Functional role of dimerization and CP190 interacting domains of CTCF protein in Drosophila melanogaster

Abstract

Background: Insulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man.

Results: In the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF-CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity.

Conclusions: Our results indicate that dCTCF-CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF-dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.

Fig. 1

a Domain structure of the dCTCF protein. b Sephacryl S200 size-exclusion chromatography of dCTCF terminal domains. (N-terminal domain is thioredoxin-tagged.) Positions of molecular weight markers are shown. c Cross-linking of dCTCF N-terminal thioredoxin-tagged deletion derivatives using increasing concentrations of glutaraldehyde (GA). Proteins were separated in a 5–12 % gradient SDS-PAGE gels and visualized with silver-staining. d Summary of the results from chemical cross-linking mapping experiments and limited proteolysis of the dCTCF–NTD multimerization domain. For further experiments see Additional file 2: Figure S2. e Superdex 200 size-exclusion chromatography of dCTCF 1–163 amino acids without thioredoxin. f Analysis of dCTCF protein N-terminal dimerization using yeast two-hybrid assay. Relative N- or C- terminal position of AD/BD is shown. AD GAL4 activation domain, BD GAL4 DNA binding domain

Fig. 2

a Schematic drawing of luciferase reporter constructs. b Firefly luciferase expression from the five reporters shown in a when co-transfected with empty vector, with a vector encoding a 3xFLAG-tagged-(nuclear localization signal)-LexA fusion protein, or with a vector encoding the 3xFLAG-tagged N-terminal dCTCF-(nuclear localization signal)-LexA fusion protein. A plasmid encoding the Renilla luciferase under the control of the actin promoter was used to correct for variations in transfection efficiency, and expression of the firefly luciferase was normalized in each case to Renilla luciferase. Each transfection experiment was performed in three independent biological replicates and each lysate was measured in four technical replicates. Error bars show standard deviations of measurements of all summarized replicates. c Chromatin immunoprecipitation of S2 cells co-transfected with the 4xLesA TATA-box reporter or the basic promoterless reporter and either of two fusion protein expression constructs, the 3xFLAG-tagged (nuclear localization signal) LexA construct or the 3xFLAG-tagged-N-terminal dCTCF-(nuclear localization signal)-LexA construct. Fixed and processed S2 chromatin samples were immunoprecipitated with antibodies directed against (as indicated) the dCTCF N-terminus, the dCTCF C-terminus, CP190, or FLAG, and then assayed for the presence of sequences corresponding to the 4xLexA TATA reporter or the basic reporter constructs as indicated. Each chromatin immunoprecipitation experiment was performed in three independent biological replicates. Error bars show standard deviations of summarized biological replicates after quadruplicate PCR measurements in each experiment. The results are presented as a percentage of input DNA. Basic no promoter, bla basic promoterless reporter, Hsp70 firefly luciferase with an hsp70 promoter, TATA firefly luciferase with a minimal TATA-box promoter, 4xlex bs, firefly luciferase with four copies of the LexA recognition sequence, 4xlex bs + TATA, firefly luciferase with four copies of the LexA recognition sequence linked to a minimal TATA-box promoter

Fig. 3

a Domain structure of the Drosophila CP190 protein. b Mapping dCTCF and CP190 interaction modules using the yeast two-hybrid assay. c Analysis of interactions between purified recombinant GST-dCTCF-CTD and 6xHis-CP190 by GST-pull-down assay. GST-dCTCF-CTD bound to glutathione agarose beads was incubated with bacterially expressed 6xHis-CP190. After successive washes, the GST-dCTCF-CTD protein was eluted from the beads with excess glutathione. d Analysis of interactions between recombinant GST-dCTCF-CTD and CP190 from Drosophila S2 cells nuclear lysate by GST-pull-down assay. An S2 nuclear extract was incubated with recombinant GST-dCTCF-CTD bound to glutathione agarose beads. After washing and elution with excess glutathione, CP190 and GAF association was assayed by western blotting. e Immunoprecipitation of FLAG-tagged dCTCF full-length and deletion mutants with CP190 antibodies. f Mapping of CTCF-interaction region within CP190 protein using GST-pull-down assay. AD activating domain, BD binding domain, S2 Schneider 2 cells

Fig. 4

a Analysis of complexes between dCTCF-CTD and thioredoxin-tagged CP190-BTB mixed at a molar ratio of 1:2 using the chemical cross-linking reagent glutaraldehyde. Proteins were visualized by Coomassie staining. a indicates position of CP190 BTB-domain monomer, b position of dCTCF-CTD, c dimer of CP190 BTB, d complex between CP190 BTB dimer and dCTCF-CTD. b Analysis of stoichiometry of interaction between dCTCF-CTD and CP190-BTB mixed in different molar ratios, and cross-linked with 0.2 % glutaraldehyde after 1 h incubation, visualized by silver-staining. a indicates position of CP190 BTB-domain monomer, b position of CTCF-CTD, c dimer of CP190 BTB, d complex between CTCF-CTD and CP190 BTB in molar ratio 1:2, and e higher order complex between CTCF-CTD and CP190 BTB with unknown stoichiometry. GA glutaraldehyde

Fig. 5

a Schematic diagram showing the GE24185 transposon insertion into the dCTCF gene. b Western blots of protein extracts prepared from wild-type and homozygous GE24185 mutant flies. c Schematic representation of dCTCF constructs used to rescue the GE24185 mutation. d Abdomen and cuticle preparations (bottom row) of wild-type and homozygous GE24185 mutant flies in the absence or presence of the hsp83:dCTCF transgenes as indicated. Arrows in GE24185 and dCTCF ^ΔN;GE21485 indicate the presence of a rudimentary A7 tergite and hairs on the A6 sternite. Arrows in dCTCF ^ΔC;GE24185 indicate an A5 to A4 transformation of the tergite. wt wild type, A4-A7 abdominal segments 4-7

Fig. 6

Histograms show dCTCF or CP190 occupancy in chromatin isolated from mid-late pupa at sequences containing the BX-C insulators Fab-3, Fab-4, Mcp, Fab-6, Fab-8, the Abd-D promoter, and several previously defined dCTCF insulators (9A1, 21E2, 24C4, 27B2 and 57B4R). Cross-linked chromatin prepared from wild-type (WT) (y ¹ w ¹¹¹⁸) pupae and homozygous GE24185 (GE/GE) mutant pupae was immunoprecipitated with antibodies directed against the N-terminal domain of dCTCF and CP190. Sequences from tub, rpl32, and 62D regions were used as negative controls for dCTCF and CP190 association. 62D is an example of a sequence in which CP190 occupancy is independent of dCTCF. The left axis shows the scale for dCTCF enrichment, while the right axis shows the scale for CP190 enrichment. Each chromatin immunoprecipitation experiment was performed in at least two independent biological replicates. Error bars show standard deviations of quadruplicate PCR measurements. The results are presented as a percentage of input DNA

Fig. 7

Histograms show dCTCF or CP190 occupancy in chromatin from mid-late pupa at sequences containing the BX-C insulators Mcp, Fab-6, Fab-8, the Abd-B promoter, and several other previously defined dCTCF insulators (9A1, 21E2, 24C4, 27B2, 57B4R). Chromatin was isolated from homozygous GE24185 mutant pupae that also carry the hsp83:dCTCF ⁺, hsp83:dCTCF ^ΔN , or hsp83:dCTCF ^ΔC transgenes. The tub sequence was used as the negative control. The left axis shows the scale for dCTCF enrichment, while the right axis shows the scale for CP190 enrichment. Each ChIP experiment with 2- to 3-day pupae was performed in at least two independent biological replicas. Error bars show standard deviations of quadruplicate PCR measurements. The results are presented as a percentage of input DNA. WT wild type

Fig. 8

Expression of Abd-B and Insv in stage 10/11 wild-type and dCTCF ^m-z- embryos. Stage 11 wild-type and stage 10 dCTCF ^m-z- (from cross of homozygous GE24185 parents) embryos were probed with antibodies directed against Abd-B (mouse monoclonal 1A2E9 from Developmental Studies Hybridoma Bank) and Insv (a rabbit polyclonal: gift of Tsutomu Aoki) and visualized by confocal microscopy. Parasegments are indicated in the Fig. Arrows in panels F and G indicate Abd-B expression in PS12. a Wild type: merged image. b Wild type: Abd-B. c Wild type: Abd-B. d WT: Insv. e dCTCF ^m-z-: merged image. f dCTCF ^m-z-:Abd-B. g dCTCF ^m-z-:Abd-B. h dCTCF ^m-z-: Insv. Red/Gray Abd-B, Blue Insv

Fig. 9

Expression of Abd-B, Insv, and En in stage 12 wild-type and dCTCF ^m-z- embryos. Stage 12 wild type (a-d) and dCTCF ^m-z- (e-h) were probed with antibodies directed against Abd-B (panels a,b e, and f), Insv (panels c and g), and En (panels d and h). Arrows in panel f point to Abd-B protein expression in PS12, PS11, and PS10 in the stage 12 dCTCF ^m-z- embryo. By contrast, arrows in panel b indicate that little or no Abd-B was detected in PS12 or PS11 of the wild-type embryo. See text for details