Download 20000 human genes - Howell Foundation

2/22/2016
The human genome sequence sets off a genomics revolution
Understanding the genome
What’s Next?
10,000 experiments at a time
Sonia Sharma, Ph.D
Assistant Professor, Division of Cellular Biology
20,000 human genes
Proteins
Director, The Functional Genomics Center
5,000 Disease-related Proteins
260 Drug Targets
Citations
4
1
2/22/2016
Proteins
??? Disease-related Proteins
??? Drug Targets
5,000 Disease-related Proteins
260 Drug Targets
Why are we looking under
the lamp post?
Citations
5
Fosters better communication
6
Fosters better communication
Creates common interests
Creates common interests
Promotes resource sharing
Promotes resource sharing
colors the results…creates a bias.
7
8
2
2/22/2016
Bias limits our ability to discover
Our Lab’s Mission
To recognize the potential of the human genome project
through large-scale, unbiased experiments.
To apply these approaches towards understanding the
immune system and immune-related diseases such as
lupus, rheumatoid arthritis and type I diabetes.
9
RNAi and CRISPR work through a process of gene elimination
RNAi and CRISPR work through a process of gene elimination
3
2/22/2016
Robots enable us to perform thousands of experiments at once
??? Disease-related Proteins
??? Drug Targets
Proteins
Large robotic liquid handlers mix cells with RNAi
thousands of times with speed and precision
5,000 Disease-related Proteins
260 Drug Targets
Citations
1
4
RNAi works through a process of elimination
20,000 genes
(whole genome)
1. Deliver RNAi to cells (40,000 experiments)
2. Wait for gene depletion (3 days)
3. Analyze cellular immune functions
That’s the technology. What about the biology?
Identified 350 new genes
associated with immune
function
4
2/22/2016
Women are generally more susceptible to auto-immune disease
than men
What accounts for the bias?
1) Hormones: in general, testosterone is immuno-suppressive while
estrogen is immuno-active.
What accounts for the bias?
1) Hormones: in general, testosterone is immuno-suppressive while
estrogen is immuno-active or inflammatory.
2) Incomplete X chromosome deactivation: females carry two
copies of X chromosomes, which contain some prominent
immuno-regulatory genes.
5
2/22/2016
What accounts for the bias?
What accounts for the bias?
1) Hormones: in general, testosterone is immuno-suppressive while
estrogen is immuno-active or inflammatory.
2) Incomplete X chromosome deactivation: females carry two copies
of X chromosomes, which contain some prominent immunoregulatory genes.
3) Microchimerism: during pregnancy and birth, there is significant
exchange of cells, which may activate the Mother’s immune
system.
1) Hormones: in general, testosterone is immuno-suppressive while
estrogen is immuno-active or inflammatory.
2) Incomplete X chromosome deactivation: females carry two copies
of X chromosomes, which contain some prominent immunoregulatory genes.
3) Microchimerism: during pregnancy and birth, there is significant
exchange of cells, which may activate the Mother’s immune
system.
4) Immune system: Women tend to have higher immune reactivity,
and higher numbers of certain immune cell populations.
What accounts for the bias?
1) Hormones: in general, testosterone is immuno-suppressive while
estrogen is immuno-active or inflammatory.
2) Incomplete X chromosome deactivation: females carry two copies
of X chromosomes, which contain some prominent immunoregulatory genes.
3) Microchimerism: during pregnancy and birth, there is significant
exchange of cells, which may activate the Mother’s immune
system.
4) Immune system: Women tend to have higher immune reactivity,
and higher numbers of certain immune cell populations.
5) Environmental factors: UV/sun exposure, specific chemicals
(cosmetics, etc).
Barriers
Innate Immunity
Adaptive Immunity
HUMORAL
intact
skin
C-reactive
protein
HUMORAL
complement
antibodies
histamine
mucus &
ciliary
clearance
neutrophil
mast cell
low pH
stomach
T cell
NK cell
macrophage
enzymes in
tears &
saliva
B cell
dendritic cell
CELLULAR
CELLULAR
2
4
6
2/22/2016
Barriers
Innate Immunity
Adaptive Immunity
HUMORAL
intact
skin
C-reactive
protein
Innate immunity: early immune response to pathogens
HUMORAL
complement
antibodies
histamine
mucus &
ciliary
clearance
INFLAMMATORY signals/receptors interlock and coordinate the
neutrophil
pieces
mast cell
dendritic cell
low pH
stomach
T cell
macrophage
B cell
NK cell
enzymes in
tears &
saliva
CELLULAR
dendritic cell
CELLULAR
2
5
Innate immunity is a common genetic signature
in many autoimmune diseases
Systemic Lupus
-IRAK1
- STAT4
-IRF5
- TLR8
-IRF7
- TREX1
Rheumatoid Arthritis
-ISG15
- RSAD2
-MX1
- STAT4
-MX2
Sjogren’s syndrome
-IRF5
-IKBa
-STAT4
Psoriasis
-CARD14
-CARM1
-DDX58
- ELMO1
- IFIH1
- KLF4
Irritable Bowel
Disease
-CARD9
-ITNL1
-NOD2
-REL
Our lab is interested in the link between
innate immunity and auto-immune diseases
Type 1 Diabetes
-IFIH1
A plethora of human genetics data
7
2/22/2016
Cells of the immune system are leukocytes
Cells of the immune system are leukocytes
The “professional” innate immune cells.
…but what about other cells?
many different cell types can trigger “early” innate immunity
…and an alternative immune cell?
Vascular endothelial cells are not passive bystanders,
they contribute to inflammation
8
2/22/2016
1) Vascular endothelial cells are
among the first to encounter innate
immune triggers in blood
Vascular endothelial cells mount robust immune
responses
2) Innate immune responses in these
cells are important for protective
immunity to sepsis, but have also
been shown to drive atherosclerosis
3) We can demonstrate remarkably
robust innate immune responses in
primary human endothelial cells
Endothelial
Vascular endothelial cells as bona fide innate immune cells?
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
Immune cells
Genome wide RNAi for innate immune responses in
vascular endothelial cells
20,000 genes
(whole genome)
1. Deliver RNAi to primary human vECs
2. Analyze innate immune responses
ALTERNATIV
E
Identified 133 genes
regulating innate immunity in primary human vECs
9
2/22/2016
Genome wide RNAi for innate immune responses in
vascular endothelial cells
20,000 genes
(whole genome)
Genome wide RNAi for innate immune responses in
vascular endothelial cells
20,000 genes
(whole genome)
1. Deliver RNAi to primary human vECs
2. Analyze innate immune responses
1. Deliver RNAi to primary human vECs
2. Analyze innate immune responses
Most genes have not been associated
with innate immunity
Many genes are functionally uncharacterized
Genome wide RNAi for innate immune responses in
vascular endothelial cells
Genome wide RNAi for innate immune responses in
vascular endothelial cells
20,000 genes
(whole genome)
20,000 genes
(whole genome)
1. Deliver RNAi to primary human vECs
2. Analyze innate immune responses
1. Deliver RNAi to primary human vECs
2. Analyze innate immune responses
Several genes are cell-type specific for
vascular endothelial cells
Many of these genes have been associated with autoimmune diseases
10
2/22/2016
Vascular endothelial cells as bona fide innate immune cells?
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
Vascular endothelial cells are bona fide innate immune cells
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
ALTERNATIV
E
Vascular endothelial cells as bona fide innate immune cells?
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
2) Important for blood-borne infections, particularly the hemorrhagic
fevers, which compromise the blood vessels: Ebola, Lassa,
Marburg, Dengue, Zika
Vascular endothelial cells are bona fide innate immune cells
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
2) Important for blood-borne infections (hemorrhagic fevers: Ebola,
Lassa, Marburg, Dengue)
3) Many auto-immune diseases involve innate immunity and vascular
inflammation:
- Cardiovascular disease/atherosclerosis
- Lupus
- Rheumatoid Arthritis
- Type 1 Diabetes
- Cancer
11
2/22/2016
Protein levels in SLE patient vs healthy serum samples
Vascular endothelial cells are bona fide innate immune cells
1) Our observations suggest that the cells lining your blood vessels
are an important new type of immune cell
2) Important for blood-borne infections (hemorrhagic fevers: Ebola,
Lassa, Marburg, Dengue)
3) Many auto-immune diseases involve innate immunity and vascular
inflammation:
- Cardiovascular disease/atherosclerosis
- Lupus
- Rheumatoid Arthritis
- Type 1 Diabetes
- Cancer
***
Acknowledgements
Funding
Mitchell Kronenberg, LJI
Vijay Pandurangan, LJI
Joel Linden, LJI
Unrestricted
Funding
Sharma Lab, LJI
Amnon Altman, LJI
Jenny Huang
Mariko Takahashi
Nikita Sharma
Rekha Dhanwani
Melody Diep
Lynn Hedrick, LJI
Karolinska Institute
Roland Nilsson
Lars Klarrskog
UCSD
The Functional Genomics Center, LJI
Mohit Jain
Christopher Oh
Yulia Kushnareva
Jeremy Day
Uppsala University
Lars Ronblomm
12