CG920 Genomics
Lesson 7
Protein Interactions in Gene Regulations
Jan Hejátko
Functional Genomics and Proteomics of Plants,
CEITEC - Central European Institute of Technology
And
National Centre for Bimolecular Research,
Faculty of Science,
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
2
 Literature sources for Chapter 06:
 Wilt, F.H., and Hake, S. (2004). Principles of Developmental Biology. (New York ;
London: W. W. Norton).
 Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, E., and Carson, J.H.
(1993). Transport and localization of exogenous myelin basic protein mRNA microinjected
into oligodendrocytes. J Cell Biol 123, 431-441.
 Alberts, B. (1998). The cell as a collection of protein machines: preparing the next
generation of molecular biologists. Cell 92, 291-294.
 Grefen, C., Stadele, K., Ruzicka, K., Obrdlik, P., Harter, K., and Horak, J. (2008).
Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene
receptor family members. Molecular Plant 1, 308-320.
 Hu, C.D., and Kerppola, T.K. (2003). Simultaneous visualization of multiple protein
interactions in living cells using multicolor fluorescence complementation analysis. Nat.
Biotechnol. 21, 539-545.
 Shahbabian, K., and Chartrand, P. (2012). Control of cytoplasmic mRNA localization.
Cellular and molecular life sciences : CMLS 69, 535-552.
 Van Leene, J., Witters, E., Inze, D., and De Jaeger, G. (2008). Boosting tandem affinity
purification of plant protein complexes. Trends Plant Sci 13, 517-520.
 Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D.,
Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of
protein interactions in living plant cells using bimolecular fluorescence complementation.
Plant J 40, 428-438.
Literature
3
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
4
 Functional importance of specific protein
interactions
 Most of the proteins in the cell exist in the
form of complexes which may further
interact with each other
 Proteasome
 protein complex responsible for the
degradation of obsolete proteins in
the cell
Importance of Protein
Interactions
5
Proteasome
 Consisting of a core, also being
designated as 20S and regulatory
portions (19 or 11S)
 Allows targeted degradation of
proteins labelled by a specific marker small
polyppetide (76 aa) called
ubiquitin
The importance of
protein interactions
6
Proteasome –targeted proteolysis
7
 Functional importance of specific protein interactions
 Chromatin structure
Importance of Protein
Interactions
8
Regulation by histone
acetyl transferases or
histone deacteylases
9
CpG or CpNpG
CpNpNp
CpG
DNA methylation in animals vs. in plants
methylation status methylation status
Cell-specific methylation allows maintain of
tissue-specific gene expression profiles
Mechanism of transcriptional regulation by
DNA methylation mostly unknownImprinting and “cell memory”
10
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
Importance of Protein
Interactions
11
Initiation of Transcription
12
13
Positive TFs
Negative TFs
Initiation of Transcription
14
Transcriptional Regulation by
TAFs
Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
15
Multifactorial Promoters Control
ProENDO16:REPORTER (sea urchin)
Deletion
mutagenesis Positive, interaction with
TAFs
Upregulation in the
presence of A and B
Developmental specificity
Combinatorial control
Midgut
Primary or skeletogenic
mesenchyme cells
Vegetal plate
16
Regulation of β-globin type of
hemoglobin chains expression
Locus control region
Development-dependent
activation by LCR
•Acetylation of H3?
•Involvement of other
genes?
Cca 50 kbp
Multifactorial Promoters Control
17
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
Importance of Protein
Interactions
18
BICOID mRNA NANOS mRNA
 Importance of mRNA localization
 Control over spatiotemporal
localization of gene product (protein)
 Asymmetric cell division during
development
 Embryo polarization
mRNA localization
Shahbabian and Chartrand, 2012
ASH1 mRNA
19
 Role of mRNA localization
 Attenuating the expression of
potentially toxic proteins
 Localization of
expression of MYELIN
BASIC PROTEIN (MBP)
into myelination regions
of nerve cells
mRNA localization
Ainger et al., 1993
MBP mRNA
20
Shahbabian and Chartrand, 2012
 Diffusion and entrapment of mRNA
 During the early stages of Xenopus
oogenesis, Xcat-2 mRNA is restricted to
a specific structure in the cytoplasm
called the mitochondrial cloud (MC,
Balbiani body)
 MC movement is partly dependent on
the depolymerization of microtubuls (socalled
„molecular motor“)
 Entrapment on the vegetal pole via
interaction of MC and ER
mRNA localization
Mechanisms
21
 Localized mRNA degradation
 During embryogenesis in
Drosophila m. Hsp83 mRNA is
localized at the posterior pole of
embryo, similarly to NANOS mRNA
 Hsp83 mRNA is localized in the
whole embryo, however, it is
destabilized by cis elements both
in 3’UTR (HDE) and in coding
region (HIE).
 HIE elements are recognized by SMAUG protein, which
mediates binding of degradation complex CCR4/POP2/NOT
 In the posterior pole the Hsp83 mRNA is protected from the
effects of SMAUG by the so-called HPE element in 3’UTR;
mechanism of this protection is still unknown
Shahbabian and Chartrand, 2012
mRNA localization
Mechanisms
22
Shahbabian and Chartrand, 2012
 Active transport of mRNA
 Asymmetric Synthesis of HO1
(ASH1) is represor of the HO
endonuclease in S. cereviseae;
inhibition of HO results in inhibition
of mating-type switching in daughter
cells
 ASH1 mRNA is actively transported
by „molecular motors“ associated
with actin
 ASH1 mRNA contains 4 cis
elements (3 in the coding
sequence and 1 in the
3’UTR), which are recognized
by RNA-binding protein SHE2
 SHE2 interacts with SHE3, an
adaptor protein, which links
SHE2 to the molecular motor
MYO4, which then binds to
actin and allows transport of
ASH1 mRNA into the
daughter cell
Shahbabian and Chartrand, 2012
ASH1 mRNA
mRNA localization
Mechanisms
23
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
Importance of Protein
Interactions
24
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
Importance of Protein
Interactions
25
Jing and Strader, Plant Structural Biology, Hormonal Regulations (2018)
Auxin Signalling
26
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
 Signal transduction
Importance of Protein
Interactions
27
 PI and signal transduction
 through G protein and
phospholipase C
 Signalling cascades using cAMP
Signal transduction
28
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
Outline
29
PI in vivo
Co-immunoprecipitation
 Isolation of protein complexes using
antibodies recognizing one of the interacting
proteins
CKI1
MY
C
CKI1
HA
αMYC
αHA
CKI1
MY
C
AHK3
HA
αMYC
CKI1-MYC
CKI1-HA
AHK3/4-HA
CKI1-HA
30
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
Outline
31
PI in vivo
Tandem affinity purification (TAP-tag)
 Isolation of protein complexes using recombinant
proteins fused with two different binding domains -
tags
 Isolated protein complexes are separated using
1D ELFO and then identified by MS
 calmodulin-binding protein (CBP)
 IgG binding domains of protein A (ProtA)
 TEV (tobacco etch virus) protease
recognition site
POI
 Advantage: using two independent protein
domains for affinity purification -> therefore high
specifity
32
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
Outline
33
PI in vivo
Yeast two-hybrid assay (Y2H)
 Isolation of protein complexes using recombinant
proteins, each fused to a part of Gal4 transcription
factor
 One of the proteins (bait) fused to DNAbinding
domain of Gal4 (Gal4-BD)
 The other protein (prey) fused to
activation domain of Gal4 (Gal4-AD)
 Protein interactions enable reconstitution of
binding domains with activation domain and
triggers the expression of a reporter gene
 Visual detection (blue color, LacZ)
 Auxotrophic selection (growth on
medium lacking histidine, His)
 Method used for searching for interaction partners
in expression libraries of individual organisms
34
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
Outline
35
 Protein interaction is detected by
reassociation of the fluorescent protein
 Each of the potential interaction
partners is fused to one of the
subunits of the fluorescent protein,
e.g. YFP
 In case of interaction, the fluorescence
appears
 Apart from identification of the interaction,
this method allows you to localize the
interaction within the cell
PI in vivo
Bimolecular fluorescence
complementation (BiFC)
36
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
Outline
37
PI in vivo
Membrane Recruitment Assay (MeRA)
 Method for identification of interactions of
cytoplasmic proteins with the membrane proteins
 Membrane protein is fused with a
fluorescecnt protein
 Potential interaction partner is fused
with another fluorescent protein with
different emission spectra
 In case of interaction the localization
of the cytoplasmic protein is changed
– it is colocalized on the membrane
with the membrane protein
38

PI in vivo
Membrane Recruitment Assay (MeRA)
39
GFP
GFPGFPGFP
P35S::ERS1:RFP + P35S::ΔTM-ETR2:GFP
PI in vivo
Membrane Recruitment Assay (MeRA)
40
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
41
D’Agostino et al., Plant Phys, 2000
Signal Transduction via MSP
NUCLEUS
CYTOKININ
PM
AHK sensor histidine kinases
• AHK2
• AHK3
• CRE1/AHK4/WOL
REGULATION OF TRANSCRIPTION
INTERACTION WITH EFFECTOR PROTEINS
HPt Proteins
• AHP1-6
Response Regulators
• ARR1-24
CK primary response genes
- Type-A ARRs expression
Recent Model of the CK Signaling via Multistep Phosphorelay (MSP)
Pathway
42
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Is there any specificity in plant
MSP?
□ Is there a signalling specificity of MSP in plants?
43
Specificity of CKI1 signalling
□ CKI1 interacts in vivo with only subset of AHPs
BiFC Y2H
AHP1
AHP2
AHP3
AHP4
AHP5
AHP6
CKI1 CKI1RD
Pekárová et al., Plant Journal (2011)
CYTOKININ
44
Specificity of CKI1 Signalling
□ Specificity of CKI1 interaction was confirmed in vitro
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250
AHP concentration [nM]
A450
AHP2 AHP3 AHP5
AHP3: Kd ~ 10,5 nM
AHP2: Kd ~ 9,17 nM
AHP5: Kd ~ 108 nM
Pekárová et al., Plant Journal (2011)
45
Structure of CKI1RD
□ X-ray crystallography revealed conserved (α/β)5 structural fold
of CKI1RD
Pekárová et al., Plant Journal (2011)
46
Dynamics of CKI1RD
□ Mg2+binding leads to remodelling of active centre of
CKI1RD
Pekárová et al., Plant Journal (2011)
47
CKI1RD structural changes are
associated with its binding specificity
□ Mg2+- and BeF3
--induced structural changes fine-tune
binding specificity of CKI1RD
Pekárová et al., Plant Journal (2011)
48
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Model Suggestion
□ YES, there is signalling specificity of MSP in plants.
P
P
P
P
P
P
P
49
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Summary
50
Discussion
51
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
Outline
52
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
TATA box
Iniciation of transcription
promoter
5’ UTR
ATG…ORF of reporter gene
Transcriptional Fusion
53
 Identification and cloning of the promoter region of the gene
 Preparation of recombinant DNA carrying the promoter and
the reporter gene (uidA, GFP)
 Preparation of transgenic organisms carrying this
recombinant DNA and their histological analysis
Transcriptional Fusion
54
GUS Reporter in Mouse
Embryos
55
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
Outline
56
 Identification and cloning of the promoter and coding region of
the analyzed gene
 Preparation of a recombinant DNA carrying the promoter and
the coding sequence of the studied gene in a fusion with the
reporter gene (uidA, GFP)
TATA box
promoter
5’ UTR
ATG…ORF of analysed gene…..….ATG…ORF of reporter gene….….....STOP
Translational Fusion
57
 Preparation of transgenic organisms carrying the
recombinant DNA and their histological analysis
 Compared to transcriptional fusion, translation fusion allows
analysis of intercellular localization of gene product (protein)
or its dynamics
Histone 2A-GFP in Drosophila embryo by PAMPIN1-GFP in Arabidopsis
Translational Fusion
58
Translational Fusion
59
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
Outline
60
Databases
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
61
Databases
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
62
Databases
□ Analysis of expression using ePlant
63
Databases
□ Analysis of expression using ePlant
64
□ Analysis of expression using Genevestigator (AHP1 and
AHP2, Arabidopsis, Affymetrix ATH 22K Array)
Databases
65
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
Outline
66
Fluorescence-Activated Cell Sorting (FACS)
□ High-Resolution Expression Map in Arabidopsis Root
Expression Maps - RNA
Brady et al., Science, 2007
67
□ High-Resolution Expression Map in Arabidopsis Root
Expression Maps - RNA
Brady et al., Science, 2007
68
□ High-Resolution Expression Map in Drosophilla
Expression Maps - RNA
Nikos Karaiskos et al. Science 2017;science.aan3235
69
Expression Maps - Proteins
Ponten et al., J Int Med, 2011
□ Human Protein Atlas
70
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
71
□ Human Protein Atlas
(http://www.proteinatlas.org/)
Expression Maps - Proteins
72
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
Outline
73
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
Outline
74
 Method, which provides quick comparison of a large
number of genes/proteins between the test sample and
control
 Oligo DNA chips are used the most
 There are commercialy available kits for the whole genome
 company Operon (Qiagen), 29.110 of 70-mer oligonucleotides
representing 26.173 genes coding proteins, 28.964 transcripts and 87
microRNA genes of Arabidopsis thaliana
 Possibility of use for the preparation of photolithography chips – facilitation
of oligonucletide synthesis e.g. for the whole human genome (about 3,1 x
109 bp) jit is possible to prepare 25-mers in only 100 steps, by this
technique
Affymetrix ATH1 Arabidopsis genome array
 Chips not only for the analysis of
gene expression, but also for e.g.
Genotyping (SNPs, sequencing
with chips, …)
DNA Chips
75
 For the correct interpretation of the results, good knowledge of
advanced statistical methods is required
 Control of accuracy of the measurement (repeated
measurements on several chips with the same
sample, comparing the same samples analysed on
different chips with each other)
 It is necessary to include a sufficient number of controls and
repeats
 Control of reproducibility of measurements
(repeated measurements with different samples
isolated under the same conditions on the same
chip – comparing with each other)
Che et al., 2002
 Identification of reliable measurement treshold
nespolehlivé
spolehlivé
 Finally comparing the experiment with the control
or comparing different conditions with each other >
the result
 Currently there‘s been a great number of results of various
experiments in publicly accessible databases
DNA Chips
76
 Protein chips
 Chips with high density containing 104 proteins
 Analysis of protein-protein interactions, kinase substrates
and interactions with small molecules
 Possibility of using antibodies – more stable than proteins
Protein Chips
77
 Identification of proteins interacting with integrin αIIbβ3
cytoplasmic domain of platelets
 Expression of cytoplasmic part as a
fusion peptide biotin-KVGFFKR
 Analysis of binding to the protein
chip containing 37.000 clones of E.
coli expressing human recombinant
proteins
 Confirmation of interaction by pulldown
analysis of peptides and by
coprecipitation of whole proteins as
well (e.g. chloride channel Icln)
 Other use: e.g. in the identification of
kinase substrates, when substrates
are bound to the chip and exposed
to kinases in the presense of
radiolabeled ATP (786 purified
proteins of barely, of which 21 were
identified as CK2α kinase
substrates; Kramer et al., 2004)
Lueking et al., 2005
Protein Chips
78
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
Outline
79
WT hormonal mutant
Next Gen Transcriptional
Profiling
□ Transcriptional profiling via RNA sequencing
mRNA
Sequencing by Illumina and
number of transcripts determination
mRNA
cDNA cDNA
80
Results of –omics Studies vs
Biologically Relevant Conclusions
□ Transcriptional profiling yielded more then 7K differentially
regulated genes…
gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
AT1G07795 1:2414285-2414967 WT MT OK 0 1,1804 1.79769e+308
1.79769e+3
08 6.88885e-05
0,00039180
1 yes
HRS1 1:4556891-4558708 WT MT OK 0 0,696583 1.79769e+308
1.79769e+3
08 6.61994e-06
4.67708e-
05 yes
ATMLO14 1:9227472-9232296 WT MT OK 0 0,514609 1.79769e+308
1.79769e+3
08 9.74219e-05
0,00053505
5 yes
NRT1.6 1:9400663-9403789 WT MT OK 0 0,877865 1.79769e+308
1.79769e+3
08 3.2692e-08
3.50131e-
07 yes
AT1G27570 1:9575425-9582376 WT MT OK 0 2,0829 1.79769e+308
1.79769e+3
08 9.76039e-06 6.647e-05 yes
AT1G60095 1:22159735-22162419 WT MT OK 0 0,688588 1.79769e+308
1.79769e+3
08 9.95901e-08
9.84992e-
07 yes
AT1G03020 1:698206-698515 WT MT OK 0 1,78859 1.79769e+308
1.79769e+3
08 0,00913915 0,0277958 yes
AT1G13609 1:4662720-4663471 WT MT OK 0 3,55814 1.79769e+308
1.79769e+3
08 0,00021683 0,00108079 yes
AT1G21550 1:7553100-7553876 WT MT OK 0 0,562868 1.79769e+308
1.79769e+3
08 0,00115582 0,00471497 yes
AT1G22120 1:7806308-7809632 WT MT OK 0 0,617354 1.79769e+308
1.79769e+3
08 2.48392e-06
1.91089e-
05 yes
AT1G31370 1:11238297-11239363 WT MT OK 0 1,46254 1.79769e+308
1.79769e+3
08 4.83523e-05
0,00028514
3 yes
APUM10 1:13253397-13255570 WT MT OK 0 0,581031 1.79769e+308
1.79769e+3
08 7.87855e-06
5.46603e-
05 yes
AT1G48700 1:18010728-18012871 WT MT OK 0 0,556525 1.79769e+308
1.79769e+3
08 6.53917e-05
0,00037473
6 yes
AT1G59077 1:21746209-21833195 WT MT OK 0 138,886 1.79769e+308
1.79769e+3
08 0,00122789 0,00496816 yes
AT1G60050 1:22121549-22123702 WT MT OK 0 0,370087 1.79769e+308
1.79769e+3
08 0,00117953 0,0048001 yes
Ddii et al., unpublished
AT4G15242 4:8705786-8706997 WT MT OK 0,00930712 17,9056 10,9098 -4,40523 1.05673e-05 7.13983e-05 yes
AT5G33251 5:12499071-12500433 WT MT OK 0,0498375 52,2837 10,0349 -9,8119 0 0 yes
AT4G12520 4:7421055-7421738 WT MT OK 0,0195111 15,8516 9,66612 -3,90043 9.60217e-05 0,000528904 yes
AT1G60020 1:22100651-22105276 WT MT OK 0,0118377 7,18823 9,24611 -7,50382 6.19504e-14 1.4988e-12 yes
AT5G15360 5:4987235-4989182 WT MT OK 0,0988273 56,4834 9,1587 -10,4392 0 0 yes
81
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
Outline
82
 Methods for identification of gene function using gain-of-function
approaches
 T-DNA activation mutagenesis
 Method enabling isolation of dominant mutants by random
insertion of constitutive promoter, resulting in
overexpression of the gene and therefore in corresponding
phenotypic changes
 First step: preparation of mutant library prepared by
tansformation of a strong constitutive promoter or enhancer
 Next step: search of interesting phenotypes
 Identification of the affected gene, e.g. by plasmid-rescue
Gain-of-Function Approaches
83
TF TF
TF
40S
60S
TF TF TF TF
40S
60S
40S
60S
40S
60S
40S
60S
TF TF
TF
Activation Mutagenesis
84
Isolation of CKI1 Gene
- Isolation of the gene using activation mutagenesis
- Mutant phenotype is a phenocopy of
exogenous application of cytokinins (CKI1,
CYTOKININ INDEPENDENT 1)
*- Tatsuo Kakimoto, Science 274 (1996), 982-985 *
*
no hormones
t-zeatin
K1 K2plasmid
rescue
35S::CK
1 cDNA
85
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
Outline
86
35S LhG4
pOP
TATA
CKI1
activator
reporter
activator x reporter
x
Regulated Expression
Systems
87
35S LhGR
pOP
TATA
CKI1
activator
reporter
activator x reporter
DEX DEX
+DEX DEX
DEX
DEX
x
Regulated Expression
Systems
88
35S LhGR
pOP
TATA
CKI1
activator
reporter
activator x reporter
DEX DEX
+DEX DEX
DEX
DEX
x
pOP
TATA
GUS
DEX DEX
wt Col-
0
4C
Regulated Expression
Systems
89
 UAS system
http://www.plantsci.cam.ac.uk/Haseloff/
 Regulatable gene expression systems
 Time- or site-specific regulation of gene expression, leading
to a change in phenotype and thereby identification of the
natural function of the gene
 pOP system
Regulated Expression
Systems
90
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
 Chemical Genetics
Outline
91
 New trends
 „chemical genetics“ – more than 50.000/120.417 records in PubMed
database (16.10. 2008/15.11. 2018, an increase of >240 %)
Chemical Genetics
92
 New trends
 „chemical genetics“ – more than 50.000/130.437 records in PubMed
database (16.10. 2008/24.10. 2019, an increase of >260 %)
Chemical Genetics
 Like in the case of genetics, there are also „forward“ and
„reverse“ genetics approaches
 Unlike in „classical“ genetics approaches, the subject of
study is not a gene, but a protein
 Chemical genetics tries to identify either the target protein
after a chemical treatment and after following phenotypic
changes („forward“ chemical genetics) or chemicals able
to interact with protein of interest („reverse“ chemical
genetics)
 For that purpose there are carried out searches in the
libraries of various chemicals (thousands of entries,
comercially available)
 example: analysis of endomembrane transport in plants
93
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 In plants cells there occurr very dynamic processes mediated mainly
by endomembrane transport
Chemical Genetics
GFP targeted to the ER
94
 Endomembrane transport is an important regulatory mechanism in
signal transduction and regulation of cellular processes
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 In plants cells there occurr very dynamic processes mediated mainly
by endomembrane transport (see film, GFP targeting to the ER)
Chemical Genetics
95
Richter et al., E J Cell Biol (2010)
Anterograde
transport
Retrograde
transport
Trans-Golgi
network
Multivesicular
bodies-late
endosome
(prevacuolar
compartment)
Recycling
endosome
Huang et al., 2010
PIN1-GFP
96
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 By searching in the „library“ of chemicals there were
identified those, that lead to the secretion of enzyme
(carboxypeptidase Y) in yeast (S. cerevisiae) – this
enzyme is normally transported to the vacuole via the
endomembrane transport
 Analysis of changes in secretion using dotblot
and immunodetection of
carboxypeptidase Y in the culture medium
with monoclonal antibodies
Zouhar et al., 2004
Chemical structure of sortins
Detection of vacuole phenotype (tonoplast shape) of
yeast by staining with a specific color (MDY-64)
Immunodetection of carboxypeptidase
Chemical Genetics
97
 Analysis of mechanisms of endomembrane transport by
chemical genetics approaches
 Identified compounds („sortins“) were able to induce
similar changes in Arabidopsis as well – transport
mechanisms are conserved in yeast and in plants
 For detailed identification of the molecular proces
affected by one of the identified „sortins“, the analysis
of its influence on a secretion of a marker protein
(AtCPY) was performed – sortin 1 specifically inhibits
only this secretory pathway
 Identifcation of mutants with altered sensitivity to
sortin 1 (hyper- or hypo-sensitive mutants) by EMS
mutagenesis
Zouhar et al., 2004
Shape of plant vacuoles using EGFP:-TIP
Phenotype of seedlings in the presence of sortins
Sortin 1
Sortin 2
 By searching in the „library“ of chemicals there were
identified those, that lead to the secretion of enzyme
(carboxypeptidase Y) in yeast (S. cerevisiae) – this
enzyme is normally transported to the vacuole via the
endomembrane transport
 Analysis of changes in secretion using dotblot
and immunodetection of
carboxypeptidase Y in the culture medium
with monoclonal antibodies
Chemical Genetics
98
 Analysis of mechanisms of endomembrane
transport by chemical genetics approaches –
summary
 GFP::d-TIP vacuole membrane
(tonoplast) labelling and identification
of mutations leading to altered
tonoplast morphology
 Chemical genetics in combination
with classical genetics – identification
of proteins participating in regulation
of endomembrane transport
 Proteomics approaches –
identification and analysis of vacuole
proteome
99
 Methods of gene expression analysis
 Qualitative analysis of gene expression
 Preparation of transcriptional fusion of promoter of analysed gene
with a reporter gene
 Preparation of translational fusion of the coding region of the
analysed gene with reporter gene
 Use of the data available in public databases
 Tissue- and cell-specific gene expression analysis
 Quantitative analysis of gene expression
 DNA and protein chips
 Next generation transcriptional profiling
 Regulation of gene expression in the identification of
gene function by gain-of-function approaches
 T-DNA activation mutagenesis
 Ectopic expression and regulated gene expression systems
 Chemical Genetics
Summary
100
Discussion