The histone H3-K27 demethylase Utx regulates HOX gene expression in Drosophila in a temporally restricted manner

Abstract

Trimethylation of histone H3 at lysine 27 (H3-K27me3) by Polycomb repressive complex 2 (PRC2) is a key step for transcriptional repression by the Polycomb system. Demethylation of H3-K27me3 by Utx and/or its paralogs has consequently been proposed to be important for counteracting Polycomb repression. To study the phenotype of Drosophila mutants that lack H3-K27me3 demethylase activity, we created Utx(Δ), a deletion allele of the single Drosophila Utx gene. Utx(Δ) homozygotes that contain maternally deposited wild-type Utx protein develop into adults with normal epidermal morphology but die shortly after hatching. By contrast, Utx(Δ) homozygotes that are derived from Utx mutant germ cells and therefore lack both maternal and zygotic Utx protein, die as larvae and show partial loss of expression of HOX genes in tissues in which these genes are normally active. This phenotype classifies Utx as a trithorax group regulator. We propose that Utx is needed in the early embryo to prevent inappropriate instalment of long-term Polycomb repression at HOX genes in cells in which these genes must be kept active. In contrast to PRC2, which is essential for, and continuously required during, germ cell, embryonic and larval development, Utx therefore appears to have a more limited and specific function during development. This argues against a continuous interplay between H3-K27me3 methylation and demethylation in the control of gene transcription in Drosophila. Furthermore, our analyses do not support the recent proposal that Utx would regulate cell proliferation in Drosophila as Utx mutant cells generated in wild-type animals proliferate like wild-type cells.

Keywords: H3-K27me3; PcG; Polycomb; Trithorax; Utx; trxG.

Fig. 1.

Analysis of Utx protein in Utx mutants and requirement for Utx in Drosophila development. (A) Domain architecture of the Drosophila Utx protein showing the tetratricopeptide repeats (TPR) and the catalytic JmjC domain. The lesions of the previously described Utx¹ allele (Herz et al., 2010) and of the Utx^Δ deletion allele are indicated; in Utx^Δ, a Ser503>stop mutation was introduced and a large portion of the JmjC coding region was deleted. (B) Total extracts from imaginal disc and CNS tissues from wild-type (wt, lane 1), homozygous Utx¹ (lane 2) and homozygous Utx^Δ (lane 3) larvae probed with an antibody against Utx_420-633. Note the lack of the 145 and 80 kDa bands (arrowheads) in the Utx¹ and Utx^Δ mutant extracts. Each lane contained the extract from ten third instar larvae and non-specific cross-reacting bands detected by the antibody provide an additional control for loading of comparable amounts of extract. (C) Survival of wild type and Utx mutants of the indicated genotypes to adults; Df Utx indicates Df(2L)BSC143 and mat+ zyg-indicates that these animals were obtained from Utx¹ and Utx^Δ heterozygous mothers. mat- zyg- animals were obtained from mothers with Utx^Δ germline clones; they arrest development before the pupal stage (no adults were obtained, asterisk) (see main text). For each genotype, three independent batches with 100 first instar larvae each were collected, transferred into vials and the number of eclosed adults was determined. Error bars indicate s.d.

Fig. 2.

Bulk H3-K27me3 and H3-K27ac levels are unchanged in Utx mutants. (A) Western blots of serial dilutions (4:2:1) of total extracts from imaginal disc and CNS tissues of wild-type (wt, lanes 1-3), Utx¹/Df(2L)BSC143 (lanes 4-6) and Utx^Δ/Df(2L)BSC143 (lanes 7-9) larvae, probed with antibodies against H3-K27me3, H3-K27ac and H3-K4me1. In each case, the membrane was simultaneously probed with an antibody against histone H4 to control for extract loading and western blot processing but was exposed to film for a shorter period of time in order not to saturate the H4 signal. Note that H3-K27me3 and H3-K27ac levels are unchanged in Utx mutant extracts but that H3-K4me1 levels are very slightly (less than one-third) decreased in the mutants. (B) Wing imaginal discs with MARCM clones of Utx^Δ homozygous cells that are marked by the presence of GFP, stained with H3-K27me3 or H3-K27ac antibodies and Hoechst (for DNA). Note that the H3-K27me3 and H3-K27ac immunofluorescence signals in Utx^Δ/Utx^Δ clone cells are comparable to those in the neighbouring Utx^Δ/+ cells. Discs were analysed 96 hours after clone induction.

Fig. 3.

Utx¹ and Utx^Δ clones do not show an overproliferation phenotype. Eye-antenna imaginal discs with MARCM clones of Utx¹ or Utx^Δ homozygous cells marked by the presence of GFP and stained with Hoechst. As reference, clones of a wild-type 2L chromosome arm (wt) were generated the same way and are also positively marked by GFP (top). In all cases, discs were analysed 96 hours after clone induction. Note that the clone sizes are comparable in all three genotypes and that the clone cells populate a comparable area of disc tissue.

Fig. 4.

Trithorax-like homeotic transformations and loss of HOX gene expression in Utx^Δ mutants. (A) Portions of the dorsal abdomen of adult males of the indicated genotype, showing the tergites of abdominal segments A4, A5 and A6. Note the patchy loss of pigmentation in the anterior part of A5 of the Utx^{Δmat- zyg+} male (arrowheads), suggesting a transformation of this tissue to A4 segment identity. (B) Ventral views of stage 16 embryos stained with antibodies against the HOX proteins Ubx and Abd-B. The Utx^{Δmat- zyg-} genotype refers to Utx^Δ/Df(2L)BSC143 embryos derived from females with a Utx^Δ germ line. Ubx expression is indistinguishable from wild type (wt). Abd-B expression is strongly reduced in parasegment (ps) 10 (arrowhead) but appears largely undiminished in more posterior parasegments; a vertical bar marks the anterior margin of the normal Abd-B expression domain in ps 10 in the CNS. (C) Haltere imaginal discs from third instar larvae stained with Ubx antibody and Hoechst. Mutant genotypes are Utx^Δ/Df(2L)BSC143 derived from a Utx^Δ heterozygous mother (Utx^{Δ mat+ zyg-}, middle) and from a female with a Utx^Δ germ line (Utx^{Δ mat- zyg-}, bottom). Note the complete loss of Ubx expression in a large fraction of cells in the Utx^{Δmat- zyg-} disc (asterisk) and that Ubx expression in the Utx^{Δmat+ zyg-} disc is indistinguishable from the wild type. The loss of Ubx expression is not compartment specific and always occurs in a clone-like pattern with large patches of Ubx-negative cells.

Fig. 5.

Lack of Utx function does not exacerbate loss of HOX gene expression in ash1 mutants. Haltere imaginal discs from third instar larvae with clones of ash1²² homozygous cells were stained with Ubx antibody and Hoechst. ash1²² mutant cells are marked by the absence of GFP and were induced in a wild-type genetic background (ash1²², top) or in the background of Utx^{Δmat+ zyg-} homozygous larvae (Utx^Δ, ash1²², bottom). Discs were analysed 96 hours after clone induction. In each sample, the boundary of one clone is outlined. Note that in both genotypes a fraction of cells in the clone shows complete loss of Ubx expression (open arrowheads), whereas other cells maintain high to moderate levels of Ubx expression (white arrowheads).