...
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This document was uploaded on 03/27/2014.

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