Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster

Abstract

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.

FIG. 1

white P-element transformation vectors. Symbols: E, the upstream regulatory region of white that contains the eye- and testes-specific enhancers; mw, mini-white cDNA expression cassette which includes the proximal promoter, with the white transcription start site depicted by the arrow; S′, scs′; S, scs; 3′, apoB 3′ MAR; 5′, apoB 5′ MAR; ATR, ATR MAR; apo, apoB transcribed sequence; black rectangles at the ends of each transposon, 5′ and 3′ P elements. Vectors: a, EmwS′; b, SEmwS′; c, apoB3′MEmwS′; d, mwS′; e, SmwS′; f, apoB3′mwS′; g, ATRMmwS′; h, apoB5′MmwS′; i, apoBmwS′; j, apoB3′MEmw; and k, apoB3′Mmw.

FIG. 2

Copy number determinations for the P-element transformants. (A) mwS′ transformants. Genomic DNA was isolated, digested with XbaI, separated on an agarose gel, and probed with a labeled, ∼400-bp EcoRI/BamHI DNA fragment containing scs′. The endogenous scs′ DNA fragment of ∼ 10 kb is indicated by the arrow. Each transformant contained, in addition, one (lanes 1 to 4) or two (lanes 5 and 6) additional scs′ fragments, corresponding to single or double transgene insertions. The marker (M) lane contains DNA fragments of 5, 10, 15, and 20 kb. (B) apoB5′MmwS′ transformants. SpeI-digested genomic DNAs were probed with a labeled, ∼1.0-kb XbaI/XhoI DNA fragment containing the apoB 5′ MAR. Single (lanes 2, 4, 5, 6, 9, 10, 11, and 12) and double (lanes 1, 3, 7, and 8) transgene insertions were obtained.

FIG. 3

Eye color phenotypes of Drosophila transformants expressing mini-white from white enhancer-containing vectors. Eyes of 4-day-old females heterozygous for each of the different, single-copy P-element insertions were classified as being light orange (phenotypic class III), orange (IV), red (V), or dark red (VI). A representative of each of the phenotypes obtained with the different white vectors is shown, and the number of independent transformed lines with that phenotype is indicated below each picture. (a) EmwS′ transformants had eye color phenotypes that varied widely. (b) SEmwS′ transformants had dark red (VI) eyes. (c) apoB3′MEmwS′ transformants had primarily red (V) or dark red (VI) eyes. (d) apoB3′MEmw transformants had red (VI) eyes.

FIG. 4

Eye color phenotypes of Drosophila transformants expressing mini-white from white enhancerless vectors, series 1. Four-day-old females heterozygous for each of the different, single-copy P-element insertions were classified as having light yellow (phenotypic class I), yellow (II), light orange (III), orange (IV), or red (V) eyes. A representative of each of the phenotypes obtained with the different white vectors is shown, and the number of transformed lines with that phenotype is indicated below each picture. (a) mwS′ transformants were widely distributed in all five phenotypic classes. (b) SmwS′ transformants all had light orange (III) eyes. (c) apoB3′MmwS′ transformants had primarily yellow (II) or light orange (III) eyes. (d) Most apoB3′Mmw transformants had yellow (II) eyes, but flies with light orange (III), orange (IV) and red (V) eyes were also obtained.

FIG. 5

Eye color phenotypes of Drosophila transformants expressing mini-white from white enhancerless vectors, series 2. Four-day-old females heterozygous for each of the different, single-copy P-element insertions were classified as having light yellow (phenotypic class I), yellow (II), light orange (III), orange (IV), or red (V) eyes. A representative of each of the phenotypes obtained with the different white vectors is shown, and the number of transformed lines with that phenotype is indicated below each picture. (a) ATRmwS′ transformants had yellow (II) or light orange (III) eyes. (b) apoB5′MmwS′ transformants and (c) apoBmwS′ transformants were widely distributed in the light yellow (I), yellow (II), light orange (III), and red (V) phenotypic classes.

FIG. 6

Eye pigment expression in the different phenotypic classes. Eye pigment was extracted from pools of each phenotypic class and quantitated spectrophotometrically as described in Materials and Methods. The mean optical density at 480 nm per head is indicated. The phenotypic classes are light yellow (I), yellow (II), light orange (III), orange (IV), red (V), and dark red (VI).