How Do Biologists Study Gene
Regulatory Networks?
Journal Club 01/27/05, presented by Hong Lan
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Introduction to technologies
Introduction to HNF4α
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)
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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
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EBNA Extract
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Unlabeled EBNA DNA
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Unlabeled Oct-1 DNA
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www.pierce.com
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
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HNF4α Network
In pancreatic β-cells
HNF3β = Foxa2
In liver
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)
http://homepages.strath.ac.uk/~dfs97113
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The MODY Genes
Chromosome
2: 164Mb
11: 58Mb
5: 112Mb
(Pdx-1)
5: 144Mb
11: 83Mb
2: 79Mb
http://techunix.technion.ac.il/~rimma/mainpage.html
Mapping Liver Gene Expression in
(B6 × BTBR)F2-ob/ob Mice
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An Example of a trans-regulation
Candidate Genes in the
Chromosome 2 QTL Region
Gene_Symbol
Gene_Name
Body fat QTL 1
Body length QTL 3
Body growth late QTL 2
Body weight and fat QTL 2
(Foxa2 , forkhead box A2)
Bfg1
Bdln3
Bglg2
Bwfg2
Hnf3 β
Ifld2 (Nipk, Trb3 ) Induced in fatty liver dystrophy 2
QTL Blood glucose level 1
Bglu1
Hnf4 α
Hepatic nuclear factor 4 alpha
C/EBP β
CCAAT/enhancer binding protein beta
Mob5
Multigenic obesity 5
Location
81.0
81.7
84.0
84.0
84.0
86.0
87.0
94.0
95.5
95.5
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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%.
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HNF4α results: antibody specificity or errors?
• Essentially identical results were obtained with two
different antibodies that recognize different portions of
HNF4α.
• Western blots showed that the HNF4α antibodies are
highly specific.
• They verified binding at more than 50 randomly selected
targets of HNF4α in hepatocytes by conventional genespecific ChIP.
• When antibodies against HNF4α 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 HNF4α was largely a
subset of those bound by RNA polymerase II.
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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
Construction of the graph
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Test PANDORA with D2Mit263 List
Input: 92
SwissProt IDs
52 acceptable
by PANDORA
Keyword
type
Keyword
Amo
unt
Sensiti
vity
Specifi
city
Pvalu
e
Corrected Pvalue
Interpro:
Family
GNS1/SUR4
membrane protein
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0.019
0.038
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.
HNF1α, HNF6, and HNF4α are at the center of
tissue-specific transcriptional regulatory networks
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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
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Examples of regulatory network
motifs in hepatocytes
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PCK1 = phosphoenolpyruvate carboxykinase
RARβ = 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 )
– HNF1α
– HNF6
– HNF4α
293 genes
314 genes
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
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Zhang et al, J.Biol. 2004
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