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

Abstract

BackgroundInsulators 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0168-7) contains supplementary material, which is available to authorized users.

Three-Dimensional Genome Organization and Function in Drosophila

“…Recent studies suggest that the DNA binding proteins CTCF, Dwg, Pita, and ZIPIC can homodimerize. This may also contribute to interactions between insulator elements (Bonchuk et al 2015; Zolotarev et al 2016). The Cp190 and Mod(mdg4) proteins, themselves, are biochemically and functionally different.…”

Boundaries of loop domains (insulators): Determinants of chromosome form and function in multicellular eukaryotes

“…While fly dCTCF appears to form tetrameric complexes [98], only dimeric complexes have been reported for mammalian CTCF [99, 100]. Thus the predicted topology of loops anchored by convergent CTCF sites would be a stem-loop, like that containing the eve locus in flies [65]).…”

Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

“…Are all the three proteins involved in the bithorax complex regulation? If so, do they act redundantly, cooperate, or have distinct roles?Of the three proteins, CTCF is the most studied, although reports do not always agree in details (Gerasimova et al 2007;Mohan et al 2007;Bonchuk et al 2015). All studies conclude that loss of zygotic CTCF function results in posterior-toanterior transformations of abdominal segments A8-A6, which is caused by the reduced expression of the Abd-B gene.…”

“…Flies homozygous for the CTCF GE24185 allele ( Figure 1B) or heterozygous for the combination of CTCF y+1 /CTCF GE24185 alleles are viable but exhibit homeotic transformations similar to those seen in flies with the loss of CTCF function (Table 1, Figure 3). Recently, Bonchuk and coauthors proposed that CTCF GE24185 is a null allele and argued that the lethality of other CTCF alleles is due to additional unrelated mutations (Bonchuk et al 2015). It is hard to imagine that the three CTCF alleles (CTCF y+1 , CTCF P35.2 , and CTCF 9 ) all carry the same lethal mutation.…”