Opleiding: Biologie en Medisch Laboratoriumonderzoek, richting Research
Towards functional analysis of cardio desmosome components using
the Yeast Two-Hybrid assay
Anne-Bart Seinen
Cardiogenetics Section, Department of Genetics, University Medical Center Groningen (UMCG) Hanzeplein
1, 9713 GZ Groningen
Research supervisors: Dr. J.D.H. Jongbloed and L.G. Boven, M.Eng
School supervisor: Dr. A. L. Drayer
Background
ARVC is a dominantly inherited cardiomyopathy, which is mainly localized in the right ventricle. It is
characterized by a gradual loss of right ventricular myocardium and replacement by adipose and fibrous
tissue. The phenotype of ARVC is highly variable; often the first and only symptom is sudden death.
Although an important function for PKP2 in the cardiac desmosome has been shown, mutations in this
gene causing ARVC have not yet been precisely determined. Human genetic studies of the desmosome
have identified mutations in almost every known component of the complex in ARVC. However, most
mutations are found in desmocollin-2 (DSC2), desmoglein-2 (DSG2), plakophilin-2 (PKP2), plakoglobin
(PG) and desmoplakin (DSP). Nearly 70% of the patients with familial ARVC have a mutation in the PKP2
gene(1), however pathogenicity of most mutations has not yet been determined.
Aim
The main goal was to set-up the yeast two-hybrid assay. Next, with the use of this assay interactions
between PKP2a were verified. After verifying the interactions, a mutation found in ARVC can be
introduced in PKP2a and studied for it pathogenicity.
Methods
The yeast two-hybrid assay exploits the ability of a pair of interacting proteins to bring a transcription
activation domain into close proximity with a DNA-binding site that regulates the expression of a reporter
gene (figure VI-VII). Two proteins of interest (X and Y) are expressed in a yeast strain as hybrids fused to a
DNA-binding domain (DB-X) or an activation domain (AD-Y). The DNA-binding domain targets the hybrid
protein to its binding site, however, because most proteins lack an activation domain, this DNA-binding
hybrid protein does not activate transcription of the reporter gene. Likewise, the hybrid protein that
contains the activation domain does not activate expression of the reporter gene because it cannot bind
the upstream activation sequence (UAS) of the reporter gene. However, when both hybrid proteins are
present the interaction of X and Y activates transcription.
AD
DB
Upstream Activation Sequence
Transcription
LacZ gene
Figure I. Normal transcription. Binding of the activation domain (AD) to the DNA-binding domain (DB) results in stable
transcription of the LacZ gene.
Opleiding: Biologie en Medisch Laboratoriumonderzoek, richting Research
AD
Y
X
Transcription
DB
Upstream Activation Sequence
LacZ gene
Figure II. Two-hybrid transcription. Interaction between protein Y and protein X results in transcription of the LacZ gene.
In these experiments the pACT2-AD and pGBKT7-DB vectors served as a backbone for the insertion of the
coding sequences of desmosome proteins. The pACT2-AD vector contains the leucine (Leu) selection
marker for selection in yeast. The pGBKT7-DB vector however contains the tryptophan (Trp) selection
marker. Cells transformed with only the pGBKT7-BD fusion construct can only grow on SD media lacking
tryptophan, due to the tryptophan selection marker. Whereas cells transformed with pACT2-AD fusion
construct can only grow on SD media lacking leucine, due to the leucine selection marker. Hence, clones
transformed with a pGBKT7-BD and a pACT2-AD fusion construct can grow on both plates. For the TwoHybrid assay the S. cerevisiae AH109 strain was used. This strain has two reporter genes downstream of
the UAS, the histidine3 (HIS3) and β-galactosidase (LacZ). If there is interaction between the two proteins
of interest, a transcription factor is formed by the binding domain and activation domain and
transcription of the two reporter genes is achieved. This makes it possible for the yeast to grow on plates
lacking histidine, whereas the transcription product of LacZ facilitates the cleavage of X- α-Gal, producing
a bluish color.
Results
S. cerevisiae AH109 cells were transformed as shown in table I. After transformation each construct was
plated onto Selective Dextrose (SD) medium lacking leucine (SD/-Leu), lacking tryptophan (SD/-Trp),
lacking leucine and tryptophan (SD/-Leu/-Trp) and lacking leucine, tryptophan and histidine (SD/-Leu/Trp/-His). X- α-Gal was added to the SD/-Leu/-Trp/-His plates to screen for blue colonies. Clones on the
SD/-Leu/-Trp represent weak or transient interactions of the pACT2-AD fusion construct with the
pGBKT7-PKP2a vector. To screen for the actual interactions between the proteins, cells were also plated
onto SD/-Leu/-Trp/-His with X- α-Gal. This final step identifies the pACT2- AD fusion proteins that
interact with PKP2a, and all false positive interactions were virtually eliminated by this screen. As a
positive control the combination KIF5b and KIAA was used. The 13 blue colonies show that the twohybrid assay is working and that the results are legitimate. 5 white colonies were seen for DSC2a and
PKP2a, the white color suggests a false positive, probably due to a “leaky” histidine reporter gene. The 4
blue colonies for DPNTP and PKP2a suggests a strong affinity between the two proteins.
Table I. S. cerevisiaeAH 109 growth results after transformation
pGBKT7PKP2a
pGBKT7-KIAA
pGBKT7PKP2a
pGBKT7-
+
+
-
SD/-Leu/Trp
-
SD/-Leu/-Trp/-His + X-αGal
-
-
>300
-
-
-
>300
-
-
>300
-
-
-
>300
-
-
-
>300
-
-
-
>300
>300
-
-
-
>300
>300
19
5 white colonies
SD/-Leu
SD/-Trp
-
-
pACT2DSC1a
pACT2DSC2a
pACT2DPNTP
pACT2-DSG2
pACT2-KIF5b
pACT2DSC1a
pACT2-
Opleiding: Biologie en Medisch Laboratoriumonderzoek, richting Research
PKP2a
pGBKT7PKP2a
pGBKT7PKP2a
pGBKT7-KIAA
+
DSC2a
pACT2DPNTP
+
pACT2-DSG2
+
pACT2-KIF5b
>300
>300
14
4 blue colonies
>300
>300
21
13 blue colonies
Conclusion
The yeast two-hybrid assay was set-up and interaction between DPNTP and PKP2a was verified.
Interactions between DSC1a, DSC2a or DSG2 and PKP2a were not verified. Constructs with mutations
introduced in PKP2a were made using site-directed mutagenesis, however, these constructs could not be
tested.
1. Plakophilin-2 mutations are the major determinant of familial Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy. Van
Tintelen, J. Peter; Entius, Mark M.; Bhuiyan, Zahurul A.; Jongbloed, Roselie; Wiesfeld, Ans C.P.; Wilde, Arthur A.M.; van der Smagt, Jasper;
Boven, Ludolf G.; Mannens, Marcel M.A.M.; van Langen, Irene M.; Hofstra, Robert M.W.; Otterspoor, Luuk C.; Doevendans, Pieter A.F.M.;
Rodriguez, Luz-Maria; van Gelder, Isabelle C.; Hauer, Richard N.W. 2006, Circulation, pp. 1650-1658.