CG920 Genomics
Lesson 6
Protein Interactions in Gene Regulations
Jan Hejátko
Functional Genomics and Proteomics of Plants,
Mendel Centre for Plant Genomics and Proteomics,
Central European Institute of Technology (CEITEC), Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.muni.cz
 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
 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
 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
 Functional importance of specific protein interactions
 Chromatin structure
Importance of Protein
Interactions
Regulation by histone
acetyl transferases or
histone deacteylases
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”
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
Importance of Protein
Interactions
Initiation of Transcription
Positive TFs
Negative TFs
Initiation of Transcription
Transcriptional Regulation by
TAFs
Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
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
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
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
Importance of Protein
Interactions
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
 Role of mRNA localization
 Attenuating the expression of
potentially toxic proteins
 Localization of
expression of MBP into
myelination regions of
nerve cells
mRNA localization
Ainger et al., 1993
MBP mRNA
Shahbabian and Chartrand, 2012
 Diffusion and recruitment 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“)
 Recruitment on the vegetal pole via
interaction of MC and ER
mRNA localization
Mechanisms
 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 has been still unknown
Shahbabian and Chartrand, 2012
mRNA localization
Mechanisms
Shahbabian and Chartrand, 2012
 Active transport of mRNA
 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
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
Importance of Protein
Interactions
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
Importance of Protein
Interactions
Jing and Strader, Plant Structural Biology, Hormonal Regulations (2018)
Auxin Signalling
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
 Signal transduction
Importance of Protein
Interactions
 PI and signal transduction
 through G protein and
phospholipase C
 Signalling cascades using cAMP
Signal transduction
 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
PI in vivo
Co-immunoprecipitation
 Isolation of protein complexes using
antibodies recognizing one of the interacting
proteins
CKI1
MYC
CKI1
HA
αMYC
αHA
CKI1
MYC
AHK3
HA
αMYC
 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
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
 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
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
 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
 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)
 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
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

PI in vivo
Membrane Recruitment Assay (MeRA)
GFP
GFPGFPGFP
P35S::ERS1:RFP + P35S::ΔTM-ETR2:GFP
PI in vivo
Membrane Recruitment Assay (MeRA)
 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
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
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?
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
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)
Structure of CKI1RD
□ X-ray crystallography revealed conserved (α/β)5 structural fold
of CKI1RD
Pekárová et al., Plant Journal (2011)
Dynamics of CKI1RD
□ Mg2+binding leads to remodelling of active centre of
CKI1RD
Pekárová et al., Plant Journal (2011)
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)
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
 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
Discussion