Measuring Protein-DNA interactions
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- Alexandra Conley
- 5 years ago
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1 Measuring Protein-DNA interactions
2 How is Biological Complexity Achieved? Mediated by Transcription Factors (TFs) 2
3 Transcription Factors are genetic switches 3
4 Regulation of Gene Expression by Transcription Factors TF trans-acting factors TF TF TF TF activation TF TF cis-regulatory elements Gene repression 4
5 Transcription Factors are genetic switches 5 Wasserman & Sandelin Nature Reviews Genetics 2004
6 The big point is: how these TFs orchestrate the expression of thousands of genes in a genome to create such a spectrum of biological diversity remains a mystery Several methods have been developed in the last several years to study TF-DNA interactions and to understand the function of TFs. 6
7 HTP Methods for studying TF-DNA interactions Yeast-1-Hybrid (Y1H) Bacterial-1-Hybrid (B1H) Chromatin Immunoprecipitation followed by chip (ChIP- Chip) or followed by Sequencing (ChIP-Seq) 7
8 Yeast-2-Hybrid 8
9 Yeast-2-Hybrid 9
10 Y1H and B1H 2 µ ori pact II' PADH1 GAL4 AD transformation cdna library TADH1 yeast reporter strain (leu2, his3) LEU2 GAL4 activation domain (AD) vector pint1 vector library protein bait sequence Gal4p AD TATA transcription if hybrid protein interacts with bait sequence HIS3 selection gene selection for positive growth on CM -His -Leu 3-AT plates Figure Both are modifications of the Yeast-2-hybrid system. - In Y1H, we screen several TFs (prey) against a fixed promoter (bait). - In B1H, we screen milions of promoters (prey) against a fixed TF (bait) 10
11 CHIP-CHIP
12 Chromatin immunoprecipitation (ChIP) 12
13 Protein-DNA interactions by ChIP-chip 13
14 Protein-DNA interactions by ChIP-chip 14
15 Protein-DNA interactions by ChIP-chip 15
16 Protein-DNA interactions by ChIP-chip 16
17 ChIP-Chip analysis 17 Harbison C, Gordon B, et al. Nature 2004
18 ChIP-Chip analysis 18 Harbison C, Gordon B, et al. Nature 2004
19 ChIP-Chip analysis!"#$%$&'&()*+%("&, -.!"#$$%%%&!"!'#$()*(!"*(!*&$+$!"!!)$'&'!"!&%'!&%)!"!)$)%)%$!"!!#$!$'$!"!#!+#$$) /.!"%)&#$)*(!"*%)+)'%(!"!!#)+!'!"*&*!+%$'!"!!'!(&$%!"!))()##)!"!!')!*!#!")!*#$%)# 0.!")!*#$%)#!"!!')!*!#!"!))()##)!"!!'!(&$%!"*&*!+%$'!"!!#)+!'!"*%)+)'%(!"%)&#$)*( 1.!"!#!+#$$)!"!!#$!$'$!"!)$)%)%$!"!&%'!&%)!"!!)$'&'!"*(!*&$+$!"!'#$()*(!"#$$%%%& bits MXL ! 1 CAG A CACATCAGTTGT CG GT T ! 19 Harbison C, Gordon B, et al. Nature 2004
20 ChIP-Chip analysis 20 Harbison C, Gordon B, et al. Nature 2004
21 Profiled 204 transcription factors in normal conditions Another 148 TFs in 13 additional growth perturbing conditions 21
22 Predicting the DNA binding sites 22 Harbison C, Gordon B, et al. Nature 2004
23 Different promoter architectures 23 Harbison C, Gordon B, et al. Nature 2004
24 Different conditions activate TFs 24 Harbison C, Gordon B, et al. Nature 2004
25 Example genome annotations based on chipchip 25 Harbison C, Gordon B, et al. Nature 2004
26 PROBLEMS WITH CHIP-CHIP 26
27 What do you get from the IP? 27
28 1 - biased approach 28
29 2 Loss of information 29
30 2 Loss of information 30
31 Alternative! ChIP-Seq!!! - Uses the same Chromatin Immunoprecipitation step but will sequence the retrieved DNA rather than hybridising it to an array: 1. No promoter bias 2. Will pick up all binding events, even the ones that are far, far away - Still, ChIP-Seq will have its own set of problems and issues to deal with. 31
32 32
33 Histone modifications Unmethylated DNA Methylated DNA H3K9Ac H3K4me H3K9me3 H3K27me3 Emes R D, Farrell W E J Mol Endocrinol 2012;49:R19-R27 33
34 Histone modifications Type of Histone modification H3K4 H3K9 H3K14 H3K27 H3K79 H4K20 H2BK5 monomethylation activation activation activation activation activation activation di-methylation repression repression activation tri-methylation activation repression repression activation, repression repression acetylation activation activation! H3K4me3 is found in actively transcribed promoters, particularly just after the transcription start site.! H3K9me3 is found in constitutively repressed genes.! H3K27me is found in facultatively repressed genes.! H3K36me3 is found in actively transcribed gene bodies.! H3K9ac is found in actively transcribed promoters.! H3K14ac is found in actively transcribed promoters. 34