Transcript KOX1, KAP1

Extra Slides
DATA TO SUPPORT
STATEMENTS IN
PRESENTATION
Outline of talk
General background
Introduction to the project
Experimental design
Experiments and results
Conclusions
Future experiments
Prior demonstrations
of KAP1 complexes
Proteins involved
in complex
Method
1
HP1a, KAP1
Yeast 2 Hybrid
2
KOX1, KAP1
Yeast 2 Hybrid
3
NuRD/HDAC, KAP1
4
KIP21, KAP1
5
KIP41, KAP1
6
KOX1, KAP1, HP1a
Summary
Reference
HP1a was used as a bait and
TIF1b (KAP1) was isolated
Le Douarin et al
1996
KRAB domain of KOX1 was used
as a bait and KAP1 was
isolated
Moosmann et al.
1996
Yeast 2 Hybrid
Mi2a (= NuRD/HDAC) was shown
interact with KAP-1PHD/BROMO but not Mi2b
Schultz et al.
2001
Yeast 2 Hybrid
KIP21 and KIP41 (=SETDB1) were
shown interact with KAP-1PHD/BROMO
Schultz et al.
2002
Gel Electrophoretic
Mobility Shift
assay (in vitro)
DNA+GAL4-KRAB+KAP1 ternary
complex was shown to
complex with HP1a
Ryan et al. 1999
Structure of CSD complex
Epigenetic Gene Silencing
Epigenetic effects are those changes in
gene function which are heritable
through mitosis and/or meiosis and are
not due to changes in DNA sequence.
Main types of epigenetic information:
– Cytosine DNA Methylation
– Genomic impritning
– Histone modifications
CSD interacting proteins
Chromatin based epigenetics
Controls chromosome domains and also helps
in cell differentiation
X –inactivation (XIST locus. Genomic
imprinting, parent-of origin-specific allele
silencing)
Developmental reprogramming of cell lineages
Plasticity of stem cells
Implications  human biology and disease,
including cancer and aging
Expression Vectors
Gal4 DBD
HP1a
pBridge HP1 KAP1
KAP1
TRP1
BAIT
9525 bp
MET25
HA/NLS
HP1a
(BD-HP1a)-(HA-KAP1)
Gal4
DBD
KAP1
GAL4 AD
HA
PREY
pACT2 Hela cDNA Lib
10114 bp
LEU2
Gene Library
(AD-?)
?
Gal4
AD
HeLa cell cDNA library
Source of human genes (cDNA).
Human cervical carcinoma cell line
Estimated number of Independent
Clones: 3.5 x 106
– Average insert (cDNA) size: 2.0 kb
– cDNA size range: 0.5 – 4.0 kb
HeLa cancer cells
Hybrid proteins used in this
study
BAITS
–
–
–
–
–
BD-HP1a
(BD-HP1A)-(HA-KAP1)
BD-KAP1
(BD-KAP1)-(HA-HP1)
(BD-HP1A)-(HACAF1p150)
PREYS
– AD-? (HeLa cell cDNA
library; Clontech)
–
–
–
–
–
AD-KIP21 (SETDB1)
AD-KIP41 (SETDB1)
AD-Mi2b
AD-KOX1
AD-Y2H6.2 (POGZ)
Prior demonstrations
of KAP1 & HP1 complexes
Proteins involved
in complex
Method
1
HP1a, KAP1
Yeast 2 Hybrid
2
KOX1, KAP1
Yeast 2 Hybrid
3
NuRD/HDAC, KAP1
4
KIP21, KAP1
5
KIP41, KAP1
6
7
KOX1, KAP1, HP1a
HP1a, POGZ
(Y2H6.2)
Summary
Reference
HP1a was used as a bait and
TIF1b (KAP1) was isolated
Le Douarin et al
1996
KRAB domain of KOX1 was used
as a bait and KAP1 was
isolated
Moosmann et al.
1996
Yeast 2 Hybrid
Mi2a (= NuRD/HDAC) was shown
interact with KAP-1PHD/BROMO but not Mi2b
Schultz et al.
2001
Yeast 2 Hybrid
KIP21 and KIP41 (=SETDB1) were
shown interact with KAP-1PHD/BROMO
Schultz et al.
2002
Gel Electrophoretic
Mobility Shift
assay (in vitro)
DNA+GAL4-KRAB+KAP1 ternary
complex was shown to
complex with HP1a
Co-immunoprecipitation
and Yeast Two
Hybrid genetic
screen
Novel prey (POGZ / Y2H6.2)
shown to interact with HP1a
Ryan et al. 1999
Lechner et al,
Unpublished
Histone Code
HP1 gene silencing
Strahl et al. Nature 2000
Long term Goals
Build complete HP1 network with all its
partner proteins and to build a
chromatin network database !
Map both the temporal and Spatial
formation of these complexes
Get in depth and complete
understanding of gene regulation in cells
Statement of Problem
Studies show that HP1a binds with
several proteins and presumably
performs different activities (mainly in
regulating chromatin structure and
function).
But, little is known about what
complexes exist in vivo and what
controls their formation
Assumptions
The main assumption in this experiment is
that there are regulatory factors that help in
the formation of HP1 complexes.
Yeast system does not interfere with any of
the interactions. (relatively ‘safe’; most of the
initial HP1 interactions were done in yeast
screens.)
Feasibility of this screen can be
demonstrated by showing a ternary complex
formation with positive controls.
Limitations
Yeast might inactivate or destroy the foreign
(human) proteins.
Some of these human proteins might be toxic
to yeast.
Functional homology might be present
between yeast proteins and the human
proteins.
Presence of false positives or false negatives.
Bait proteins that auto-activate cannot be
used.
Cannot directly extrapolate the results to
higher eukaryotes without further
Histone H3 tail bound to
Chromo Domain of Drosophila
HP1a
Yelow: Me2k9
Red/Orange: Me3k9
Y : Tyrosine
W: Tryptophan
E : Glutamine
T : Threonine
K : Lysine
V : Valine
N : Asparagine
S : Serine
A : Alanine
Jacobs and Khorasanizadeh (2002), Science express
Mock transformation
Number of
Transformants = ~ 2 x 106
per ml;
For a 5ml final volume of
transformed cells
number of library
colonies screened is
around ~107 !
Large enough to cover all
clones in the library.
(Number of independent
clones in the library: 3.5
x 106; clontech )
HIS3 expression seems to be
Leaky on –L/T/H plates
S.
No
PARENT
PLATE
PREY
1
SD - LT
pACT-Mi2b
2
SD - LT
pACT-KIP21
3
SD - LT
pACT-KIP41
4
SD - LT
pACT
TO
COLONIES
SD - LT
10 -20
SD -LTH
0
SD - LT
100-150
SD -LTH
20 -30
SD - LT
100-150
SD -LTH
1
SD - LT
Lawn
SD -LTH
20 -30
All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).
BAIT and empty pACT seem to grow on –L/T/H but not –
L/T/H/A (observation from mock transformation).
HIS3 titration with competitive
inhibitor 3-AT
This assay is to determine the amount of 3-AT needed to reduce
noise (growth when there is no interaction) on selection plates.
S.no
-- L/T/H with 3-AT @ different
concentrations
plasmid
0mM
1
pACT
~60
2
KIP21
TNC
3
KIP41
SV40T
+ p53
positive
control
1mM
~24
3mM
5mM
10mM
15mM
3
No colonies
~100
10-20
No colonies
TNC
~100
0
No colonies
Lawn
not
plated
Lawn
Lawn
Lawn
Law
n
-L/T/H/A
Lawn
NOTE : 3-AT is 3-AMINO-1,2,4-TRIAZOLE, a competitive inhibiotor
of HIS3 gene product.
TNC means too numerous to count
All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).
Cell Lysate prepared for
Western blot used for Co-IPs
Anti-HA used for IMMUNOPRECIPITATION
Harsh conditions used to obtain cell lysate
Blot demonstrates that the Anti-HA antibody is not as sensitive as expected.
Who's calling the shots?
Some transcription factors have, or recruit proteins that have, histone
modification and remodeling activities (Fig. 1). Presumably, gene
activation requires at least one such factor that can bind its
recognition sequence within 'inactive' chromatin and recruit other
factors that collaborate in altering local chromatin structure. These
altered regions of chromatin would then expose binding sites for other
factors, including the basal transcription machinery3. Histone
modifications may also be necessary to allow RNA polymerase to transit
across nucleosomal DNA sequences7.
Also, whereas acetylation can be reversed by various histone
deacetylases, there are no known histone demethylases. Therefore,
once a genomic region is methylated, modified nucleosomes must be
replaced rather than altered to remove this epigenetic mark. Further
work will be required to understand the mechanisms responsible for the
spread of local histone modifications and the impact of these
modifications on chromatin structure and transcriptional regulation.
Nature Genetics 36, 438 - 440 (2004)
Figure 1. Transcription factors recruit chromatin
modification enzymes, which in turn regulate
chromatin structure in the vicinity of the
promoter.
Dashed lines indicate spread of chromatin changes
outward from the promoter region. In this model,
changes are reversible until nucleosomes are modified
by histone methylation. Ac, acetylated; Me,
methylated.
Fig. 3. Model to explain the role of positive and negative factors in heterochromatin and
euchromatin. Methylated amino acids in the histone H3 tail are indicated by red lettering, and
acetylated residues are shown in blue. The underlying sequence of the satellite repeats promotes
the formation of a regular array of stable nucleosomes, which are favoured substrates for
methylation at H3 lysine 9 (K9) by SUVAR39H1. Binding of HP1 to long arrays of nucleosomes
containing H3 methylated at K9 promotes the formation of the higher-order heterochromatin
structure. Transcriptionally active euchromatin is generated by transcription factors binding to
clustered recognition sequences resulting in the formation of DNase I hypersensitive sites (HS).
The HS generate and maintain the open structure of the euchromatin by promoting H3 K9
acetylation and K4 methylation of neighbouring nucleosomes. They can also act as barriers
preventing the spread of heterochromatin into neighbouring euchromatin.
Trends Genet. 2002 May;18(5):252-8.
How to identify different classes
of interaction?
My Interests
How do cells in different tissues have
different functions when they have the
same genome?
Is entire human genome expressed?
– NO
There is approximately one gene every ~75,000
base pairs.
And only a fraction (~2%)of this part codes for
polypeptides.
Global gene expression patterns?