How Do Biologists Study Gene Regulatory Networks Journal Club by bzs14448


									     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
         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)
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

                                        Lane   1    2   3   4

                               EBNA Extract    -    +   +   +

                         Unlabeled EBNA DNA    -    -   +   -

                         Unlabeled Oct-1 DNA   -    -   -   +

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

              HNF4a Network
   In pancreatic b-cells                           In liver

HNF3b = Foxa2              Kulkarni and Kahn, Science 303: 1311-1313, 2004
        DNA-Binding Domain of
         Transcription Factors
• Helix-loop-helix (homeodomain)
• Zn-Fingers (at least two)
• Basic-Lucine Zipper (works as dimers)

          The MODY Genes

                                                          2: 164Mb

                                                          11: 58Mb

                                                          5: 112Mb

(Pdx-1)                                                   5: 144Mb

                                                          11: 83Mb

                                                          2: 79Mb

Mapping Liver Gene Expression in
  (B6  BTBR)F2-ob/ob Mice
An Example of a trans-regulation
     Candidate Genes in the
    Chromosome 2 QTL Region
Gene_Symbol       Gene_Name                               Location
Bfg1              Body fat QTL 1                            81.0
Bdln3             Body length QTL 3                         81.7
Bglg2             Body growth late QTL 2                    84.0
Bwfg2             Body weight and fat QTL 2                 84.0
Hnf3 b            (Foxa2 , forkhead box A2)                 84.0
Ifld2 (Nipk, Trb3 ) Induced in fatty liver dystrophy 2     86.0
Bglu1               QTL Blood glucose level 1              87.0
Hnf4 a              Hepatic nuclear factor 4 alpha         94.0
C/EBP b             CCAAT/enhancer binding protein beta    95.5
Mob5              Multigenic obesity 5                     95.5
Genome-scale location analysis of
HNF regulators in human tissues
             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%.
 HNF4a results: antibody specificity or errors?
• Essentially identical results were obtained with two
  different antibodies that recognize different portions of
• 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.
        The PANDORA Tool
• Protein ANnotation Diagram ORiented
  Analysis v3.1.(http://www.pandora.cs.huji.
• 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
Construction of the graph
    Test PANDORA with D2Mit263 List

Input: 92
SwissProt IDs
52 acceptable

Keyword                          Amo    Sensiti   Specifi          Corrected P-
            Keyword                                         valu
type                             unt    vity      city             value
Interpro:   GNS1/SUR4                                       4.72
                                 2      0.019     0.038            4.20e-4
Family      membrane protein                                e-6

     This group of eukaryotic integral membrane proteins are evolutionary
     related, but exact function has not yet clearly been established.
     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
      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
  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
Zhang et al, J.Biol. 2004

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