How Do Biologists Study Gene Regulatory Networks Journal Club 012705, presented by Hong Lan

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 DNAProtein 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
Kulkarni and Kahn, Science 303 1311-1313, 2004
HNF3b 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
Chromosome
(Pdx-1)
http//techunix.technion.ac.il/rimma/mainpage.htm
l
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
(No Transcript)
15
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

16
Construction of the graph
17
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.
18
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

19
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

20
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
  Keiths 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
22
(No Transcript)