Drosophila lin-52 acts in opposition to repressive components of the Myb-MuvB/dREAM complex

Abstract

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.

Fig 1

Generation of a lin-52 null mutant and lin-52⁺ transgenes. (A) Mobilization of a P element resulted in a 1,302-bp deletion that removed the entire lin-52 gene. A wild-type, 1.6-kb genomic DNA fragment was used to create P elements capable of rescuing the lin-52⁹³ null mutant, one of which encoded a Lin-52–GFP fusion protein. (B) A Lin-52–GFP fusion protein produced under the control of a transgenic lin-52 promoter was present in the nuclei of ovarian germ line and somatic cells (left), in the nuclei of all cells of a larval eye imaginal disc (center), and in the euchromatic but not the heterochromatic regions of the nuclei of a larval eye imaginal disc (Lin-52–GFP in green, DNA in red) (right). (C) The Lin-52–GFP fusion protein was also present in polytene salivary gland nuclei and predominantly decorated the decondensed euchromatic regions of the chromosomal arms.

Fig 2

lin-52 mutant larval lethality can be rescued by loss of mip40. (A and B) Two hundred to four hundred fifty progeny per indicated cross were counted. The values listed are the percent viability averages with standard deviations from two independent experiments. mip40^EY16520 is a viable null mutation. FM7 and CyO are balancer chromosomes. FM7 is homozygous, viable, and female sterile. CyO is homozygous lethal. (C and D) Immunoblots for Lin-52 and Myb in lin-52⁹³/FM7, Kr-GFP female and lin-52⁹³/Y male embryos and larvae. Tubulin was used as a loading control. (E) Immunoblot of Mip120, Myb, Mip40, and Lin-52 protein in lin-52, mip40 double mutant adult female flies (lin-52⁹³ mip40^EY16520) or FM7 control flies.

Fig 3

Lin-52 is present exclusively in the MMB/dREAM complex and interacts with the complex via its C terminus. (A) Silver-stained SDS-PAGE of eluate from M2 (anti-FLAG IgG) beads after incubation in extract of lin-52⁹³/lin-52⁹³; FLAG–Lin-52/FLAG–Lin-52 embryos grown for 0 to 12 h. The MMB/dREAM subunits were identified by immunoblotting (data not shown). Only the peak fractions are shown. (B) Gel filtration of FLAG–Lin-52-associated proteins. Eluate shown in panel A was fractionated on a Smart Superdex 200 gel filtration column. Sequential fractions were immunoblotted for the proteins indicated at the left. (C) RNA interference was performed in Drosophila Kc cells using double-stranded RNAs targeted against the gene indicated above each lane. After 3 days, cells were harvested and immunoblot analysis was performed with the antibodies indicated on the left.

Fig 4

Absence of Lin-52 affects the levels of other MMB/dREAM components in vivo. Heat shock-FLP was used to induce FRT-mediated mitotic recombination during the development of heterozygous lin-52⁺/lin-52⁹³ ovarian follicle cells. H2avGFP marks cells bearing the wild-type lin-52⁺ allele. The absence of H2avGFP indicates cells that are homozygous for the lin-52⁹³ null allele. Doubly bright H2avGFP-positive cells represent twin spots bearing two copies of the wild-type lin-52⁺ allele. Ovaries were immunostained with the antibodies indicated on the right of each row. Dotted lines indicate the boundaries of homozygous lin-52⁹³ mutant clones.

Fig 5

lin-52 is required for normal adult eye development. The EGUF/hid method was used to generate eyes composed completely of homozygous lin-52⁹³ mutant cells (top row, center). The resulting rough-eye phenotype was rescued by the presence of a lin-52⁺ transgene (top row, right). No abnormalities were observed when the EGUF method was used with a y w control (top row, left). The different eye colors were due to the dosage of mini-white⁺ contributed by different transgenes. Scanning electron microscopy (bottom row, two left panels) and toluidine-stained sections (bottom row, two right panels) were used to further characterize the lin-52⁹³ mutant eye defects.

Fig 6

Adult eye defects caused by a lin-52 null mutant can be rescued by a mip130 null mutant. Mitotic recombination was induced via heat shock-FLP to produce mosaic eyes containing mutant clones that were homozygous for the indicated mutant combinations. Thick sections of adult eyes were then prepared and visualized by phase-contrast microscopy. The wild-type white⁺ gene is required for the formation of the pigment granules in both the photoreceptor and pigment cells. The pigment granules of the photoreceptor cells are marked by two black dots near the rhabdomeres. The pigment granules are absent (arrows) from homozygous mutant pigment cells and rhabdomeres.

Fig 7

Maternal lin-52 is required for early embryonic development. The dominant female-sterile method was used to generate female germ line clones that were homozygous for the lin-52⁹³ null mutant in the absence or presence of a lin-52⁺ transgene. w¹¹¹⁸ females were used as a control. Wild-type zygotic gene expression was provided by mating these females to w¹¹¹⁸ males. (A) Hatch rate analysis of embryos derived from females of the indicated genotype. Depletion of lin-52 in the female germ line results in a maternal-effect lethal phenotype that can be rescued by a transgene carrying a lin-52 genomic rescue construct. (B) Embryos (60 to 90 min after egg laying) were stained with propidium iodide (PI) to detect nuclei. Eggs derived from lin-52⁹³ females (C) exhibit a reduced number of nuclei compared to eggs from wild-type mothers (B). (D) Quantitation of the number of nuclei in lin-52⁹³ or wild-type eggs. (E) Wild-type embryos (0 to 30 min after egg laying) showing different stages of mitosis. (F to J) Embryos derived from the lin-52⁹³ mutant germ line (30 to 60 min after egg laying) displaying various mitotic defects. (F to H) Embryos were stained for DNA (PI, red), microtubules (alpha-tubulin, green), and a centrosome component (Cnn, blue). One or both centrosomes are often missing from mitotic spindles. (I and J) Embryos were stained for DNA (PI, red) and microtubules (alpha-tubulin, green) only. Additional defects include a broad spindle with no centrosomes (F), a branched spindle with no centrosomes (G), or an elongated spindle with no centrosomes (H). Abnormal chromosome distribution was often observed in mutant embryos (I and J).

Fig 8

Maternal-effect lethal phenotype of a lin-52 mutant can be rescued by loss of mip130 or mip120. The dominant female-sterile method was used to generate homozygous lin-52⁹³ mutant germ line clones in the presence or absence of a wild-type lin-52⁺ transgene or in the presence or absence of mutants of other genes encoding components of the MMB/dREAM complex. All mutants were homozygous unless the presence of a wild-type allele is indicated by a plus. Wild-type zygotic gene expression was provided by mating these females to w¹¹¹⁸ males. Hatch rates were calculated as described in the legend to Fig. 7. The genotypes of the female flies used for hatch rate analyses are listed in Materials and Methods.