SUMO conjugation attenuates the activity of the gypsy chromatin insulator

Abstract

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. We present evidence suggesting that two protein components of the gypsy chromatin insulator of Dorsophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation.

Figure 1

Mod(mdg4)2.2 and CP190 are modified by SUMO in vitro. The size of various protein bands is indicated in kDa. The unmodified form of Mod(mdg4)2.2 runs at 100 kDa, and the unmodified form of CP190 is 190 kDa. The sumoylated form of CP190 is 220 kDa and Mod(mdg4)2.2 shows two different sumoylated bands of 120 and 135 kDa. (A, B) In vitro sumoylation reactions with ³⁵S-labeled CP190 (A) or Mod(mdg4)2.2 (B) used as substrate, in the presence or absence of SUMO reaction components (SUMO rxm), including E1, E2 enzymes, SUMO and ATP, or of dTopors. Lane 1, CP190 alone; lane 2, CP190 with SUMO rxm; lane 3, CP190 with SUMO rxm and in vitro-generated dTopors; lane 4, Mod(mdg4)2.2 alone; lane 5, Mod(mdg4)2.2 with SUMO rxm; lane 6, Mod(mdg4)2.2 with SUMO rxm and in vitro-generated dTopors. Arrows point to sumoylated forms of CP190 and Mod(mdg4)2.2. (C) In vitro sumoylation reactions in the presence or absence of SUMO E1 and E2, SUMO, Mod(mdg4)2.2 or dTopors monitored with α-SUMO antibodies. The arrow points to the Mod(mdg4)2.2-specific SUMO-GST conjugate. The lower molecular weight band marked with an asterisk corresponds to Ubc9-SUMO-GST. (D) GST-Ubc9 or GST, bound to glutathione beads, were mixed with His₆-Mod(mdg4)2.2 in the presence or absence of His₆-dTopors. The precipitated fractions and input proteins were resolved by SDS–PAGE and Western blotted with α-Mod(mdg4)2.2 or α-dTopors antibodies.

Figure 2

Mod(mdg4)2.2 and CP190 are sumoylated in vivo. The size of various protein bands is indicated in kDa. The unmodified form of Mod(mdg4)2.2 runs at 100 kDa, and the unmodified form of CP190 is 190 kDa. The sumoylated form of CP190 is 220 kDa and Mod(mdg4)2.2 shows two different sumoylated bands of 120 and 135 kDa. (A) Protein extracts from Drosophila larvae were prepared in the presence or absence of SUMO Isopeptidase Inhibitors (SII) NEM and IAA, resolved by SDS–PAGE and Western blotted with antibodies to SUMO and indicated gypsy insulator proteins. (B) Larval protein extracts from wild-type organisms or indicated lwr mutants were prepared in the presence of SII, resolved by SDS–PAGE and Western blotted with antibodies to indicated proteins. Triangles indicate increasing amounts of extracts loaded on the gels. Asterisk indicates a non-Mod(mdg4)2.2 band recognized by the antibody (Mongelard et al, 2002). (C) Protein extracts from wild-type (−) or UAS-dTopors/ActGAL4 (+) larvae were prepared in the presence of SII, resolved by SDS–PAGE and Western blotted with antibodies to indicated proteins.

Figure 3

SUMO is associated with a fraction of gypsy protein complexes on chromatin. White arrows point to places of colocalization between SUMO and insulator proteins. DNA is stained with DAPI (blue). (A) Immunostaining of polytene chromosomes of third instar larvae with antibodies to Mod(mdg4)2.2 (red) and SUMO (green). (B) Immunostaining of polytene chromosomes with antibodies to CP190 (red) and SUMO (green).

Figure 4

Mutations in components of the SUMO conjugation pathway promote activity of the gypsy insulator. Shown are abdomens, wings and eyes of y²omb^P1-D11ct⁶; +; +, y²omb^P1-D11ct⁶; +; mod(mdg4)^u1, y²omb^P1-D11ct⁶; lwr⁵⁴⁸⁶/smt3⁴⁴⁹³; mod(mdg4)^u1 and y²omb^P1-D11ct⁶; lwr²⁸⁵⁸/smt3^k06307; mod(mdg4)^u1 female flies.

Figure 5

Sumoylation does not affect the binding of CP190 and Mod(mdg4)2.2 to chromatin. DNA is stained with DAPI (blue). (A) Immunostaining of polytene chromosomes from wild-type, UASlwr/ActGAL4 and lwr^5/5486 larvae with antibodies to Mod(mdg4)2.2 (red) and CP190 (green). (B) Immunostaining of polytene chromosomes from y²; +; mod(mdg4)^u1 and y²; lwr^5/5486; mod(mdg)^u1 larvae with antibodies to Mod(mdg4)2.2 (red) and CP190 (green). Arrows point to the y² locus (insets).

Figure 6

SUMO conjugation antagonizes nuclear coalescence of insulator proteins. (A) Immunostaining of diploid cells from brains and imaginal discs of wild-type and UASlwr/ActGAL4 larvae with antibodies to Mod(mdg4) (red). (B) Immunostaining of diploid cells of wild-type, mod(mdg4)^u1, lwr^5/5; mod(mdg)^u1 and lwr^5/5486; mod(mdg)^u1 larvae with antibodies to CP190 (red). DAPI alone is shown at left and in blue in overlay.

Figure 7

Model for the effect of SUMO modification on chromatin domain formation. Chromatin loop domains may be established through interactions via distant gypsy insulator complexes and further stabilized through the tethering function of dTopors (right). Sumoylation of CP190 and Mod(mdg4)2.2 interferes with their self-interactions, resulting in a breakdown of chromatin domains (left). Gray line represents the nuclear lamina, and yellow spheres represent nucleosomes.