Abstract

Heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1), and both are involved 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 was performed 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.

Inversion

WT

white white

ln(1)wm4

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 a euchromatic site.

HP2

Merge

HP1

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.

T588I Thr -> Ile

1 2 3 4 5 6

P2763L Pro -> Leu

8 G572

N3220I Asn -> Ile

9 230

288

Is there a picture of the Exon 8 Su(var)?

T588I Strong Su(var) High Mitotic Defects

P2763L Strong Su(var) High Mitotic Defects

N3220I Moderate Su(var) V. Low Mitotic Defects

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.

Positive interactors

Lose WT HP2 plasmid by growing in liquid culture

Transform with WT and Mutant HP2 plasmids

PCR and sequence interesting clones

Pick clones that grow on QDO with WT but not with Mutant

Yeast-2-Hybrid Screen Overview:

P2763L Pro -> Leu

3 4

Exon 8

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.
GAL4AD HP2 Bait GAL4BD GAL UAS Minimal promoter Reporter gene
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 Dropout Media (QDO), which is leucine, tryptophan, histidine, and arginine deficient.

Library Prey

TRP1

GAL4-BD

HP2 Exon 8
Bait Plasmid

GAL4-AD

Prey library

cDNA

LEU2

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.

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: Only the wild-type Bait plasmid interacted with the Prey library. Note: Growth on DDO confirms that both Prey and Bait plasmids are present.

Prey + WT DDO

Prey + WT QDO

Prey + M QDO

Prey + M DDO

3 interactors
snap syx1a cda5 cg12645 sxc mxc cact actn amph

2 interactors
pms2 prc cg8301 usnp lqfr cg9784 mod(mdg4) sns zasp66 hrs tub

ckIIalpha-i3 cg7457 tepII sinu cg31224 nudE

cher npc2a

mhc

cg32676 ten-m cg8080

droj2

HTIPs (1 interactors)
hp2

Interaction Hierarchy:

3 interactors
hrs amph droj2 tub actn cact mxc ten-m hp2 cher lqfr cg32676 cg7457 tepII

pms2 2 interactors

prc cg31224

sinu

sxc
cg9784 usnp zasp66 npc2a mod(mdg4) cg8301 syx1a snap sns HTIPs (1 interactors) cg12645 cda5 mhc ckIIalpha-i3 cg8080

Interaction Matrix:

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):

Genes with multiple interacting clones:

Conclusions and Future Studies

TBD