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How Do Biologists Study Gene Regulatory Networks? Journal Club 01/27/05, presented by Hong Lan

How Do Biologists Study Gene Regulatory Networks? Journal Club 01/27/05, presented by Hong Lan. Introduction to technologies Introduction to HNF4 a Go over the Richard Young Paper Some thoughts Brief review of Ingenuity Pathway Analysis. Biological Questions of DNA:Protein Interaction.

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How Do Biologists Study Gene Regulatory Networks? Journal Club 01/27/05, presented by Hong Lan

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  1. How Do Biologists Study Gene Regulatory Networks?Journal Club 01/27/05, presented by Hong Lan • Introduction to technologies • Introduction to HNF4a • Go over the Richard Young Paper • Some thoughts • Brief review of Ingenuity Pathway Analysis

  2. Biological Questions of DNA:Protein Interaction • Have a DNA fragment, and want to identify if it is protein binding (transcription factor), or what the binding sequence/motif is • Gel Mobility shift assay, or Electrophoretic Mobility Shift Assay (EMSA) • DNA footprinting • Have a protein (transcription factor), and want to know what DNA sequences the protein binds • Chromatin immunoprecipitation, or ChIP • Chromoatin immunoprecipitation combined with promoter microarrays (ChIP-on-chip)

  3. Gel Shift Assay, or Electrophoretic Mobility Shift Assay (EMSA) If you have a fragment of DNA sequence, and want to know if it binds proteins, use gel shift assay www.pierce.com

  4. Chromatin immunoprecipitation (ChIP): If a DNA sequence binds to a transcription factor DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. Isolate the chromatin. Shear DNA along with bound proteins into small fragments. Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody http://www.bio.brandeis.edu/haberlab/jehsite/chip.html

  5. HNF4a Network In pancreatic b-cells In liver HNF3b = Foxa2 Kulkarni and Kahn, Science 303: 1311-1313, 2004

  6. DNA-Binding Domain of Transcription Factors • Helix-loop-helix (homeodomain) • Zn-Fingers (at least two) • Basic-Lucine Zipper (works as dimers) http://homepages.strath.ac.uk/~dfs97113

  7. The MODY Genes Chromosome (Pdx-1) http://techunix.technion.ac.il/~rimma/mainpage.html

  8. Mapping Liver Gene Expression in (B6  BTBR)F2-ob/ob Mice

  9. An Example of a trans-regulation

  10. Candidate Genes in the Chromosome 2 QTL Region

  11. Genome-scale location analysis of HNF regulators in human tissues

  12. Technical Notes • A minimum of 30,000 viable islet equivalents (approximately 2 x 107 beta cells) were fixed and handled for HNF4a, HNF6, and RNA polymerase II. • HNF1a ChIP required significantly more material, typically 80,000 islets, to produce results with somewhat lower enrichment ratios than the results obtained with hepatocytes. • These results suggest that empirical rate of false positives is at most 16%.

  13. HNF4a results: antibody specificity or errors? • Essentially identical results were obtained with two different antibodies that recognize different portions of HNF4a. • Western blots showed that the HNF4a antibodies are highly specific. • They verified binding at more than 50 randomly selected targets of HNF4a in hepatocytes by conventional gene-specific ChIP. • When antibodies against HNF4a were used for ChIP in control experiments with Jurkat, U937, and BJT cells, no more than 17 promoters were identified. • When preimmune antibodies were used in hepatocytes, the number of targets identified was within the noise. • The set of promoters bound by HNF4a was largely a subset of those bound by RNA polymerase II.

  14. The PANDORA Tool • Protein ANnotation Diagram ORiented Analysis  v3.1.(http://www.pandora.cs.huji.ac.il/) • Developed by Noam Kaplan, Dr. Avishay Vaaknin and Prof. Michal Linial • Kaplan N, Vaaknin A and Linial M. (2003). Nucleic Acids Research 31 5617-5626. • hierarchical clustering of the SwissProt database

  15. Construction of the graph

  16. Test PANDORA with D2Mit263 List Input: 92 SwissProt IDs 52 acceptable by PANDORA This group of eukaryotic integral membrane proteins are evolutionary related, but exact function has not yet clearly been established.

  17. HNF1a, HNF6, and HNF4a are at the center of tissue-specific transcriptional regulatory networks • SHP = Src homology 2 domain phosphatase • GABPA = GA binding protein transcription factor, alpha • NR2C2 = nuclear receptor subfamily 2, group C, member 2 • RAMP = RA-regulated nuclear matrix-associated protein

  18. Examples of regulatory network motifs in hepatocytes • PCK1 = phosphoenolpyruvate carboxykinase • RARb = retinoic acid receptor, beta • HGFAC = hepatocyte growth factor activator • HNMT = histamine N-methyltransferase • NR1D1 = nuclear receptor subfamily 1D1

  19. A Proposal to Keith and Mark • Identify motif in promoters of HNF targets created by ChIP-on-chip (up to 16% false positives ) • HNF1a 293 genes • HNF6 314 genes • HNF4a 2323 genes • Identify true positives and false positives using Keith’s motif-finding program • Re-verify these genes experimentally using ChIP (back to Richard Young?) • Validation of the motif-finding program

  20. Zhang et al, J.Biol. 2004

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