Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BACKGROUND: Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. RESULTS: Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. CONCLUSION: Our studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility

In Drosophila, female gonadal cells repress male-specific gene expression in XX germ cells.

In Drosophila, the sex of XX germ cells is determined by somatic signals. Whether sex-specific genes, however, are activated or repressed by somatic signals is not known. We have used mgm1, a germline-marker which is specifically expressed in male germ cells to analyze sex-specific gene expression of embryonic germ cells. We found that XX and XY germ cells that do not contact gonadal tissue can express mgm1. In contrast, XX germ cells that were associated with female somatic gonadal cells never expressed mgm1. Our results suggest that XX germ cells express male-specific genes, unless these genes are repressed by feminizing short range signals produced by the somatic cells of the prospective ovary

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in -8

Reporter: 4xMRE from the metallothionein B (MtnB) promoter [] fused to firefly luciferase; reference: tubulin promoter fused to renilla luciferase []. Dpy-30L1 and Dpy-30L2 expression constructs were under the control of the actin promoter. 72 hours after transfection, cells were treated with the indicated concentrations of heavy metals for 24 hours. B) Expression level of green fluorescent protein in transgenic larvae that carry an MtnA-YFP reporter construct. Transgenic flies were allowed to lay eggs on normal food or food supplemented with different heavy metals.<p><b>Copyright information:</b></p><p>Taken from "Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in "</p><p>http://www.biomedcentral.com/1471-213X/8/68</p><p>BMC Developmental Biology 2008;8():68-68.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2478662.</p><p></p

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in -2

To firefly luciferase; reference, tubulin promoter fused to renilla luciferase. Dpy-30L1 and the mouse ortholog were under the control of the actin promoter. 72 hours after transfection, the medium in half of the plates was supplemented with 40 μM cadmium chloride for 24 hours, while the others served as controls. A) Mouse MTF-1 was co-transfected in the indicated samples; B) MTF-1 was co-transfected in the indicated samples.<p><b>Copyright information:</b></p><p>Taken from "Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in "</p><p>http://www.biomedcentral.com/1471-213X/8/68</p><p>BMC Developmental Biology 2008;8():68-68.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2478662.</p><p></p

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in -5

2A D variant (H2AvD). Arrow, H2AvD-GFP in degradation; arrowheads, H2AvD-GFP degraded. Incorporation of protamine B and Mst77F was analyzed in knockout males that carry either a transgene of protamine B fused to eGFP or of Mst77F fused of eGFP. During the "canoe" and "post-canoe" stages of spermatid development, ProtamineB-eGFP and Mst77F-eGFP incorporation in the spermatid nucleus appeared to be normal in knockout males. Any (diffuse) YFP signal from the promoter was not filtered out.<p><b>Copyright information:</b></p><p>Taken from "Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in "</p><p>http://www.biomedcentral.com/1471-213X/8/68</p><p>BMC Developmental Biology 2008;8():68-68.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2478662.</p><p></p

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in -1

Reporter: 4xMRE from the metallothionein B (MtnB) promoter [] fused to firefly luciferase; reference: tubulin promoter fused to renilla luciferase []. Dpy-30L1 and Dpy-30L2 expression constructs were under the control of the actin promoter. 72 hours after transfection, cells were treated with the indicated concentrations of heavy metals for 24 hours. B) Expression level of green fluorescent protein in transgenic larvae that carry an MtnA-YFP reporter construct. Transgenic flies were allowed to lay eggs on normal food or food supplemented with different heavy metals.<p><b>Copyright information:</b></p><p>Taken from "Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in "</p><p>http://www.biomedcentral.com/1471-213X/8/68</p><p>BMC Developmental Biology 2008;8():68-68.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2478662.</p><p></p

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in -6

females were allowed to mate with wild type or mutant males, the females were then dissected and checked for a GFP signal in their reproductive system. 30 minutes after mating, 20% of wild type sperm had accumulated in female storage organs. However, mutant sperm remained in the uterus and failed to be transferred to seminal receptacle and spermathecae, the female sperm storage organs. Ov – ovaries; spt – spermatheca; src – seminal receptacle; ut – uterus; ovd – oviduct.<p><b>Copyright information:</b></p><p>Taken from "Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in "</p><p>http://www.biomedcentral.com/1471-213X/8/68</p><p>BMC Developmental Biology 2008;8():68-68.</p><p>Published online 27 Jun 2008</p><p>PMCID:PMC2478662.</p><p></p