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DATA TO SUPPORT DATA TO SUPPORT STATEMENTS IN STATEMENTS IN PRESENTATIONPRESENTATION
Outline of talkOutline of talk
General backgroundGeneral background
Introduction to the projectIntroduction to the project
Experimental designExperimental design
Experiments and resultsExperiments and results
ConclusionsConclusions
Future experimentsFuture experiments
Proteins Proteins involved in involved in
complexcomplexMethodMethod SummarySummary ReferencReferenc
ee
11 HP1HP1, KAP1, KAP1 Yeast 2 HybridYeast 2 Hybrid HP1HP1 was used as a bait and was used as a bait and TIF1TIF1 (KAP1) was isolated (KAP1) was isolated
Le Douarin et Le Douarin et alal
19961996
22 KOX1, KAP1KOX1, KAP1 Yeast 2 HybridYeast 2 Hybrid KRAB domain of KOX1 was KRAB domain of KOX1 was used as a bait and KAP1 used as a bait and KAP1
was isolatedwas isolated
Moosmann et Moosmann et al.al.
19961996
33NuRD/HDAC, NuRD/HDAC,
KAP1KAP1Yeast 2 HybridYeast 2 Hybrid
Mi2Mi2 (= NuRD/HDAC) was (= NuRD/HDAC) was shown interact with KAP-1-shown interact with KAP-1-PHD/BROMO but not Mi2PHD/BROMO but not Mi2
Schultz et al.Schultz et al.20012001
44 KIP21, KAP1KIP21, KAP1Yeast 2 HybridYeast 2 Hybrid
KIP21 and KIP41 (=SETDB1) KIP21 and KIP41 (=SETDB1) were shown interact with were shown interact with
KAP-1-PHD/BROMOKAP-1-PHD/BROMO
Schultz et al.Schultz et al.2002200255 KIP41, KAP1KIP41, KAP1
66KOX1, KAP1, KOX1, KAP1,
HP1HP1
Gel Electrophoretic Gel Electrophoretic Mobility Shift Mobility Shift
assay (in vitro)assay (in vitro)
DNA+GAL4-KRAB+KAP1 DNA+GAL4-KRAB+KAP1 ternary complex was ternary complex was
shown to complex with shown to complex with HP1HP1
Ryan et al. Ryan et al. 19991999
Prior demonstrations Prior demonstrations of KAP1 complexesof KAP1 complexes
Structure of CSD complexStructure of CSD complex
Epigenetic Gene SilencingEpigenetic Gene Silencing
Epigenetic effects are those changes in Epigenetic effects are those changes in gene function which are heritable gene function which are heritable through mitosis and/or meiosis and are through mitosis and/or meiosis and are not due to changes in DNA sequence.not due to changes in DNA sequence.
Main types of epigenetic information:Main types of epigenetic information:– Cytosine DNA MethylationCytosine DNA Methylation– Genomic impritningGenomic impritning– Histone modificationsHistone modifications
CSD interacting proteinsCSD interacting proteins
Chromatin based Chromatin based epigeneticsepigenetics
Controls chromosome domains and also Controls chromosome domains and also helps in cell differentiation helps in cell differentiation
X –inactivation (XIST locus. Genomic X –inactivation (XIST locus. Genomic imprinting, parent-of origin-specific allele imprinting, parent-of origin-specific allele silencing)silencing)
Developmental reprogramming of cell Developmental reprogramming of cell lineageslineages
Plasticity of stem cellsPlasticity of stem cells
Implications Implications human biology and human biology and disease, including cancer and agingdisease, including cancer and aging
Expression VectorsExpression Vectors
pBridge HP1 KAP19525 bp
HP1a
KAP1
Gal4 DBD
TRP1
MET25
HA/NLS
Gal4DBD
BAIT HP1
pACT2 Hela cDNA Lib10114 bp
Gene Library
GAL4 AD
LEU2
HA
Gal4AD??
PREY
KAP1
(BD-HP1a)-(HA-KAP1)
(AD-?)
HeLa cell cDNA libraryHeLa cell cDNA library
Source of human genes (cDNA).Source of human genes (cDNA).
Human cervical carcinoma cell lineHuman cervical carcinoma cell line
Estimated number of Independent Estimated number of Independent Clones: Clones: 3.5 x 103.5 x 1066
– Average insert (cDNA) size: 2.0 kbAverage insert (cDNA) size: 2.0 kb– cDNA size range: 0.5 – 4.0 kbcDNA size range: 0.5 – 4.0 kb
HeLa cancer cells
Hybrid proteins used in this Hybrid proteins used in this studystudy
BAITSBAITS– BD-HP1aBD-HP1a– (BD-HP1A)-(HA-(BD-HP1A)-(HA-
KAP1)KAP1)– BD-KAP1BD-KAP1– (BD-KAP1)-(HA-HP1)(BD-KAP1)-(HA-HP1)– (BD-HP1A)-(HA-(BD-HP1A)-(HA-
CAF1p150)CAF1p150)
PREYSPREYS– AD-?AD-? (HeLa cell cDNA (HeLa cell cDNA
library; Clontech)library; Clontech)
– AD-KIP21 (SETDB1)AD-KIP21 (SETDB1)– AD-KIP41 (SETDB1)AD-KIP41 (SETDB1)– AD-Mi2bAD-Mi2b– AD-KOX1AD-KOX1– AD-Y2H6.2 (AD-Y2H6.2 (POGZPOGZ))
Proteins Proteins involved in involved in
complexcomplexMethodMethod SummarySummary ReferencReferenc
ee
11 HP1HP1, KAP1, KAP1 Yeast 2 HybridYeast 2 Hybrid HP1HP1 was used as a bait and was used as a bait and TIF1TIF1 (KAP1) was isolated (KAP1) was isolated
Le Douarin et Le Douarin et alal
19961996
22 KOX1, KAP1KOX1, KAP1 Yeast 2 HybridYeast 2 Hybrid KRAB domain of KOX1 was KRAB domain of KOX1 was used as a bait and KAP1 used as a bait and KAP1
was isolatedwas isolated
Moosmann et Moosmann et al.al.
19961996
33NuRD/HDAC, NuRD/HDAC,
KAP1KAP1Yeast 2 HybridYeast 2 Hybrid
Mi2Mi2 (= NuRD/HDAC) was (= NuRD/HDAC) was shown interact with KAP-1-shown interact with KAP-1-PHD/BROMO but not Mi2PHD/BROMO but not Mi2
Schultz et al.Schultz et al.20012001
44 KIP21, KAP1KIP21, KAP1Yeast 2 HybridYeast 2 Hybrid
KIP21 and KIP41 (=SETDB1) KIP21 and KIP41 (=SETDB1) were shown interact with were shown interact with
KAP-1-PHD/BROMOKAP-1-PHD/BROMO
Schultz et al.Schultz et al.2002200255 KIP41, KAP1KIP41, KAP1
66KOX1, KAP1, KOX1, KAP1,
HP1HP1
Gel Electrophoretic Gel Electrophoretic Mobility Shift Mobility Shift
assay (in vitro)assay (in vitro)
DNA+GAL4-KRAB+KAP1 DNA+GAL4-KRAB+KAP1 ternary complex was ternary complex was
shown to complex with shown to complex with HP1HP1
Ryan et al. Ryan et al. 19991999
77HP1HP1, POGZ , POGZ
(Y2H6.2)(Y2H6.2)
Co-Co-immunoprecipitatimmunoprecipitat
ion and Yeast ion and Yeast Two Hybrid Two Hybrid
genetic screengenetic screen
Novel prey (POGZ / Y2H6.2) Novel prey (POGZ / Y2H6.2) shown to interact with shown to interact with
HP1HP1
Lechner et al,Lechner et al,UnpublishedUnpublished
Prior demonstrations Prior demonstrations of KAP1 & HP1 complexesof KAP1 & HP1 complexes
Histone CodeHistone Code
Strahl et al. Nature 2000
HP1 gene silencing
Long term GoalsLong term Goals
Build complete HP1 network with all Build complete HP1 network with all its partner proteins and to build a its partner proteins and to build a chromatin network database !chromatin network database !
Map both the temporal and Spatial Map both the temporal and Spatial formation of these complexesformation of these complexes
Get in depth and complete Get in depth and complete understanding of gene regulation in understanding of gene regulation in cellscells
Statement of ProblemStatement of Problem
Studies show that HP1Studies show that HP1 binds with binds with several proteins and presumably several proteins and presumably performs different activities (mainly performs different activities (mainly in regulating chromatin structure and in regulating chromatin structure and function).function).
But, little is known about what But, little is known about what complexes exist complexes exist in vivoin vivo and what and what controls their formationcontrols their formation
AssumptionsAssumptions
The main assumption in this experiment is The main assumption in this experiment is that there are regulatory factors that help that there are regulatory factors that help in the formation of HP1 complexes. in the formation of HP1 complexes.
Yeast system does not interfere with any Yeast system does not interfere with any of the interactions. (relatively ‘safe’; most of the interactions. (relatively ‘safe’; most of the initial HP1 interactions were done in of the initial HP1 interactions were done in yeast screens.)yeast screens.)
Feasibility of this screen can be Feasibility of this screen can be demonstrated by showing a ternary demonstrated by showing a ternary complex formation with positive controls.complex formation with positive controls.
LimitationsLimitations
Yeast might inactivate or destroy the foreign Yeast might inactivate or destroy the foreign (human) proteins. (human) proteins. Some of these human proteins might be toxic to Some of these human proteins might be toxic to yeast. yeast. Functional homology might be present between Functional homology might be present between yeast proteins and the human proteins.yeast proteins and the human proteins.Presence of false positives or false negatives.Presence of false positives or false negatives.Bait proteins that auto-activate cannot be used.Bait proteins that auto-activate cannot be used.Cannot directly extrapolate the results to higher Cannot directly extrapolate the results to higher eukaryotes without further verification.eukaryotes without further verification.
Y : TyrosineW: TryptophanE : GlutamineT : ThreonineK : LysineV : ValineN : AsparagineS : SerineA : Alanine
Histone H3 tail bound to Histone H3 tail bound to Chromo Domain of Chromo Domain of DrosophilaDrosophila HP1 HP1
Yelow: Me2k9Red/Orange: Me3k9
Jacobs and Khorasanizadeh (2002), Science express
Mock transformationMock transformation
Number of Transformants Number of Transformants = = ~ ~ 2 x 102 x 1066 per ml; per ml;
For a 5ml final volume of For a 5ml final volume of transformed cells number transformed cells number of library colonies of library colonies screened is around screened is around ~~101077 ! !
Large enough to cover all Large enough to cover all clones in the library. clones in the library. ((Number of independent Number of independent clones in the library: clones in the library: 3.5 3.5 x 10x 1066; clontech; clontech ) )
HIS3 expression seems to be HIS3 expression seems to be Leaky on –L/T/H platesLeaky on –L/T/H plates
S.No
PARENTPLATE PREY TO COLONIES
1
SD - LT
pACT-Mi2b
SD - LT 10 -20
SD -LTH 0
2
SD - LT
pACT-KIP21
SD - LT 100-150
SD -LTH 20 -30
3
SD - LT
pACT-KIP41
SD - LT 100-150
SD -LTH 1
4
SD - LT
pACT
SD - LT Lawn
SD -LTH 20 -30
All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).KAP1).
BAIT and empty pACT seem to grow on –L/T/H but not BAIT and empty pACT seem to grow on –L/T/H but not –L/T/H/A (observation from mock transformation).–L/T/H/A (observation from mock transformation).
HIS3HIS3 titration with competitive titration with competitive inhibitor 3-ATinhibitor 3-AT
S.no
plasmid
-- L/T/H with 3-AT @ different concentrations --
L/T/H/A0mM 1mM 3mM
5mM
10mM
15mM
1 pACT ~60 ~24 3 No colonies
2 KIP21 TNC ~100 10-20 No colonies
3 KIP41 TNC ~100 0 No colonies
positivecontrol
SV40T + p53
Lawn
notplated
Lawn
Lawn Lawn
Lawn
Lawn
All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).
NOTE : 3-AT is 3-AMINO-1,2,4-TRIAZOLE, a competitive inhibiotorof HIS3 gene product.
TNC means too numerous to count
This assay is to determine the amount of 3-AT needed to reduce This assay is to determine the amount of 3-AT needed to reduce noise (growth when there is no interaction) on selection plates.noise (growth when there is no interaction) on selection plates.
Cell Lysate prepared for Cell Lysate prepared for Western blot used for Co-IPsWestern blot used for Co-IPs
Harsh conditions used to obtain cell lysateHarsh conditions used to obtain cell lysateBlot demonstrates that the Anti-HA antibody is not as sensitive as expected. Blot demonstrates that the Anti-HA antibody is not as sensitive as expected.
Anti-HA used for IMMUNOPRECIPITATION
Who's calling the shots?Who's calling the shots?Some transcription factors have, or recruit proteins that have, Some transcription factors have, or recruit proteins that have, histone modification and remodeling activities (histone modification and remodeling activities (Fig. 1Fig. 1). ). Presumably, gene activation requires at least one such factor that Presumably, gene activation requires at least one such factor that can bind its recognition sequence within 'inactive' chromatin and can bind its recognition sequence within 'inactive' chromatin and recruit other factors that collaborate in altering local chromatin recruit other factors that collaborate in altering local chromatin structure. These altered regions of chromatin would then expose structure. These altered regions of chromatin would then expose binding sites for other factors, including the basal transcription binding sites for other factors, including the basal transcription machinerymachinery33. Histone modifications may also be necessary to allow . Histone modifications may also be necessary to allow RNA polymerase to transit across nucleosomal DNA sequencesRNA polymerase to transit across nucleosomal DNA sequences77..Also, whereas acetylation can be reversed by various histone Also, whereas acetylation can be reversed by various histone deacetylases, there are no known histone demethylases. deacetylases, there are no known histone demethylases. Therefore, once a genomic region is methylated, modified Therefore, once a genomic region is methylated, modified nucleosomes must be replaced rather than altered to remove this nucleosomes must be replaced rather than altered to remove this epigenetic mark. Further work will be required to understand the epigenetic mark. Further work will be required to understand the mechanisms responsible for the spread of local histone mechanisms responsible for the spread of local histone modifications and the impact of these modifications on chromatin modifications and the impact of these modifications on chromatin structure and transcriptional regulation.structure and transcriptional regulation.
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.
Nature GeneticsNature Genetics 3636, 438 - 440 (2004) , 438 - 440 (2004)
Trends Genet. 2002 May;18(5):252-8. Trends Genet. 2002 May;18(5):252-8.
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.
How to identify different How to identify different classes of interaction?classes of interaction?
My InterestsMy Interests
How do cells in different tissues have How do cells in different tissues have different functions when they have different functions when they have the same genome? the same genome? Is entire human genome expressed?Is entire human genome expressed?– NONO
There is approximately one gene every There is approximately one gene every
~~75,000 base pairs.75,000 base pairs.
And only a fraction (And only a fraction (~~2%)of this part codes 2%)of this part codes for polypeptides. for polypeptides.
Global gene expression patterns?Global gene expression patterns?
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