Mutations in Su(var)205 and Su(var)3-7 suppress P-element-dependent silencing in Drosophila melanogaster

Abstract

In Drosophila melanogaster, the w(+) transgene in P[lacW]ci(Dplac) is uniformly expressed throughout the adult eye. However, when other P elements are present, this w(+) transgene is randomly silenced and this produces a variegated eye phenotype. This P-element-dependent silencing (PDS) is limited to w(+) transgenes inserted in a specific region on chromosome 4. In a screen for genetic modifiers of PDS, we isolated mutations in Su(var)205, Su(var)3-7, and two unidentified genes that suppress this variegated phenotype. Therefore, only a few of the genes encoding heterochromatic modifiers act dose dependently in PDS. In addition, we recovered two spontaneous mutations of P[lacW]ci(Dplac) that variegate in the absence of P elements. These P[lacW]i(Dplac) derivatives have a gypsy element inserted proximally to the P[lacW]ci(Dplac) insert. The same mutations that suppress PDS also suppress w(+) silencing from these P[lacW]ci(Dplac) derivative alleles. This indicates that both cis-acting changes in sequence and trans-acting P elements cause a similar change in chromatin structure that silences w(+) expression in P[lacW]ci(Dplac). Together, these results confirm that PDS occurs at P[lacW]ci(Dplac) because of the chromatin structure at this chromosomal position. Studying w(+) variegation from P[lacW]ci(Dplac) provides a model for the interactions that can enhance heterochromatic silencing at single P-element inserts.

Figure 1.—

Photographs depicting mutations in Su(var)205 and Su(var)3-7 suppressing w⁺ variegation from P{lacW}ci^Dplac when P{ry⁺ SalI}89D is present. Photographs were taken of male progeny from a cross between y w; P{ry⁺ SalI}(89D); P{lacW}ci^Dplac females and a male with the indicated mutation/balancer. Male progeny that inherited the mutation were compared to progeny that inherited the balancer to determine if the variegation is suppressed. All eye pigment production is dependent on the P{lacW}ci^Dplac chromosome. Alleles tested for a Su(PDS) phenotype are as follows: (A) CyO, (B) Su(var)205^P4, (C) Su(var)205^P5, (D) Su(var)205², (E) Su(var)205⁴, (F) Su(var)205⁵, (G) wg^Sp-1 Bl¹ L^rm Bc¹ Pu² Pin^B, (H) Su(var)205⁵* L^rm Bl¹, (I) TM6B, (J) Su(var)3-7^P9, (K) Su(var)3-7^P25, (L) Su(var)3-7¹⁴, (M) Su(var)3-7⁹, (N) Su(var)3-7^7.1, (O) Su(PDS)P80, and (P) Su(PDS)P86. The CyO and TM6B balancers produce a wild-type phenotype and the eye is predominately white.

Figure 2.—

Increasing the Su(var)3-7⁺ gene dose acts to silence w⁺ expression from P{lacW}ci^Dplac when P{ry⁺ SalI}89D is present. (A) Genetic cross to produce the progeny observed in B and C. (B) Pigment accumulation when P{ry⁺ SalI}89D and P{lacW}ci^Dplac is present at 21°. As indicated at the bottom of the graph, Su(var)3-7⁺ dosage increases from 1, 2 (transgene), 2, to 3 doses. Measurements are taken from three samples with 10 male heads each. Error bars represent standard error. (C) Photographs of eyes from flies with increasing Su(var)3-7⁺ dosage. Each row of eyes is collected from flies raised at the rearing temperature indicated. The Su(var)3-7⁺ dosage increases in each column from 1, 2 (transgene), 2, and 3 as indicated in B.

Figure 3.—

Mutations in Su(var)3-7 do not interrupt P{ry⁺ SalI}89D suppression of vg^21-3 or repression of P-lacZ. (A) w; vg^21-3/vg^B; Sb P{ry⁺ SalI}89D/TM6B, Ubx. (B) w; vg^21-3/vg^B; Su(var)3-7^P9/TM6B, Ubx. (C) w; vg^21-3/vg^B; Sb P{ry⁺ SalI}89D/Su(var)3-7^P9. (D) Wing imaginal discs were stained for β-galactosidase activity to determine if mutations in Su(var)3-7 affect P-lacZ expression from P{lacW}ci^Dplac. Changes in genotype of the wing imaginal discs are indicated at the top of each column and to the left of each row. All imaginal discs inherited P{lacW}ci^Dplac. The imaginal discs on the left (+) are from progeny of w; P{lacW}ci^Dplac females while the imaginal discs on the right (P{ry⁺ SalI}89D) are from progeny of w; P{ry⁺ SalI}89D; P{lacW}ci^Dplac females. These females were crossed with Su(var)3-7⁺, Su(var)3-7^P9, or Su(var)3-7^P13 males balanced with TM6B, Antp^Hu e Tb. Imaginal discs were collected from Tb+ larvae, which contain the indicated mutation.

Figure 4.—

Su(var)3-7^P9 suppresses w⁺ silencing caused by KP elements at P{lacW}ci^Dplac . Genotypes are indicated above each column and at the left of each row. All flies inherited a single P{lacW}ci^Dplac chromosome. Eyes on the left inherited the Su(var)3-7^P9 allele and have one functional copy. Eyes on the right inherited two functional copies. Eyes in the top row inherited KP-U and in the bottom row inherited KP-D. The flies that inherited KP-U chromosome were produced by crossing w; TM6B, Ant^Hu e Tb KP-U/Ly females to w; e/Su(var)3-7^P9; P{lacW}ci^Dplac males. The flies that inherited KP-D chromosome were produced by crossing w; Cy Bl vg KP-D/l(2)NS females to w; TM6B/Su(var)3-7^P9; P{lacW}ci^Dplac males.

Figure 5.—

The effect of Su(var)3-7⁺ dosage on pigment production from a variety of w⁺ transgenes in the absence of P elements. (A) The insert tested is indicated at the bottom of each set of bars. The insert P{hsp26-pt-T}2-M010.R is shortened to 2-M010.R. The Su(var)3-7⁺ dosage increases from 1 to 2 (transgene), 2, and 3 doses from left to right for each w⁺ transgene. The average pigment value was obtained from an average of three samples each containing 20 heads from flies reared at 21°. Error bars indicate standard error. Asterisk above data set indicates ANOVA analysis found a significant (P < 0.05) difference between the samples taken at the three different Su(var)3-7⁺ dosages. (B) Pigment production is dependent on the w⁺ transgene indicated to the left. From left to right the Su(var)3-7⁺ dosage increased by 1, 2 (transgene), 2, and 3 as indicated in A above each column.

Figure 6.—

Mutations in Su(var)205 and Su(var)3-7 suppress the variegated P{lacW}ci^E alleles. The P{lacW}ci^E allele being tested is indicated to the left. The Su(var) mutation being tested is indicated at the top. These male flies were generated by crossing w; P{lacW}ci^E females to males with mutant/balancer.

Figure 7.—

Diagram of P{lacW}ci^E alleles. P{lacW}ci^E1 has a gypsy element inserted between a 4-bp duplication (TACA) starting at 81,294 (AE003854). P{lacW}ci^E2 has a gypsy element inserted between a 4-bp duplication (TATA) starting at nucleotide at 80,747 (AE003854). Arrows indicate the direction of transcription.

Figure 8.—

Trans-silencing between P{lacW}ci derivatives and other w⁺ inserts in the ci region. (A) The P{lacW}ci^E alleles trans-silence w⁺ expression from P{lacW}ci^Dplac. Pigment produced depended solely on the P{lacW} allele(s) inherited. The abbreviation for each P{lacW} allele is shown at the bottom. Plus indicates a wild-type chromosome 4 that does not have a P{lacW} insertion. The different males were produced from a cross between w; P{lacW}ci^Dplac females and males with a P{lacW}ci^E1, P{lacW}ci^E2, or wild-type chromosome 4. (B) P{lacW}ci^E1 does not trans-silence w⁺ expression from the P{lacW}ci^Dplac translocation, T(3;4)DB63E, as strongly as the original P{lacW}ci^Dplac allele. Flies in the first two columns inherited P{ry⁺ SalI}89D and those in the last two columns inherited TM3, Ser. The translocation is in combination with either ey^D or P{lacW}ci^E1 as indicated. Eyes are from male siblings in a cross between w; T(3;4)DB63E/TM6B males and w; Sb P{ry⁺ SalI}89D/TM3; P{lacW}ci^E1/ey^D females. (C) P{lacW}ci^E2 does not trans-silence expression from the P{lacW}ci^Dplac translocation, T(3;4)DB63E, as strongly as the original P{lacW}ci^Dplac allele. Genotypes and labeling are the same as in B but P{lacW}ci^E2 is tested rather than P{lacW}ci^E1. Eyes are from male siblings from a cross between w; T(3;4)DB63E/TM6B males and w; Sb P{ry⁺ SalI}89D/TM3; P{lacW}ci^E2/ey^D females. (D) Trans-silencing between P{lacW}ci^2-M1021.R and P{lacW}ci alleles with and without P{ry⁺ SalI}89D present. Flies in the first two columns inherited P{ry⁺ SalI}89D and flies in the last two columns inherited TM3, Ser. The chromosome 4 combinations are indicated at the top. The inherited P{lacW}ci allele is indicated to the left. Eyes shown in each row are from siblings produced from a cross between w; Sb P{ry⁺ SalI}89D/TM3; P{lacW}ci^2-M1021.R/M^57g females and w; P{lacW}ci allele/ey^D, where the P{lacW}ci allele is P{lacW}ci^Dplac, P{lacW}ci^E1, or P{lacW}ci^E2 as listed to the left.

Figure 9.—

Pigment analysis testing whether the Su(PDS) mutants suppress In(1)w^m4 variegation in males. The crosses compare pigment production between siblings that inherit a mutation against those that inherit a CyO or TM2, Ubx balancer chromosome (solid bars). As a control, these balancer chromosomes (black bar) were compared to wild-type (Oregon-R) chromosomes (shaded bar) in the TM2, Ubx and CyO labeled columns. The balancer chromosomes did not cause a substantial increase in pigment production. The other pairs of columns compare a mutant (shaded bar) to balancer (solid bar). Measurements are the average of three samples each containing 20 heads. Error bars show the standard error between the three samples. Asterisk indicates the mean value is significantly different from that of the siblings that inherit the balancer (P < 0.05) according to a t-test.

Figure 10.—

Transgene arrays are also sensitive to Su(var)3-7 dosage and insensitive to Su(var)3-9 dosage. (A) Increasing the Su(var)3-7⁺ dose enhances variegation from T1 and BX2. Pigment production is dependent on either T1 or BX2 as indicated to the left. The Su(var)3-7⁺ dosage increased from 1, 2 (transgene), 2, and 3 doses as indicated at the top. The male progeny depicted were generated by crossing w; Bl/Su(var)3-7^+t6.5; Su(var)3-7^P9/TM2, Ubx males to females that had the indicated P{lacW} array. (B) Testing whether Su(var)3-9 mutations suppress variegation from w⁺ transgenes that are affected by PDS. The transgene tested is indicated at the left and the Su(var)3-9 allele is indicated at the top. The male progeny depicted were generated by crossing males with the indicated Su(var)3-9 allele to females with the indicated w⁺ reporter gene(s).