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Abstract
Heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1), and both areinvolved in the condensation of DNA into heterochromatin. Most mutations in HP2 result in the
suppression of position effect variegation (PEV) in D. melanogaster, indicating a potential role in
heterochromatin formation. In previous experiments, ethyl methyl sulfunate (EMS) was used in a
genetic screen that recovered 17 mutants of HP2 (14 nonsense, three missense). Most isolated
mutations cause mitotic defects and all are homozygous lethal. The three missense mutations
identified, 288, P2763L, and 230, are on exons 6, 8, and 9, respectively. Because each missense
mutation induces a large phenotypic shift with just a single amino acid substitution, it is possible
the mutations affect binding sites for protein partners of HP2. A Yeast-2-Hybrid screen was
implemented to find proteins that bind wild-type HP2 exon 8, with a second screen to elucidate
those that display reduced association with exon 8 containing the P2763L mutation. 75 of 189
(38%) of interacting clones showed diminished association with the mutant exon 8, supporting
the hypothesis that certain protein partners can discriminate in binding between wild-type and
mutant HP2. Computational analysis of the proposed primary interactors with HP2 wasperformed to predict secondary and tertiary interactors and potential protein interaction
complexes. Further investigation of these protein interactors will use genetic and cytological
(polytene chromosome staining) tests to identify those that interact and/or co-localize with HP2.
Identification of partner proteins may reveal new information on HP2s structural role in
heterochromatin formation.
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white
white
WT
ln(1)wm4
Inversion
Position Effect Variegation (PEV) as Phenotypic Indicator: The reporter gene,
in this case white, responsible for deposition of red pigment in the eye of
Drosophila, acts as a phenotypic indicator for the juxtaposition of the white
gene to a heterochromatic region. PEV is observed in the ln(1)wm4 line where
the white genes location is inverted to a heterochromatic region from aeuchromatic site.
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HP1
HP2Merge
Interaction and Colocalization of HP1 and HP2: Heterochromatin protein one
(HP1) is involved in packaging DNA into heterochromatin. Heterochromatin
protein two (HP2), isolated in a previous Yeast-2-Hybrid screen as an interactor
of HP1, displays a nearly identical binding pattern to HP1 on polytene
chromosome immunofluorescence studies.
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T588I
Thr -> Ile
P2763L
Pro -> Leu
N3220I
Asn -> Ile
T588I
Strong Su(var)
High Mitotic Defects
N3220I
Moderate Su(var)
V. Low Mitotic Defects
P2763L
Strong Su(var)
High Mitotic Defects
1 2 3 4 5 6 8 9288 230G572
Is there a picture of the
Exon 8 Su(var)?
Su(Var) (Suppression of Variegation) Effect in HP2 Mutants: A reduced display
of the PEV pheontype occurs in individuals possessing one of the three
missense mutations of HP2, supporting HP2s role in heterochromatin
formation.
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Positive interactors
Pick clones that grow
on QDO with WT but
not with Mutant
Transform with WT and
Mutant HP2 plasmids
PCR and sequenceinteresting clones
Lose WT HP2 plasmid
by growing in
liquid culture
Yeast-2-Hybrid Screen Overview:
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GAL4BD
HP2
Bait
GAL UAS Minimal promoter Reporter gene
GAL4AD
Library
Prey
1 2 3 4 5 6 8 9
Exon 8
P2763L
Pro -> Leu
Exon 8 and Library Clone Interaction Results in Reporter Gene Expression: The Gal4 DNA Binding
Domain (GAL4BD) portion of the Bait plasmid peptide binds to the Upstream Activating Sequence
of the reporter gene. If there is an interaction between the exon 8 portion of the Bait peptide and
the clone portion of the Prey library, the Gal4 Activating Domain (GAL4AD) portion of the library
will initiate expression of the reporter gene by recruiting transcription machinery to the minimal
promoter. The reporter gene in this screen enables yeast to synthesize histidine and arginine. The
expression of both plasmid and yeast reporter genes allows for growth on Quadruple DropoutMedia (QDO), which is leucine, tryptophan, histidine, and arginine deficient.
Region of Exon 8 Screened: The portion of HP2 exon 8 that contains the
missense mutation, P2763L, that was screened is highlighted in red above.
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Bait Plasmid
TRP1GAL4-BD
HP2
Exon 8Prey library
LEU2
GAL4-AD
cDNA
Bait and Prey Plasmids: The Bait plasmid synthesizes a peptide containing a
portion of exon 8 and the Gal4 DNA Binding Domain (green). The Prey library
synthesizes a peptide containing a cDNA transcript and the Gal4 Transcriptional
Activating Domain (red). Both plasmids contain a genetic growth marker that
enables synthesis of certain amino acids(tryptophan for the Bait plasmid,
leucine for the Prey library). Thus, the presence of both plasmids allows for
yeast growth on Double Dropout Media (DDO), which is both tryptophan and
leucine deficient.
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Prey + WT
DDO
Prey + WT
QDO
Prey + M
QDO
Prey + M
DDO
Analyzing Wild-Type and
Mutant Bait Reinsertion:
(a) No growth on QDO for
both WT and Mutant:
neither wild-type nor
mutant interaction with the
Prey Library. (b) Growth on
QDO for both WT and
Mutant: wild-type and
mutant Bait plasmids
displayed interaction with
the Prey library. (c) Growth
on QDO only for WT: Onlythe wild-type Bait plasmid
interacted with the Prey
library. Note: Growth on
DDO confirms that both Prey
and Bait plasmids are
present.
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hp2
cg7457
cherckIIalpha-i3
cg32676
npc2a
tepII
mhc
droj2
ten-m
cg9784lqfrusnp
zasp66mod(mdg4)
cg8080
snshrs tub
pms2
actn amphcactcda5
cg12645 mxcsnap
sxcsyx1a
sinu cg31224
prc
HTIPs
(1 interactors)
2 interactors
3 interactors
nudE
cg8301
Interaction Hierarchy:
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cher
hp2
ckIIalpha-i3
cg32676
npc2a
cg7457
tepII
mhc
droj2
ten-m
cg9784
lqfr
usnp
zasp66
mod(mdg4)
cg8080
sns
hrs
tub
pms2
actn
amph
cact
cda5cg12645
mxc
snap
sxc
syx1a
sinu
cg31224
prc
cg8301
2 interactors3 interactors
HTIPs
(1 interactors)
Interaction Matrix:
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A3-3
bves
CG11781
CG12004
CG1516
CG15386
CG1696 CG17490
CG31224
CG3217
CG32373
CG32676
CG32690
CG32767
CG33978
CG42371 CG4271
CG7457
CG8080
CG8177
cher
dpr7
Droj2
l(3)mbn
Mhc npc2a
NTPase
nudE
Rab8
rdx
sinu
Smg6
Ten-m
TepII TER94
Yellow indicates multiple hits
Heterochromatin Protein Two Interacting Proteins (HTIPs):
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Genes with multiple
interacting clones:
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Conclusions and Future Studies
TBD