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Master
gene
regulators
Mapping the New Frontier: Human Regulatory Circuitry
Gene regulation
- control of cell state
- master gene regulators
Mapping gene regulatory circuitry
Importance of maps
- human biology
- future medicine
Gene Regulation is Key to Human Development
Master gene regulators establish gene expression program
Cell state is a product of that gene expression program
Master Gene Regulators are Generally
Transcription Factors
Transcription Factor
Binds specific DNA sequence
in control region of gene
DNA
TFIIH
Transcription
RNA
Translation
Protein
Master Gene Regulators are Known
for Many Cell Types
Embryonic stem cells
OCT4, NANOG, SOX2
PcG
Brain and Spinal Cord
SOX1-18, PAX6, OCT6,
NGN, NEUROD
Cerebrum
Cerebellum
Ganglia & nerves
Circulatory System
Myocardin, GATA4, TBX5,
NKX2.5, MEF2, HAND
Heart
Vascular system
Digestive System
HNF1, HNF4, HNF6, CBP,
PGC1, FOXA, PDX1
Esophagus
Stomach
Intestines
Liver
Pancreas
Urinary System
HNF1B, HNF4, CDX, FTF
C/EBP, FOXA, GATA3
Kidney
Urinary tract
Respiratory System
HNF-3, NKX2.1 and GATA6
Airways
Lungs
Reproductive Organs
ERα, SF1, DAX1, C/EBPβ
Ovary
Uterus
Breast
Testis
Skeletal and Muscular
MYOD, MEF2, MRF4, MYF5
Myogenin
Bone
Muscle
Cartilage
Hematopoietic System
TAL1, LMO1, LMO2, E2A,
XBP1, AML1, MLL1, PU.1,
C/EBPα
Bone marrow
Blood
Embryonic Liver
Immune System
STAT1, STAT3 STAT5,
IRF1, IRF3, IRF5,
NFκB family, FoxP3
Thymus
Spleen
Lymph nodes
Sensory Organs
SOX1-18, OCT6, PAX3,
PAX6, NGN, SKIN1
Eye
Ear
Olfactory
Skin
Tongue
Master Gene Regulators Can Reprogram Cell State
Oct4, Sox2,
KLF4, cMyc
iPS cells
MyoD
Takahashi Cell (2006)
Maherali CellSC (2007)
Okita Nature (2007)
Wernig Nature (2007)
Davis Cell (1987)
Weintraub PNAS (1989)
Weintraub Science (1991)
Fibroblasts
Skeletal muscle
Neurod1
Kojima Nature (2003)
CEBPβ
Shen NatureCB (2003)
Hepatocytes
Pancreatic-like cells
Mutations in Master Regulators Cause Major Diseases
Diabetes
Hnf1α, Hnf1β, Hnf4α, Pdx1, NeuroD1
Coronary Heart Disease and Hypertension
NKX2.5, MEF2, NR3C2, GCCR
Cancer
TAL1, LMO1, LMO2, E2A, AML1, p53, PLZF, PML, Rb, WT1
Developmental Defects
GATA1, VDR, CRX, CBP, MeCP2
Autoimmunity and Immunological Disorders
FoxP3, RFX5, WHN
Central Nervous System Disorders
PAX3, EGR-1, EGR-2, OCT6, SOX family
Obesity
PPARγ, SIM1
Gene Regulation I: Transcription Factors Control
RNA Polymerase, Which Produces RNA Transcripts
H3K4 methyase
HATs
Active Gene
H3K36 methylase
RNA Polymerase
Transcription factors
Initiation
Elongation
Inactive Gene
Repressors
We understand how only a few genes are regulated by transcription factors
H3K9 methylase
PcG
HDACs
Human Gene Regulation
Human genome
• 25,000 genes
• 1500 genes encode transcription factors
• 3 times more DNA devoted to gene control
than to protein-coding
• regulation is understood for only a few genes
Transcriptional Regulatory Circuitry:
How 1500 Transcription Factors Regulate 25,000 Genes
Transcription Factors
The mapping of transcriptional regulatory
circuitry will:
• reveal how cell state, differentiation and
response to stimuli are controlled
Target
Genes
• suggest new strategies to combat
disease
Mapping the New Frontier: Human Regulatory Circuitry
Summary so far:
Master regulators (transcription factors) establish gene expression program
Master regulators are powerful - they can reprogram gene expression
Defects in master regulators can cause disease
Basic principles of gene regulation established
How 1500 transcription factors regulate 25,000 genes is not known
Map of transcriptional regulatory circuitry should reveal how cell state is
controlled and suggest new strategies to combat disease
Mapping the New Frontier: Human Regulatory Circuitry
Gene regulation
- control of cell state
- master gene regulators
Mapping gene regulatory circuitry
Importance of maps
- human biology
- future medicine
Where Do We Start?
ES Cells Can Differentiate into Most Any Cell Type
Founding Circuitry of Human Development
ES Cells
Technology: Mapping Transcription Factor - DNA
Interactions Genome-wide
Crosslink
protein to DNA
binding sites in
living cells
Harvest cells
and fragment
DNA
Enrich for
protein-bound
DNA fragments
with antibodies
Differentially
label the two
pools of DNA
Hybridize to
microarray
Obtain
binding ratio
Identification of All Genes Regulated by a
Transcription Factor
Transcription Factor 1
Gene A
TFIIH
Gene B
Gene C
Gene D
This can be repeated for each transcription factor
to build a map showing all target genes for all transcription factors
Map of Core Regulatory Circuitry of Human Embryonic
Stem Cells
Oct4, Sox2 and Nanog:
• Master regulators of
embryonic stem cells
• Regulate their own genes
• Regulate regulators
of development
Mapping is a Big Problem: Master Regulators
Change with Each Cell State
Challenge is to map all regulators in all cell states
NIH is funding major new efforts to do this
This is Great, But We Have Noticed a Problem…
Transcription factors typically recognize
6 base pair DNA sequence
A 6 base pair DNA sequence should
occur every 4n base pairs (46)
So we should observe one binding
event every ~4000bp
Actually see much lower frequency:
perhaps one binding event every
~1,000,000bp
Why?
The Genome Packaging Problem
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
Most human cells are roughly 20 um across,
with nuclei about 10 um (10-5 m)
Human Genome Packaging
Nucleosome Structure
Nat Rev Cancer 1:194 (2001)
Chromatin
Hamkalo and Foe
in Essential Cell Biology (1998)
Human Chromosomal Karyotype
(NIH)
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
Quiz: What is the total length of DNA fiber in your body?
One cell:
Each base pair in DNA double helix is 3.4 x 10-10 m
Human genome is 6 billion base pairs
(3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m
DNA fiber length in Human:
We have about ~10 trillion cells
1013 cells x 2.4m = 2.4 x 1013 m
Is this:
1) Distance around earth?
2) Distance to moon?
3) Distance to sun?
4) Diameter of solar system?
4 x 107 m
4 x 108 m
1.5 x 1011 m
1.6 - 2.4 x 1013 m
So… Transcription Factors Have to Overcome
Constraints Due to Human Genome Packaging
Nucleosome Structure
Nat Rev Cancer 1:194 (2001)
Chromatin
Hamkalo and Foe
in Essential Cell Biology (1998)
Human Chromosomal Karyotype
(NIH)
Gene Regulation II: Transcription Factors and
Chromatin Regulators Collaborate
H3K4 methyase
HATs
H3K36 methylase
Active Gene
Transcription factors
Initiation
Elongation
Inactive Gene
Repressor
H3K9 methylase
PcG
HDACs
Regulatory Circuitry of Human Cells
Environmental Stimulus
Signal Transduction
Signal transduction
Transcription
Factors
Transcription factors
Chromatin regulators
miRNA regulation
Chromatin
Regulators
miRNA
Target Genes
Mapping the New Frontier: Human Regulatory Circuitry
Gene regulation
- control of cell state
- master gene regulators
Mapping gene regulatory circuitry
Importance of maps
- human biology
- future medicine
Maps Are Key to Development of Therapeutics But
Only Protein Pathways Have Been Mapped
Signaling Pathways
Regenerative Medicine: Reprogramming
With Maps and Without Viral Vectors
• Small molecule drugs
• Artificial transcription factors
• RNAi