Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster

Abstract

Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3' MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed.

FIG.1.

Factors affecting insulation of mini-white transgenes by the human apoB 3′ MAR. Transgenic Drosophila lines containing the transgenes shown above each panel were generated by P-element transformation as described in Materials and Methods. Each transformed line was assigned to one of five eye color phenotypes—1, light yellow; 2, yellow; 3, light orange; 4, orange; or 5, red—based on the eye colors of 4-day-old females homozygous for the P-element. Examples of each eye color phenotype are shown in the photographs below panels A and B. The colored bars represent the number of independent transformed lines exhibiting that particular eye color phenotype. Key: 3′, apoB 3′ MAR; mini-., mini-white expression cassette; s′, scs′; 3′ (inverted), apoB 3′ MAR in the reverse orientation; Δ, 5′ and 3′ P-element ends. (A) Mini-white flanked by the apoB 3′ MAR at the 5′ end and scs' at the 3′ end; (B) mini-white containing only scs′ at the 3′end; (C) mini-white containing only the apoB 3′ MAR at the 5′ end; (D) mini-white flanked by the apoB 3′ MAR in the reverse orientation at the 5′ end and scs′ at the 3′ end; (E) mini-white flanked by the apoB 3′ MAR both 5′ and 3′. The MAR downstream of mini-white is in the inverted 3′-to-5′ orientation. (F) Mini-white containing two copies of the apoB 3′ MAR upstream of the transcription unit.

FIG. 2.

Excision of the apoB 3′ MAR after integration does not alter mini-white transgene expression. The transgenes in each collection of transformants are illustrated within each panel of the figure, where mini-. is the mini-white expression cassette, s′ is scs′, 3′ is apoB 3′ MAR, the blue boxes are the flp recombinase sites, and triangles indicate the 5′ and 3′ P-element ends. (A) The mwS′ lines used as controls in thisfigure are the same as those shown in Fig. 1A. The colored bars represent the number of independent transformed lines exhibiting that particular eye color phenotype, as follows: 1, light yellow; 2, yellow; 3, light orange; 4, orange; or 5, red. (B) The MARFRT transformants containing the mini-white transgene flanked by the apoB 3′ MAR at the 5′ end and scs′ at the 3′ end. Transgenic Drosophila lines containing the MARFRT transgene were prepared by P-element transformation as described in Materials and Methods. (C) −MARFRT lines were generated from the original +MARFRT transformants shown in panel B by precise excision of the apoB 3′ MAR by using flp recombinase as described in Materials and Methods.

FIG. 3.

Mini-white transgenes flanked by the apoB 3′ MAR and scs′ maintain insulation when mobilized to new locations in the Drosophila genome; transgenes without the apoB 3′MAR do not. P-element transgenes in the MAR-containing transformants MARFRT-D and MARFRT-F and their −MAR derivatives, −MARFRT-D and −MARFRT-F, respectively, were mobilized by expression of P-element transposase to create lines with novel transgene insertions sites, as illustrated schematically in panel A. Each line represents a particular Drosophila chromosome, the asterisks denote novel insertion sites, and the number below each line indicates the chromosome into which the P-element inserted after mobilization. (B) Eye color phenotypes of collections of Drosophila transformants created by mobilizing the P-elements in MARFRT-D (left) and MARFRT-F (right). The mobilized MARFRT and −MARFRT transgenes are illustrated inside each panel, where the blue boxes indicate the flp recombinase recognition sequences, 3′ is the apoB 3′ MAR, mini-. is the mini-white expression cassette, s′ is scs′, and the “▵” symbols indicate the 5′ and 3′ P-element ends. The colored bars represent the number of lines with novel MARFRT-D (left) and MARFRT-F (right) insertions exhibiting that eye color phenotype as follows: 1, light yellow; 2, yellow; 3, light orange; 4, orange; and 5, red. (C) Eye color phenotypes of collections of Drosophila transformants created by mobilizing the P-elements in −MARFRT-D (left) and −MARFRT-F (right). Panel C is organized as described above for panel B.

FIG. 4.

Models for mechanisms of transgene insulation by the human apoB 3′ MAR. (A) Nonrandom targeting of the .-element transgene. The P-element containing either two copies of the apoB 3′ MAR or the apoB 3′ MAR and scs′ flanking the mini-white cassette associate with the nuclear matrix before integration of the P-element into the Drosophila genome. (B) Epigenetic chromatin alteration. The apoB 3′ MAR and scs′ cause an epigenetic change in local chromatin structure that results in insulation of the transgene that can be transmitted from one generation to the next.

FIG. 5.

The human apoB 3′ MAR binds to the Drosophila nuclear matrix in vivo. (A) Schematic of a P-element inserted into the genome of the transgenic Drosophila line MARFRT-D; the blue boxes indicate flp recombinase recognition sequences, 3′ is the apoB 3′ MAR, mini-. is the mini-white expression cassette, s′ indicates the scs′, and the “▵” symbols indicate the 5′ and 3′ P-element ends. (B) An in vivo MAR assay showing partitioning of the human apoB 3′ MAR with the Drosophila nuclear matrix. Nuclei were isolated from 0 to 18 h old Drosophila embryos, chromatin proteins were extracted with LIS, and the resulting nuclear halos of DNA were digested with PstI, BamHI, BglII, XhoI, HindIII, and NotI. Matrix-associated (P, pellet) and released (S, supernatant) DNA fragments were separated by centrifugation and purified, and 5 μg of DNA of each fraction was analyzed by Southern hybridization with the human apoB 3′ MAR fragment as a probe. Total (T) genomic DNA was purified from MARFRT-D embryos and used as a control.

FIG. 6.

MAR-Wiz predictions of the locations of Drosophila MARs around P-element insertion sites. The arrows represent the different insertion sites of transgenes among all of the +MAR and −MAR transformants; each “M” represents the location of the nearest predicted MAR in the Drosophila genome. Predicted MAR sequences were identified in the vicinity of P-element insertion sites in both +MAR and −MAR transformants.