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Comparative Study
. 2004 Jul 15;18(14):1667-80.
doi: 10.1101/gad.1206604.

Dm-myb Mutant Lethality in Drosophila Is Dependent Upon mip130: Positive and Negative Regulation of DNA Replication

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Free PMC article
Comparative Study

Dm-myb Mutant Lethality in Drosophila Is Dependent Upon mip130: Positive and Negative Regulation of DNA Replication

Eileen L Beall et al. Genes Dev. .
Free PMC article

Abstract

Gene amplification at the chorion loci in Drosophila ovarian follicle cells is a model for the developmental regulation of DNA replication. Previously, we showed that the Drosophila homolog of the Myb oncoprotein family (DmMyb) is tightly associated with four additional proteins and that DmMyb is required for this replication-mediated amplification. Here we used targeted mutagenesis to generate a mutant in the largest subunit of the DmMyb complex, the Aly and Lin-9 family member, Myb-interacting protein 130 (Mip130). We found that mip130 mutant females are sterile and display inappropriate bromodeoxyuridine (BrdU) incorporation throughout the follicle cell nuclei at stages undergoing gene amplification. Whereas mutations in Dm-myb are lethal, mutations in mip130 are viable. Surprisingly, Dm-myb mip130 double mutants are also viable and display the same phenotypes as mip130 mutants alone. This suggests that Mip130 activity without DmMyb counteraction may be responsible for the Dm-myb mutant lethality. RNA interference (RNAi) to selectively remove each DmMyb complex member revealed that DmMyb protein levels are dependent upon the presence of several of the complex members. Together, these data support a model in which DmMyb activates a repressive complex containing Mip130 into a complex competent to support replication at specific loci in a temporally and developmentally proscribed manner.

Figures

Figure 1.
Figure 1.
Molecular verification of the mip130 mutant. (A) Restriction endonuclease map of the region surrounding the mip130 gene. (WT) Wild type. The normal chromosomal configuration without any mutations. (mip1301-723 + 1-36) The targeted mutation showing the duplication of mip130 and intervening P-element sequences. The arrowhead indicates the residual FLP-recombinase-binding site, the red box indicates the mini-white gene marker, and the blue box indicates the I-CreI restriction endonuclease site. (mip1301-723) The single-copy reduction event leaving the mip1301-723 allele at the locus. (mip1301-36) The single-copy reduction event leaving the mip1301-36 allele at the locus. Light blue boxes denote the Mip130 protein product that can be encoded by the locus. Distances in kilobases, between restriction endonuclease sites, are indicated at the top. (B) BamHI; (E) EcoRI; (H) HindIII; (S) SnaBI; (Xb) XbaI; (Xh) XhoI. (B) Southern blot of mip1301-723 + 1-36. Samples were analyzed in pairs for each enzyme, with digested DNA from mip1301-723 + 1-36/FM7 and wild type (WT) loaded side by side for direct comparison. Digests and expected fragments in kilobases are as follows: (lanes 2,3) EcoRI and BamHI (11.6 wild type and 7.6, 7.25, and 6.65 mutant); (lanes 4,5) EcoRI and XbaI (10.2 wild type and 12.2, 5.85, and 2.05 mutant); (lanes 6,7) XhoI (7.0 and 3.4 wild type and 9.9 mutant); (lanes 8,9) HindIII and XhoI (3.6 and 3.4 wild type and 9.9 mutant); (lanes 10,11) SnaBI (7.3 wild type and 5.8 mutant). The probe used was a 1.8-kb fragment derived from the mip130 gene spanning the internal XhoI site. M, molecular weight markers with sizes in kilobases, given at the left. (C) Southern blot of reduction alleles. Two independent lines for each allele (either mip1301-36 or mip1301-723) and wild type (FM7) were analyzed from the fly type indicated at the top. DNAs were digested with both EcoRI and BamHI, and fragments were detected with an internal mip130 probe spanning the XhoI site. Sizes expected in kilobases are as follows: mip1301-36 allele, 11.6 wild type and 6.65 and 4.95 mutant; mip1301-723 allele, 11.6 wild type and 7.25 and 4.35 mutant.
Figure 2.
Figure 2.
DmMyb levels are severely reduced in mip130 mutants. (A) Flies of the type indicated at the top of each lane were homogenized in sample buffer, and 0.5 fly per lane was examined for the presence of the protein indicated at the right of each panel by immunoblot analysis. Tubulin was used as a loading control (right panel). Mip130* represents the truncated Mip130 product produced by the mip1301-723 allele in mip1301-723 + 1-36 mutants. (B) RT-PCR analysis of RNA isolated from ovaries dissected from either mip1301-723 + 1-36 mutant or wild-type control females. Twofold serial dilutions of the input cDNA were analyzed for either Dm-myb transcripts or an unrelated transcript (control) as indicated on the right. (-) PCR performed on the isolated RNA prior to cDNA synthesis in order to ensure that there was no contaminating genomic DNA. (C) Progeny from the cross between mip1301-723 + 1-36/FM7cKrGFP (GFP-containing chromosome) females and mip1301-723 + 1-36 mutant males were sorted for GFP-containing (mip1301-723 + 1-36/FM7c) and non-GFP (homozygous mip1301-723 + 1-36 mutant) animals at the stages indicated at the top of the figure. Immunoblot analysis was performed with the antibodies indicated on the right. Tubulin was used as a loading control. (D) Same as in C except that the cross was between Dm-myb mutant MH107/FM7cAcGFP females and FM7c males.
Figure 6.
Figure 6.
Dm-myb mip130 homozygous double mutants are viable. (A) Southern blot analysis of Dm-myb mip1301-723 + 1-36 double mutants: mip130 locus determination. Two independent lines (#6 and #19), the original mip1301-723 + 1-36 mutant, and wild type (FM7) were analyzed as indicated at the top. DNAs were digested with both EcoRI and BamHI, and fragments were detected with an internal mip130 probe spanning the XhoI site. Sizes expected, in kilobases, are as follows: 11.6 for wild type and 7.6, 7.25, and 6.65 for mutant. (B) Southern blot analysis of Dm-myb mip1301-723 + 1-36 double mutants: Dm-myb locus determination. Two independent lines (#6 and #19), the original Dm-myb mutant (MH107), wild-type (FM7), and mip1301-723 + 1-36 mutants were analyzed as indicated on the top. DNAs were digested with BamHI, and fragments were detected using a probe specific for Dm-myb. Sizes expected, in kilobases, are as follows: 10.4 wild type and 14.5 mutant. (C) Immunoblot analysis of Dm-myb mip1301-723 + 1-36 double mutants. Flies (lanes 1-6) and pupae (lanes 7-12) of the type indicated at the top were blotted for Mip130 and DmMyb as indicated at the right. (-) GFP-negative pupae representing homozygous mutant animals, (+) GFP-positive control siblings. (D) Immunoblot analysis of the Dm-myb mip1301-723 and Dm-myb mip1301-36 double mutants. Flies of the type indicated at the top of each panel were blotted for Mip130 and DmMyb as indicated at the right. (Upper and lower panels, lane 1) double mutant/FM7 female. (Lane 2) Homozygous double mutant male. (Lane 3) Heterozygous mip1301-36 1A2/FM7 (upper) or mip1301-723 1A7/FM7 (lower) female. (Lane 4) Homozygous mip1301-36 1A2 (upper) or mip1301-723 1A7 (lower) mutant male. (Lane 5) FM7 control male. (E) Immunoblot analysis of the mip1301-36 mutant complementation experiment. Transgenic lines (TG) containing the Mip130 cDNA under the control of Gal4 binding sites were used to complement the mip1301-36 mutant allele for female sterility. Gal4 was under the control of the e22c enhancer (driver), which expresses Gal4 in the ovary as well as at other stages in development. Females were homogenized in sample buffer, and 0.5 fly per lane was analyzed for Mip130 and DmMyb as indicated on the right. (Lane 1) Flies that were mip1301-36 homozygous mutant and carried the e22c driver only. (Lane 2) Flies that were mip1301-36 homozygous mutant and carried both the e22c driver and the mip130 cDNA transgene-1. (Lane 3) Heterozygous mip1301-36/FM7 sisters that carried the e22c driver only. The flies carrying both the driver and Mip130 transgene (lane 2) resulted in expression of both Mip130 and DmMyb.
Figure 5.
Figure 5.
BrdU incorporation occurs throughout the nucleus in mip130 mutants. (A) Stage 10 egg chambers from either mip1301-36/FM7a or mip1301-36 homozygous mutants. (Red) Anti-Myb; (green) anti-BrdU; (blue) DAPI. (B) Stage 10 egg chambers from mip1301-723 + 1-36/FM7 (left), mip1301-723 + 1-36 homozygous mutant (center), and Dm-myb mip1301-723 + 1-36 homozygous double mutants (right). (Red) Anti-Orc2; (green) anti-BrdU; (blue) DAPI.
Figure 3.
Figure 3.
RNAi analysis of the Myb complex. (A) RNA-interference was performed on Drosophila S2 cells with the double-stranded RNAs targeted towards the transcript indicated above each lane. Cells were harvested after 3 d, and immunoblot analysis was performed on the cell pellets with the antibodies indicated on the left. Tubulin was used as a loading control. SK+, RNAi using a nonspecific control RNA against a fragment of the Blue-Script plasmid. S2, mock-treated cells. (B) RT-PCR analysis on RNA isolated from RNAi-treated cells as indicated above each line. Shown is an ethidium bromide-stained agarose gel containing PCR products from reactions performed with primers targeted to the specific transcript indicated above each lane. For each RNAi sample, selective depletion of the transcript for which the dsRNA was targeted was observed. (C) Schematic diagram of the stability of each protein after RNAi analysis. Arrow strength (solid > large dash > medium dash > small dash) is indicative of the amount that each protein was reduced in the absence of the indicated complex member.
Figure 4.
Figure 4.
Absence of mip130 results in a thin chorion. Images shown are eggs laid by either wild-type (WT, left) or mip1301-723 + 1-36 mutant mothers (center and right). The wild-type (WT) egg appears very dark, due to the opacity of the thick chorion. The eggs laid by mip1301-723 + 1-36 mutant females are thin and often collapsed (center), and contain thin and fragile dorsal appendages. Images were taken using an Axiophot 373 light microscope.
Figure 7.
Figure 7.
Model for how the DmMyb complex functions in transcription and/or replication. The DmMyb complex comprised of Mip40, Caf1 p55, DmMyb, Mip120, and Mip130 (blue and green balls) is bound to many sites along the chromosome and ensures a silenced transcriptional and/or replicative state. In this model, DmMyb is a silent partner of the repressive complex and does not play a role in repression. Through some unknown mechanism, a site is targeted for replication initiation, and is subsequently bound by members of the pre-RC (orange balls). In a late step of replication initiation, DmMyb (red) converts an inactive replication origin (purple) to one that is competent for initiation (yellow), possibly after appropriate posttranslational modification(s) of DmMyb have occurred. In this way, the lethal Dm-myb mutant phenotype results from the inability to activate the repressive complex containing Mip130. In double mutant animals, the loss of the activator (DmMyb) is compensated by the loss of the repressive complex (Mips), and pre-RCs can assemble at many sites along the chromosome that serve as sites of replication initiation.

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