y2h rough panels with some captions

8/2/2019 Y2H Rough Panels With Some Captions

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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

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