Transcript A Comprehensive Two-Hybrid Analysis to Explore the Yeast Protein

A Comprehensive Two-Hybrid
Analysis to Explore the Yeast
Protein Interactome
Ito et al.
Genome-Scale In Vivo Protein Interaction Testing
Background
• So you sequenced a genome, and now
have thousands of gene candidates of
unknown function. We’ve all been there.
• When bioinformatic analysis of the open
reading frame fails to identify a gene’s
function, functional genomic techniques
are used.
• Functional genomics is a field of
techniques used to find the functions or
interactions of previously found genes.
• In vivo testing is naturally preferable to in
vitro for functional genomics.
Cytoskeleton
Miscellaneous 4% (14)
1% (4)
Unidentified
21% (73)
After sequencing and
running against databases,
47% of the genes are either
hypothetical or unidentified.
Energy metabolism
3% (9)
Extrcellular matrix
3% (10)
Housekeeping
15% (52)
Tumor suppressor
1% (2)
Transcriptional
15% (53)
Signal tranduction
8% (26)
Retinoid metabolism Neural
1% (3)
2% (7)
Hypothetical
26% (90)
Cytoskeleton
Energy metabolism
Extrcellular matrix
Housekeeping
Hypothetical
Neural
Retinoid metabolism
Signal tranduction
Transcriptional
Tumor suppressor
Unidentified
Miscellaneous
Hypothetical: there is a
predicted gene there based
on statistical analysis, but it
may not be expressed.
Unidentified genes are
known to exist
experimentally, but nothing
is known about what they
do.
Background - Methods
• As a test of two-hybrid analysis at the genome scale, Ito wanted to
analyze how all ~6000 proteins in Saccharomyces Cervesiae, baker’s
yeast, interacted with each other.
• Microarrays (in this case protein chips) are one-to-many, but not a
good representation of in vivo conditions.
• 6000 proteins * 1 microarray per protein = $$$
• Yeast two-hybrid analysis with two modified proteins was established
for large-scale use at this point, but had not been tested on the
genome scale.
Background – Why Yeast?
• How yeast breeds makes two-hybrid
analysis easy: Two haploid strains mate to
create one diploid strain, and their
plasmids mix.
• Yeast have the genders A and alpha,
which do not fuse with themselves.
• Ito used strains (a) PR69-2A for bait
plasmids and (alpha) MaV204K for prey
plasmids.
• PR69-2A used adenine and histidine
auxotrophic reporter genes, MaV204K
used uracil and histidine, all driven by the
Gal4 promoter.
Hypotheses
• This was a pilot project to show the feasibility of genome-scale
analysis of protein interactions using the two-hybrid method.
• Goals were to investigate protein interactions and compare the
results to a replicate using the same method.
• Ito hypothesized the replicate would confirm the same protein
interactions he found.
• The knowledge of protein interactions gained from this study will lead
to future experiments regarding interesting protein interactions.
The Yeast Two-Hybrid Protein Interaction Test
• First, Ito purchased yeast strains that can only survive with a certain
gene expressed: Adenine, uracil, and histidine-producing enzymes.
• He gave that ‘reporter’ gene a custom promoter with a known
sequence, which he took from the gene Gal4.
• Created a custom transcription factor for that promoter in two parts:
the DNA-binding domain and the activation domain. The promoter
will activate when both parts are interacting with each other, but they
do not interact on their own.
Cassette, homologous recombination, CRISPR-Cas9, etc.
Yeast Two-Hybrid Test: Protein Interactions
• The two halves of the transcription factor are spliced into the plasmid
along with the two proteins of interest. The plasmids are called the “bait”
and the “prey”, and each has a unique ID tag called an IST, or interaction
sequence tag.
• If the proteins interact, they will bind each other, creating a whole
transcription factor that will go on to bind the promoter for the reporter
gene. Four reporter genes were used to eliminate false positives, and the
genes were checked for activation using two methods.
• If the proteins do not interact, the TF is never formed and the reporter
gene is not activated.
• Assay is by observing the action of the reporter gene. In Ito’s case, colonies
would form in a petri dish of deficient media after the yeast had mated.
IST for ID
Prey Sequence –
another ORF
Plasmid: pGBK-RC
Bait Sequence –
one ORF
Plasmid: pGAK-RC
Gal4 Active Domain
• The bait and prey sequences
interact…
• which causes the active and
binding domains to interact,
forming a whole promoter...
• Which recruits the transcription
complex to the reporter gene.
Gal4 Binding Domain
Gal4-Homologous Promoter
RNA Polymerase
II Complex
Reporter gene: Produces a necessary amino acid
Two-Hybrid Tests at Genome Scale
• Ito made a bait plasmid and a prey plasmid out of each protein to be
tested. All baits were gender a, all preys were gender alpha.
• Now Ito was ready to observe interactions between any two proteins
by crossing any bait and prey samples, and watching for the reporter
genes.
• How could this method be automated or scaled-up from two proteins
to the whole transcriptome? By doing the tests 100 at a time.
• 3.5x107 matings were performed, 96 at a time. 62 pools of bait and
prey clones were mixed from individual strains of one gender
arbitrarily, and those pools were mated to each other in a Millipore
1225 sampling manifold.
Genome Scale Methodology
• 3,844 matings were performed by mixing a bait strain pool (with a Gal4
DNA-binding domain) and a prey strain pool(with a Gal4 activation
domain), then selecting for the reporter genes ADE2, URA3, and HIS3 on
deficient media, which all use GAL4 promoters in the custom strain of
Cervesiae used for the experiment.
• Interactions that activated the reporter genes (and so survived) were
transplanted to another plate containing 5-bromo-4-chloro-3-indolyl-a-Dgalactopyranoside, which confirmed activation of the endogenous gene
Mel1, which is normally activated by Gal4. This verifies there is no loss-offunction associated with the exogenous Gal4.
• Once an interaction is found, the IST in the plasmid was sequenced and
BLAST and the Yeast Protein Database were checked for the protein.
Results
• 3,844 mating reactions produced 15,523 surviving colonies in which
all three reporter genes were activated (Histidine+, Uracil+,
Adenine+).
• The ISTs of each clone were sequenced, reducing the dataset to
4,549 unique high-certainty interactions involving 3,278 proteins.
Some proteins were part of two or three reactions.
• The amount of interactions the screen found that are already known
(in the database) indicates the screen’s sensitivity.
Uetz
691 Interactions
88 Known
12.7% Known
Ito
841 Interactions
105 Known
12.5% Known
Results – the Bad News
• Strangely, with equally sensitive
screens, only 135 (28.3%) of all
known interactions were found
independently by both studies.
• Neither study found even a fifth
of the known interactions they
were looking for: There is
substantial randomness in this
method at the genome scale.
Results – the Good News: Protein Networks
• Protein network sizes vary wildly: One network of 417 proteins, and
132 networks of 2-12 proteins. The large network probably reflects
crosstalk inside the cell, since Ito checked the YPD to look for
networks of similar size, and found networks containing up to 1000
proteins in projects that did not use two-hybrid methods.
• This is excluding RNA polymerase proteins. Including them, 87% of all proteins
are in one cluster.
• The authors have developed a tool to query databases to indicate if
two proteins are found in the same complex, are co-expressed, their
interactions, and if those interactions have been independently
confirmed.
Interesting Protein Networks
Autophagy. Arrows in one direction
indicate an interaction from bait to prey,
arrows in both directions indicate twoway interactions. Circular interactions
indicate a protein complex.
Apg16-Mec3 is involved in the G2 DNA
damage checkpoint.
Spindle pole function. Black proteins are
hypothetical, of unknown function. The
authors would like to do knockout analysis
of these proteins: Interactions do not tell
you everything.
Conclusions
• A genome-scale two-hybrid analysis of the transcriptome of saccharomyces
Cervesiae found novel protein networks for further study.
• However, a replicate of this project failed to duplicate these results.
• Errors in PCR while amplifying ORFs.
• Different plasmids for inserting ORFs may affect folding between
replicates.
• Different reporter genes in the replicate study.
• Reporter genes activate for no apparent reason sometimes. The authors
may not have corrected for this perfectly.
• Mating may not occur between every pair of yeast strains in a pool of
200, leading to false negatives.
• Not all interactions cause reporter gene activation to the same degree,
but that is difficult to correlate to interaction strength.
Further Reading
• An earlier test of this two-hybrid method: Ito, T., et al. "Toward a
Protein-protein Interaction Map of the Budding Yeast: A
Comprehensive System to Examine Two-hybrid Interactions in All
Possible Combinations between the Yeast Proteins." PROCEEDINGS
OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF
AMERICA 97.3 (2000): 1143-147. Web. 28 Feb. 2017.
• The independent replicate of this study: Uetz, P., et al. "A
Comprehensive Analysis of Protein-protein Interactions in
Saccharomyces Cerevisiae." Nature 403.6670 (2000): 623-27. Web. 28
Feb. 2017.