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Matthews Lecture 2_final 140115

E 5 pregnane 320 dione large species variaon in ligand

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Unformatted text preview: etec/on of postransla/onal modifica/ons – histone modifica/ons, DNA modifica/ons •  /me resolu/on is minutes •  possible ar/facts due to poten/ally biased "crosslinkability"; epitope may be masked •  can be applied to most cell systems •  study protein:chroma/n interac/on on endogenous genes in intact cells 38 Coac/vators Cyclical recruitment observed by ChIP Assays ER target genes Coac/vators Corepressors Chip- chip Genome-wide coverage Genome Tiled Probes 35 bp 10 bp 7.5kb 2.45kb Probe coverage Tiled Probes on array DNA from ChIP Enriched Region Computa?onal Analysis map the exact loca?on of protein:chroma?n binding Iden?fy direct gene targets 41 ChIP- Sequencing (ChIP- Seq) 42 ChIP- chIP iden/fies distal DNA sites bound by NRs ERs regulate genes by binding to far distal enhancers elements 5` or 3` from start site DNA sequence is not sufficient for NR binding – more complex regula/on Xenobio/c Sensing Nuclear Receptors Pregnane X Receptor (PXR), NR1I2 Cons/tu/ve Androstane Receptor (CAR), NR1I3 •  regulate phase I, II and transporters 45 Pregnane X receptor (PXR), NR1I2 •  expressed in liver, small intes/ne and colon •  regulates expression of CYP3A4 – involved in metabolism of many (50%) prescrip/on drugs and endogenous steroids. •  also regulates the expression of other CYP450s, Phase II and drug transporters (mul/- drug resistant transporter) •  Binds and mediates the biological func/ons of pregnane steroids C21 steroid ring, i.e. 5β- pregnane- 3,20- dione •  Large species varia/on in ligand binding/ac/va/on i.e. mouse vs human •  Promiscuous receptor that binds a large number of xenobio/cs •  xenobio/c sensor receptor 46 PXR Mechanism of Ac/on ligand cytosol Ligand binding Nuclear translocation Hsp90 CCRP nucleus PXR PXR RXR Recruitment of Coregulators -activators - repressors Heterodimerization & DNA Binding PXR RXR XREM PXR RXR RNA...
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