Mutation of Drosophila Lsd1 disrupts H3-K4 methylation, resulting in tissue-specific defects during development

Abstract

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified. Lsd1 is highly conserved in many species, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. Here we describe the effects of Lsd1 mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development.

Figure 1. dLsd1^ΔN mutant animals have developmental defects

A, B: dLsd1^ΔN mutant flies have a held-out wing phenotype. Images of wild-type (w¹¹¹⁸) and dLsd1^ΔN flies. C, D: dLsd1^ΔN mutant ovaries are smaller than wild-type ovaries. Images of the ovaries of wild-type and dLsd1^ΔN flies. E, F: dLsd1^ΔN mutant ovaries lack proper ovariole structures. DNA staining (YOYO-1) of a wild-type ovary versus the ovary of dLsd1^ΔN flies. G, H: In dLsd1^ΔN mutant ovaries follicle cells fail to encapsulate the cyst. Wild-type and dLsd1 mutant ovarioles were stained with YOYO-1 (green) and anti-fasiclin III (red) to outline somatic cells. I, L, M, N: Egg chambers of dLsd1^ΔN mutant ovaries are abnormal at very early stages. Wild-type and dLsd1^ΔN mutant ovarioles were stained with anti-αspectrin (white), to show the fusomes (I,L) and anti-vasa (white) to label the germ cells (M,N). The germarium is indicated by arrows.

Figure 2. dLsd1 specifically affects global levels of H3-K4 mono and dimethylation

A: Western blot analysis of protein extracts from w¹¹¹⁸(wt), dLsd1^ΔN/dLsd1^ΔN (hom) and dLsd1^ΔN/TM3 (het) females using antibodies specific for different modified histone H3 and H4 residues as indicated on the right. Equal loading was assessed by blotting with either a histone H3 specific antibody or a tubulin specific antibody. B: Western blot analysis of protein extracts from w¹¹¹⁸, dLsd1^ΔN/dLsd1^ΔN and dLsd1^ΔN/TM3 males. C: dLsd1 is highly expressed in the ovary. Western blot analysis of dLsd1 expression and of the level of H3-K4 monomethylation in the ovary. dLsd1 protein level are higher in the ovary than in the rest of the body and this correlates with lower levels of H3-K4 monomethylation. D: Ectopic expression of dLsd1 affects H3-K4 methylation levels and gene expression. Ectopic expression of the pUAST-dLsd1 transgene was driven by the Actin5C-GAL4 driver. dLsd1 expression was verified by Western blot analysis. The level of mono and dimethyl H3-K4 in w¹¹¹⁸ versus dLsd1 overexpressing flies are shown. Equal loading was assessed using a tubulin specific antibody.

Figure 3. dLsd1 is a suppressor of PEV

Heterozygotes for the dLsd1^ΔN allele were crossed to either T(2;3)Sb^v or In(1)y^3P. The effects of dLsd1^ΔN on Stubble variegation of T(2;3)Sb^v are shown in A. The effects of dLsd1^ΔN on Yellow variegation are shown in B. Red asterisks indicate the yellow bristles in In(1)y^3P/+;+; Me¹/dLsd1^ΔN flies. C: Quantification of the percentage of yellow bristles in the indicated genotypes.

Figure 4. Neuron specific genes and Hox genes are misexpressed in SL2 cells depleted for dLsd1 and in dLsd1^ΔN mutant animals

A: Western blot analysis of SL2 cells treated for 8 days with white and dLsd1 dsRNA. Three different dsRNAs directed against the N-terminal (Nt), Central (Ce) or C-terminal (Ct) region target dLsd1 protein with the same efficacy while dsRNA against the white gene (wh), used as negative control, does not affect dLsd1 protein expression. qPCR analysis of the expression of the indicated genes 8 days after dsRNA treatment of SL2 cells. The expression level is normalized against the white dsRNA treated control. Experiments were performed in triplicate and standard deviation of the mean is indicated. B: Western blot analysis of wild-type (wt), dLsd1^ΔN/dLsd1^ΔN (hom) and dLsd1^ΔN/TM3 (het) flies with an antibody specific for dLsd1 and with anti-tubulin (loading control). qPCR analysis of the expression of the indicated genes in wild-type (+) and dLsd1 mutant (−) third instar larvae (L3) and adult flies respectively 1, 2 and 3 days after eclosion (A1, A2, A3). Rp49 was used as a control (data not shown). Experiments were performed in triplicate and standard deviation of the mean is indicated.