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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 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 dCTCFΔN;GE21485 indicate the presence of a rudimentary A7 tergite and hairs on the A6 sternite. Arrows in dCTCFΔC;GE24185 indicate an A5 to A4 transformation of the tergite. wt wild type, A4-A7 abdominal segments 4-7
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Fig5: 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 dCTCFΔN;GE21485 indicate the presence of a rudimentary A7 tergite and hairs on the A6 sternite. Arrows in dCTCFΔC;GE24185 indicate an A5 to A4 transformation of the tergite. wt wild type, A4-A7 abdominal segments 4-7

Mentions: A fifth predicted dCTCF allele, GE24185, has been described [55]. The viability of adults homozygous for the GE24185 mutation is reduced by a third or more, while F2 flies do not survive. The GE24185 mutation was generated by insertion of an EPS transposon in reverse orientation into the third exon of the dCTCF gene (Fig. 5a). The EPS transposon contains an hsp70 minimal promoter that drives transcription in the opposite orientation to the dCTCF gene [66]. The promoter is under control of a GAL4-responsive enhancer. As would be expected from its insertion site, the GE24185 disrupts expression of the dCTCF protein. Extracts prepared from F1 adults homozygous for the GE24185 mutation showed no dCTCF-specific bands when probed with antibodies directed against N-terminal or C-terminal regions of the dCTCF protein (Fig. 5b). Unlike the other dCTCF alleles, we found that two copies of the hsp83-dCTCF+ transgene rescued the F1 and F2 lethal phenotypes of the GE24185 mutation.Fig. 5


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 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 dCTCFΔN;GE21485 indicate the presence of a rudimentary A7 tergite and hairs on the A6 sternite. Arrows in dCTCFΔC;GE24185 indicate an A5 to A4 transformation of the tergite. wt wild type, A4-A7 abdominal segments 4-7
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: 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 dCTCFΔN;GE21485 indicate the presence of a rudimentary A7 tergite and hairs on the A6 sternite. Arrows in dCTCFΔC;GE24185 indicate an A5 to A4 transformation of the tergite. wt wild type, A4-A7 abdominal segments 4-7
Mentions: A fifth predicted dCTCF allele, GE24185, has been described [55]. The viability of adults homozygous for the GE24185 mutation is reduced by a third or more, while F2 flies do not survive. The GE24185 mutation was generated by insertion of an EPS transposon in reverse orientation into the third exon of the dCTCF gene (Fig. 5a). The EPS transposon contains an hsp70 minimal promoter that drives transcription in the opposite orientation to the dCTCF gene [66]. The promoter is under control of a GAL4-responsive enhancer. As would be expected from its insertion site, the GE24185 disrupts expression of the dCTCF protein. Extracts prepared from F1 adults homozygous for the GE24185 mutation showed no dCTCF-specific bands when probed with antibodies directed against N-terminal or C-terminal regions of the dCTCF protein (Fig. 5b). Unlike the other dCTCF alleles, we found that two copies of the hsp83-dCTCF+ transgene rescued the F1 and F2 lethal phenotypes of the GE24185 mutation.Fig. 5

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