E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

Abstract

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.

Figure 1.—

E(var)3-9 mutations suppress lt variegation in the adult eye. The eye pigmentation of 5-day-old lt^x2/lt¹; TM3/+ females was compared with that of sibling lt^x2/lt¹; E(var)3-9¹/+ and lt^x2/lt¹; E(var)3-9²/+ females. Red eye pigment was extracted from groups of 10 heads and the mean absorbance value (± one standard deviation) for 9–10 groups is shown. The pigment values for both E(var)3-9 classes were significantly higher than that of the control (P < 0.001) using a Student's t-test.

Figure 2.—

Complementation analyses with deficiencies spanning the 85 and 86 cytological intervals localize the E(var)3-9 gene to 85C1-3 or 85D2-8. (A) A drawing of cytological intervals 85 and 86 is shown above depictions of the alignment of seven deficiencies, with the approximate deleted region illustrated as a black line. The results of a complementation test between each deficiency and E(var)3-9¹, for female fertility, is shown at right. (B) The chromosome map of the 85B7–85D8 region is demarcated by numbered band and illustrated as it corresponds to the genomic sequence and genes (http://www.flybase.org, Release 5.1). Vertical dotted lines indicate the positions of six P-element insertions (illustrated as triangles) used for male recombination mapping. (Two insertions yielding no data are omitted.) The right and left block arrows projecting from each dotted line reflect the relative position of the E(var)3-9 gene with respect to that P insertion as indicated by the phenotype of the male recombinants, of the number specified by the value of n. The data from multiple experiments are combined. White block arrows depict the results of male recombination mapping with the E(var)3-9¹ allele, and black arrows depict the results of male recombination mapping with the E(var)3-9² allele. Shown below the map are depictions of seven deficiencies as they correspond to this region. The deleted sequences and results of complementation analysis with E(var)3-9¹ and/or E(var)3-9² are illustrated as in A.

Figure 2.—

Complementation analyses with deficiencies spanning the 85 and 86 cytological intervals localize the E(var)3-9 gene to 85C1-3 or 85D2-8. (A) A drawing of cytological intervals 85 and 86 is shown above depictions of the alignment of seven deficiencies, with the approximate deleted region illustrated as a black line. The results of a complementation test between each deficiency and E(var)3-9¹, for female fertility, is shown at right. (B) The chromosome map of the 85B7–85D8 region is demarcated by numbered band and illustrated as it corresponds to the genomic sequence and genes (http://www.flybase.org, Release 5.1). Vertical dotted lines indicate the positions of six P-element insertions (illustrated as triangles) used for male recombination mapping. (Two insertions yielding no data are omitted.) The right and left block arrows projecting from each dotted line reflect the relative position of the E(var)3-9 gene with respect to that P insertion as indicated by the phenotype of the male recombinants, of the number specified by the value of n. The data from multiple experiments are combined. White block arrows depict the results of male recombination mapping with the E(var)3-9¹ allele, and black arrows depict the results of male recombination mapping with the E(var)3-9² allele. Shown below the map are depictions of seven deficiencies as they correspond to this region. The deleted sequences and results of complementation analysis with E(var)3-9¹ and/or E(var)3-9² are illustrated as in A.

Figure 3.—

E(var)3-9 mutations enhance w^m4 variegation in the adult eye. (A) Extracted pigment from groups of 10 males, aged 5 days, was quantified by spectrophotometry. The mean absorbance value (± one standard deviation) for 10 groups per genotype is shown. The decrease in pigmentation for the E(var)3-9^DG08508w−, E(var)3-9^Δ149 and E(var)3-9² alleles with respect to the wild-type E(var)3-9 allele, obtained by precise excision of the P{wHy}^DG08508 element, is highly significant (P < 0.001 for each allele) as determined by a Student's t-test. Enhancement was also observed in females, which had a much higher overall pigmentation level (data not shown). (B) The extent of w^m4 variegation is illustrated for a representative male fly bearing the E(var)3-9⁺ (left) or E(var)3-9^Δ149 allele (right).

Figure 4.—

The 588 amino acid E(var)3-9 protein has multiple predicted zinc fingers and a zinc finger-associated domain. As illustrated in a diagram of the coding sequence, high confidence matches to a C2H2 type zinc finger (Zn) motif were found when the E(var)3-9 protein sequence was queried against the Pfam database (c, d, and f), the Prosite database (c, e, and f), and the SMART database (a–f). An N-terminal zinc finger-associated domain (zf-AD) was also detected in a Pfam search. The potential protein products of E(var)3-9 mutant alleles are illustrated below the full-length protein. The frameshift mutation present in the E(var)3-9¹ and E(var)3-9² alleles and the nonsense mutation present in the E(var)3-9³ allele would terminate protein synthesis prior to translation of the zinc fingers. An appendage of 24 amino acids due to out-of-frame translation is depicted for the E(var)3-9¹ and E(var)3-9² gene products. The position of the P{wHy}^DG08508 insertion with respect to the coding region is shown.

Figure 5.—

Molecularly mapped deletions and duplications isolated by P{wHy}^DG08508 mutagenesis. The chromosome 3R genomic sequence and genes (http://www.flybase.org, Release 5.1) in the region affected by the deletions and duplications isolated in this study are illustrated. The P{wHy} hybrid transposon was inserted within predicted gene CG11971, identified herein as E(var)3-9, at position 3R:4,818,459 (first nucleotide of the 8-bp duplication) in the orientation shown. As described in the results, replicative hopping of the hobo (H) element followed by a recombination between the original and transposed H elements resulted in deletions (black bars) and duplications (white bars) of sequence flanking the P{wHy} insertion. The events were detected through loss of expression of the w⁺ or y⁺ marker genes of the transposon. Isolates having a homozygous lethal [L] or female sterile [FS] phenotype are indicated. The size of the deletion or duplication for each isolate was as follows: 5 (131,065 bp); 96 (110,076 bp); 21 (30,322 bp); 12 (29,841 bp); 136 (14,739 bp); 14 (12,090 bp); 149 (1065 bp); 18 (9940 bp); 124 (5598 bp); 188 (158,344 bp); 38 (78,061 bp).