Role of mod(mdg4)-67.2 protein in interactions between su(hw)-dependent complexes and their recruitment to chromatin

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Abstract

Su(Hw) belongs to a class of proteins that organize chromosome architecture, determine promoter activity, and participate in the formation of boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms a complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study the functioning of Su(Hw)-dependent complexes, we used the previously described su(Hw)E8 mutation, with inactive seventh zinc finger, which produced the mutant protein losing the ability to bind to the consensus site. The present work shows that the Su(Hw)E8 protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)E8 protein can be recruited into Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that DNA-unbound Su(Hw)-dependent complexes can be recruited to Su(Hw)-binding sites through specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.

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L. S. Melnikova

Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: lsm73@mail.ru
Russian Federation, Moscow

V. V. Molodina

Institute of Gene Biology, Russian Academy of Sciences

Email: lsm73@mail.ru
Russian Federation, Moscow

P. G. Georgiev

Institute of Gene Biology, Russian Academy of Sciences

Email: lsm73@mail.ru
Russian Federation, Moscow

A. K. Golovnin

Institute of Gene Biology, Russian Academy of Sciences

Email: lsm73@mail.ru
Russian Federation, Moscow

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2. Fig. 1. Su(Hw) protein derivatives used in the work. a – Schematic representation of the full-length Su(Hw) protein. Domain designations: CID – domain interacting with CP190; ZF – zinc fingers; LZ – leucine zipper. The diagram shows the regions of interaction with the Mod(mdg4)-67.2 (Mod-67.2) and CP190 proteins, which are depicted as ovals. The vertical arrow indicates the su(Hw)E8 mutation. The bracket under the diagram indicates the region of the protein to which the antibodies were obtained. The amino acid residues limiting the domains and derivative forms are indicated by numbers. The names of the derivatives are indicated on the left, the size of the derivatives is indicated by segments, the dotted lines indicate internal deletions, the su(Hw)E8 mutation is indicated by an asterisk. The results obtained in DDS are shown to the right of the diagrams. “+” – presence of interaction, “–” – absence of interaction. b – Deletion derivatives used in genetic experiments and in immunostaining of polytene chromosomes

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3. Fig. 2. Effect of the Su(Hw)E8 protein on gypsy-dependent insulation. a – Schematic representation of the y2 and ct6 alleles. The exons of the yellow and cut genes are shown as rectangles. The transcription initiation sites of the genes are indicated by arrows. The gypsy retrotransposon is shown as a triangle. The rectangles at its ends denote the long terminal repeats (LTRs), the orientation of which is indicated by arrows. Designations: Su(Hw) – Su(Hw) insulator; En-K – wing enhancer, En-T – body enhancer; En-Shch – bristle enhancer; En-Kp – wing plate enhancer. b – Effect of Su(Hw) protein derivatives on the activity of the gypsy insulator in the y2 and ct6 alleles against the background of the su(Hw)v/su(Hw)2 (v/2) and su(Hw)v/su(Hw)E8 (v/E8) mutations. The names of the lines used for phenotypic analysis are shown in the right column: wt – y2ct6, Su(Hw)+ – the transgene expressed the full-length protein. The schemes and names of the remaining derivatives are shown in Fig. 1. The numbers in the y2 column indicate the level of yellow gene expression in the body and wing cuticle. The photographs show changes in the wing phenotype of the cut gene against various mutant backgrounds.

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4. Fig. 3. Binding of the Su(Hw)E8 protein to polytene chromosomes. Immunostaining of polytene chromosomes of the salivary glands of third instar larvae from the y2ct6 (wt), y2ct6 su(Hw)v/su(Hw)2 (v/2), y2ct6;su(Hw)v/su(Hw)E8 (v/E8), y2ct6;su(Hw)vmod(mdg4)u1/su(Hw)E8mod(mdg4)u1 (v-m/E8-m) lines and from the same lines expressing the Su(Hw)ΔN-FLAG or Su(Hw)+-FLAG protein. Antibodies to the FLAG epitope (αFLAG) and to the N-terminal domain of the Su(Hw) protein (αSu(Hw)-N) were used in the experiments. Arrows point to the gypsy insertion at the end of chromosome X.

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5. Fig. 4. Binding of Su(Hw)E8 protein to SBS. a – Testing of Su(Hw) protein binding in y2ct6 lines using antibodies to the N-terminal domain of Su(Hw). b – Testing of Su(Hw)ΔN protein binding in y2ct6;Su(Hw)ΔN-FLAG/Su(Hw)ΔN-FLAG lines using antibodies to the FLAG epitope. c – Testing of Su(Hw)E8 protein binding in y2ct6;Su(Hw)ΔN-FLAG/Su(Hw)ΔN-FLAG lines using antibodies to the N-terminal domain of Su(Hw). The coding region of the ras64B gene (ras) was used as a control, which does not contain Su(Hw) protein binding sites. The percentage of enrichment of immunoprecipitated DNA (Y axis) was calculated relative to the amount of loaded DNA. The names of the selected Su(Hw)-dependent sites are indicated at the bottom (X-axis). The standard deviation for three independent biological replicates is shown. Significance levels (Student's t-test) p < 0.05. Designations: wt – wild type; v-m/2-m – combination of mutations su(Hw)vmod(mdg4)u1/su(Hw)2mod(mdg4)u1; IgG – immunoglobulins. Other designations are as in Figs. 2 and 3

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6. Fig. 5. Model of recruitment of Su(Hw)-dependent complexes to SBS during DNA replication.

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