Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromatic function

Abstract

In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It is required for proper chromatin condensation, and changing its dose modifies position-effect variegation. Sumoylation is a post-translational modification shown to play a role in diverse biological processes. Here, we demonstrate that sumoylation is essential for proper heterochromatin function in Drosophila through modification of SU(VAR)3-7. Indeed, SU(VAR)3-7 is sumoylated at lysine K839; this modification is required for localization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4, and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisite for its ability to enhance position-effect variegation. Thus, these results show that the heterochromatic function of SU(VAR)3-7 depends on its own sumoylation, and unveil a role for sumoylation in Drosophila heterochromatin.

Figure 1.

SU(VAR)3-7 is sumoylated at lysine K839. (A) Sumoylation of SU(VAR)3-7. A GFP-Su(var)3-7 construct (amino acids 82–1250) or the empty vector (vect) were transfected in S2 cells together with the empty vector, 3HA-SUMO, or 3HA-ubi. Western blot analysis with an α-GFP shows the different forms of GFP-SU(VAR)3-7, namely a doublet when expressed alone, and an additional band when co-expressed with 3HA-SUMO. 3HA-SUMO and 3HA-ubi are both expressed (data not shown). (B) Profile of SU(VAR)3-7 upon co-expression of Ulp1. GFP-SU(VAR)3-7 was expressed with 3HA-SUMO or the empty vector, and with or without Ulp1. DNA amount was kept constant in transfections with empty vector. Proteins were analyzed as in A. (C) Sumoylation of the endogenous SU(VAR)3-7 protein. Total proteins were extracted from S2 cells and endogenous SU(VAR)3-7 was immunoprecipitated from 500 µg total extract with α-SU(VAR)3-7 Ab1399. One-fiftieth of the total extract (input), one-tenth of the negative control IP without antibody (neg) and one-tenth of the IP (IP) were loaded twice and analyzed by western blot with α-SU(VAR)3-7 Ab1399 or α-SUMO. The identity of the bands was verified by running in parallel extract from cells overexpressing SU(VAR)3-7 (data not shown). Square: non-modified SU(VAR)3-7. Circle: sumoylated SU(VAR)3-7. Stars: SU(VAR)3-7 with possibly several molecules of SUMO or other additional post-translational modifications (see discussion). (D) Sumoylation of the K269R mutated form. GFP-SU(VAR)3-7(WT) or (K269R) expressed with the empty vector or 3HA-SUMO were analyzed as in A. (E) Lack of sumoylation in the SU(VAR)3-7(K839R) mutant. GFP-SU(VAR)3-7(WT), with eight or nine K to R substitutions (8KR or 9KR), or with the K839R substitution, were expressed with 3HA-SUMO or the empty vector. Proteins were analyzed as in A. (F) Absence of sumoylation in the 3HA-tagged full-length SU(VAR)3-7 bearing the K839R substitution. 3HA-Su(var)3-7 full-length (amino acids 1–1250) constructs, namely WT, 9KR and K839R mutant forms, or the empty vector, were expressed in S2 cells together with the empty vector or 3HA-SUMO. Proteins were analyzed as in A., but with α-HA. (G) Expression of 3HA-SU(VAR)3-7(WT) and (K839R) in transgenic lines. Western blot analysis on brain, salivary glands, and imaginal discs (dissected together) protein extracts of control, 3HA-SU(VAR)3-7(WT) or (K839R) expressing third instar larvae. Membranes were cut and probed with α-HA and α-tubulin. Genotypes are the following: lane 1: y⁻w⁻; P{daGal4. w⁻}/+. Lane 2: y⁻w⁻;P{UAST-3HA-Su(var)3-7(WT); y⁺}/+; P{daGal4.w⁻}/+. Lane 3: y⁻w⁻; P{UAST-3HA-Su(var)3-7(K839R); y⁺}/+; P{daGal4.w⁻}/+.

Figure 2.

Sumoylation of SU(VAR)3-7 is required for localization at heterochromatin. (A) Expression and cellular localization of 3HA-SU(VAR)3-7(WT) and (K839R) proteins. Whole mount salivary glands of y⁻w⁻; P{UAST-3HA-Su(var)3-7(WT)or(K839R); y⁺}/P{UAST-3HA-Su(var)3-7(WT)or(K839R); y⁺}; Su(var)3-7^R2a8/ P{daGal4; w⁺} Su(var)3-7^R2a8 third instar larvae were stained with α-HA. First lane shows whole salivary glands, second lane enlargements of the nuclei (pictures taken on a confocal microscope). (B) Localization of 3HA-SU(VAR)3-7(WT) and HP1 on chromatin. Polytene chromosomes of larvae expressing 3HA-SU(VAR)3-7(WT) were stained with α-HA or α-HP1. DNA was stained with DAPI. Genotypes are the same as in Figure 2A. ‘C’ stands for chromocenter, ‘tel’ for telomere, ‘r31’ for region 31, and ‘chr4’ for Chromosome 4. (C) Localization of 3HA-SU(VAR)3-7(K839R) and HP1 on chromatin. Polytene chromosomes of larvae expressing 3HA-SU(VAR)3-7(K839R) were stained with α-HA and α-HP1 together. Genotypes and arrows codes are the same as in Figure 2A and B.

Figure 3.

(A) Sumoylation is required for PEV. Repression of the variegating white gene in the In(1)w^m4h line is relieved in the lwr heterozygous mutant background (left part). Data are shown for the females; the same trend is observed in males, as well as in the Heidi line (data not shown). The repression of the variegating white gene in the presence of an additional dose of Su(var)3-7 (T21A transgene) is also relived in the lwr heterozygous mutant background (right part). Pictures were taken and quantification of pigments was done 3 days after eclosion. The scale corresponds to the absorption at OD 480 nm. Genotypes, from left to right: 1) In(1)w^m4h / y⁻w⁻; + / lwr⁵ FRT40A. 2) In(1)w^m4h / y⁻w⁻; + / lwr^4-3 FRT40A. 3) In(1)w^m4h / y⁻w⁻; + / +. 4) In(1)w^m4h / y⁻w⁻; T21A / lwr⁵ FRT40A. 5) In(1)w^m4h / y⁻w⁻; T21A / lwr^4-3 FRT40A. 6) In(1)w^m4h / y⁻w⁻; T21A / +. (B) Sumoylation of SU(VAR)3-7 is required for PEV. The variegating white transgene in the Heidi line is further repressed upon expression of 3HA-Su(var)3-7(WT), but not upon expression of 3HA-Su(var)3-7(K839R). Pictures were taken 2 days after eclosion. The eye color phenotype was constant from fly to fly. Genotypes, from top: (i) y⁻w⁻; Heidi / +; P{daGal4.w⁻} / +. (ii) y⁻w⁻; Heidi / P{UAST-3HA-Su(var)3-7(WT); y⁺}; P{daGal4.w⁻} / +. (iii) y⁻w⁻; Heidi / P{UAST-3HA-Su(var)3-7(K839R); y⁺}; P{daGal4.w⁻} / +.