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First published online 29 April 2008
doi: 10.1242/jcs.018655

Journal of Cell Science 121, 1693-1703 (2008)
Published by The Company of Biologists 2008
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Interaction between the Drosophila heterochromatin proteins SUUR and HP1

Alexey V. Pindyurin1, Lidiya V. Boldyreva1, Victor V. Shloma1, Tatiana D. Kolesnikova1,2, Galina V. Pokholkova1, Evgeniya N. Andreyeva1, Elena N. Kozhevnikova1,*, Igor G. Ivanoschuk1, Ekaterina A. Zarutskaya1, Sergey A. Demakov1, Andrey A. Gorchakov1,{ddagger}, Elena S. Belyaeva1 and Igor F. Zhimulev1,2,§

1 Institute of Cytology and Genetics of Siberian Division, Russian Academy of Sciences, Novosibirsk 630090, Russia
2 Department of Cytology and Genetics, Novosibirsk State University, Novosibirsk 630090, Russia


Figure 1
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  		Fig. 1. Yeast two-hybrid analysis of interaction between SUUR and HP1. (A) Equal amounts of SKY191 cultures containing the constructs indicated in each case were planted at a density of 2x106 cells/ml (column 1) and serial tenfold dilutions (columns 2-4) on His–, Trp–, Leu– plates containing galactose and raffinose. Yeasts were grown for 7 days at 30°C. SKY191 cells containing pSH18-34 and indicated combinations of plasmids were streaked on His–, Trp–, Ura– plates containing galactose, raffinose and X-Gal (column 5) and incubated for 4 days at 30°C. (B) The SUUR bait deletion constructs were tested for interaction with the HP1 prey deletion constructs (Delattre et al., 2000Go). Minus and plus signs indicate the absence and presence of interaction between tested constructs, respectively; ND, no data. SUUR regions are shown: the homology region to the SNF2 domain is in yellow, the homology region to the bromodomain is in brown, the negatively charged amino acid clusters are in blue, the positively charged amino acid clusters are in red and the NLSs are in grey. In HP1 the chromodomain is indicated in yellow and the chromoshadow domain is in orange.

 

Figure 2
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  		Fig. 2. SUUR interacts with HP1 in vitro. The ability of the full-length SUUR and its central portion (AA 371-578) to recruit HP1 from Drosophila embryo nuclear extracts was tested by GST pull-down assays. GST alone, GST-SUUR (AA 1-962) and GST-SUUR (AA 371-578) were produced in bacteria and immobilized on glutathione-Sepharose beads. The beads were then incubated with nuclear extracts of wild-type Drosophila embryos. Bound proteins were washed, resolved by SDS-PAGE, and transferred to nitrocellulose. The blot was probed with antibodies against HP1. The amount in the input column is 5% of the amount applied to beads. SUUR constructs employed in the experiment are schematically depicted below the blot; the homology region to the SNF2 domain is in yellow, the homology region to the bromodomain is in brown, the negatively charged amino acid clusters are in blue, the positively charged amino acid clusters are in red, the NLSs are in grey.

 

Figure 3
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  		Fig. 3. Recruitment of SUUR to ectopically localized HP1. Immunolocalization of HP1 (red) and SUUR (green) in polytene chromosomes of hs-GAL4-HP1/Wink-D transheterozygotes before (A-C) and after (D-F) heat-shock-induced expression of GAL4-HP1.

 

Figure 4
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  		Fig. 4. Distributions of SUUR and HP1 on chromosomes are interdependent. Salivary gland polytene chromosomes from the indicated Drosophila strains were stained with antibodies against HP1 (column 2) and SUUR (column 4). Corresponding phase-contrast images are shown in columns 1 and 3.

 

Figure 5
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  		Fig. 5. Repeated overexpression of GAL4-HP1 results in the displacement of SUUR from chromosomes. SUUR (green) and HP1 (red) localization patterns were detected by immunofluorescence staining of polytene chromosomes from control (non-heat-shocked, A-C) and experimental (heat-shocked, D-F) hs-GAL4-HP1/Wink-D transheterozygous larvae.

 

Figure 6
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  		Fig. 6. Overexpression of HP1 leads to SuUR mutant phenotype. UR level of the region 89E in salivary glands polytene chromosomes of the hs-HP1 homozygous was measured without (lane 1) and with (lane 2) heat-shock treatments. The Southern blot was hybridized with the abd-A and rosy (control) probes; the ratios of abd-A-specific hybridization signals to rosy-specific hybridization signals are given below the blot.

 

Figure 7
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  		Fig. 7. Overexpression of full-length SUUR under the control of the Sgs3-GAL4 driver results in HP1 redistribution to the intercalary heterochromatin regions of polytene chromosomes. Sites of swellings (Zhimulev et al., 2003bGo) and the fourth chromosome are indicated.

 

Figure 8
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  		Fig. 8. Overexpressed N-terminal portion of SUUR recruits HP1 to ectopic sites. Immunofluorescent staining of polytene chromosomes with antibodies against SUUR (green) and HP1 (red) after overexpression of the N-terminal SUUR fragment rs4 (A) and of the C-terminal SUUR fragment C17 (B) under the control of the Sgs3-GAL4 driver in the SuUR mutant background. Schematics of the rs4 and C17 constructs are depicted below the corresponding panels; the homology region to the SNF2 domain is in yellow, the homology region to the bromodomain is in brown, the negatively charged amino acid clusters are in blue, the positively charged amino acid clusters are in red, the NLSs are in grey and the HA-tag is in white.

 

Figure 9
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  		Fig. 9. SuUR and Su(var)2-5 genes are expressed independently. RT-PCR analysis of SuUR, Su(var)2-5 and rp49 (control) expression levels in salivary glands of wild-type OregonR, SuUR, Su(var)2-503/Su(var)2-505 heterozygotes (HP1 null mutants), hs-SuUR (SUUR overexpression) and hs-HP1 (HP1 overexpression) strains. Minus and plus signs indicate the absence and presence of reverse transcriptase in RT-PCR reaction, respectively. Transcription of Su(var)2-5 gene in Su(var)2-503/Su(var)2-505 heterozygotes is not abolished because the Su(var)2-505 mutation is caused by a dinucleotide deletion in the coding region (Eissenberg et al., 1992Go); the molecular lesion associated with the Su(var)2-503 mutation is unknown.

 

Figure 10
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  		Fig. 10. High similarity between the localization patterns of SUUR (A) and HP1 (B) on chromosome arm 3L of a Su(var)3-9ptn/+ heterozygous larva.

 

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