1. How Do Biologists Study Gene Regulatory Networks? Journal Club 01/27/05, presented by Hong Lan Introduction to technologies Introduction to HNF4  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) Lane 1 2 3 4 EBNA Extract - + + + Unlabeled EBNA DNA - - + - Unlabeled Oct-1 DNA - - - + 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 http://www.bio.brandeis.edu/haberlab/jehsite/chip.html 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

5. HNF4  Network In pancreatic  -cells In liver Kulkarni and Kahn, Science 303: 1311-1313, 2004 HNF3  Foxa2

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 2: 164Mb 11: 58Mb 5: 112Mb 5: 144Mb 11: 83Mb 2: 79Mb Chromosome (Pdx-1) http://techunix.technion.ac.il/~rimma/mainpage.html

8. Mapping Liver Gene Expression in (B6  BTBR)F 2 -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. HNF4  results: antibody specificity or errors? Essentially identical results were obtained with two different antibodies that recognize different portions of HNF4 . Western blots showed that the HNF4  antibodies are highly specific. They verified binding at more than 50 randomly selected targets of HNF4  in hepatocytes by conventional gene- specific ChIP. When antibodies against HNF4  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 HNF4  was largely a subset of those bound by RNA polymerase II.

15. The PANDORA Tool Protein ANnotation Diagram ORiented Analysis v3.1.(http://www.pandora.cs.huji. ac.il/)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

16. Construction of the graph

17. Test PANDORA with D2Mit263 List Keyword type Keyword Amo unt Sensiti vity Specifi city P- valu e Corrected P- value Interpro: Family GNS1/SUR4 membrane protein 20.0190.038 4.72 e-6 4.20e-4 This group of eukaryotic integral membrane proteins are evolutionary related, but exact function has not yet clearly been established. Input: 92 SwissProt IDs 52 acceptable by PANDORA

18. HNF1 , HNF6, and HNF4  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

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

20. A Proposal to Keith and Mark Identify motif in promoters of HNF targets created by ChIP-on-chip (up to 16% false positives ) –HNF1  293 genes –HNF6314 genes –HNF4  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

21. Zhang et al, J.Biol. 2004