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

Bonchuk A, Maksimenko O, Kyrchanova O, Ivlieva T, Mogila V, Deshpande G, Wolle D, Schedl P, Georgiev P - BMC Biol. (2015)

Bottom Line: 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 allele.A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies.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.

View Article: PubMed Central - PubMed

Affiliation: Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

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 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 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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig1: 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

Mentions: The 11 zinc fingers of the CTCF proteins are highly conserved in bilaterian phyla [24, 25] (see schematic in Fig. 1a and Additional file 1: Figure S1A). In contrast, the NTDs and CTDs were poorly conserved and there was little sequence similarity even between proteins from different dipteran families (Additional file 1: Figure S1A). Sequence alignment of CTCF proteins from species within the Drosophila genus revealed much more extensive homology in the NTDs and CTDs, including several very well conserved sequence blocks (Additional file 1: Figure S1B). A plausible hypothesis is that these conserved sequences may serve as protein interaction modules that are important for dCTCF activities.Fig. 1


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

Bonchuk A, Maksimenko O, Kyrchanova O, Ivlieva T, Mogila V, Deshpande G, Wolle D, Schedl P, Georgiev P - BMC Biol. (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528719&req=5

Fig1: 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
Mentions: The 11 zinc fingers of the CTCF proteins are highly conserved in bilaterian phyla [24, 25] (see schematic in Fig. 1a and Additional file 1: Figure S1A). In contrast, the NTDs and CTDs were poorly conserved and there was little sequence similarity even between proteins from different dipteran families (Additional file 1: Figure S1A). Sequence alignment of CTCF proteins from species within the Drosophila genus revealed much more extensive homology in the NTDs and CTDs, including several very well conserved sequence blocks (Additional file 1: Figure S1B). A plausible hypothesis is that these conserved sequences may serve as protein interaction modules that are important for dCTCF activities.Fig. 1

Bottom Line: 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 allele.A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies.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.

View Article: PubMed Central - PubMed

Affiliation: Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.

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 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 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.

No MeSH data available.


Related in: MedlinePlus