LTR retrotransposons and the evolution of dosage compensation in Drosophila

Abstract

Background: Dosage compensation in Drosophila is the epigenetic process by which the expression of genes located on the single X-chromosome of males is elevated to equal the expression of X-linked genes in females where there are two copies of the X-chromosome. While epigenetic mechanisms are hypothesized to have evolved originally to silence transposable elements, a connection between transposable elements and the evolution of dosage compensation has yet to be demonstrated.

Results: We show that transcription of the Drosophila melanogaster copia LTR (long terminal repeat) retrotransposon is significantly down regulated when in the hemizygous state. DNA digestion and chromatin immunoprecipitation (ChIP) analyses demonstrate that this down regulation is associated with changes in chromatin structure mediated by the histone acetyltransferase, MOF. MOF has previously been shown to play a central role in the Drosophila dosage compensation complex by binding to the hemizygous X-chromosome in males.

Conclusion: Our results are consistent with the hypothesis that MOF originally functioned to silence retrotransposons and, over evolutionary time, was co-opted to play an essential role in dosage compensation in Drosophila.

Figure 1

Structure of the copia LTR-CAT construct. Position of Apa I site (274 bp) and the 5' (14 bp) and 3' (485 bp) PCR primer binding sites are shown (LTR = 5' copia long terminal repeat; ULR= copia untranslated leader region; CAT= bacterial chloramphenicol acetyltransferase reporter gene (see Methods for primer sequences).

Figure 2

PCR amplification products (copia LTR-CAT primers shown in Figure 1) of DNA prepared from intact nuclei (chromatin structure maintained) vs. DNA purified using standard procedures (chromatin structure not maintained-see Methods) digested (+) or not digested (-) with Apa I restriction enzyme. (a) The results indicate that the Apa I restriction site is accessible for digestion in nuclei preps (chromatin structure preserved) from wild-type larvae homozygous for the copia LTR-CAT construct but is not accessible for digestion in nuclei preps from larvae hemizygous for the construct in the wild-type genetic background. (b) The resistance of hemizygous copies of the copia LTR-CAT construct is lost in larvae homozygous for the mof¹ (loss-of-function) allele. All experiments were conducted with the 9-3 transformant strain. [wild type (mof +) = DNA from larvae carrying the wild-type allele at the mof locus; mutant (mof -) = DNA from larvae carrying the mof¹ allele at the mof locus; homo = DNA isolated from larvae homozygous for the copiaLTR-CAT construct; hemi = DNA isolated from larvae hemizygous for the copiaLTR-CAT construct].

Figure 3

(a). RT-PCR of mRNA isolated from 3rd instar larvae wild-type (column 1) or mutant (mof¹) (column 2) at the mof locus using primers specific for five Drosophila melanogaster LTR retrotransposons (copia, gypsy, 297, 1731, 412 and roo) and β-tubulin as a control. The results demonstrate uniformly higher levels of LTR retrotransposon expression in larvae carrying the mutant mof¹ allele (see Methods for primer sequences). (b). Northern hybridization of mRNA isolated from 3rd instar larvae wild-type (column 1) or mutant (mof¹) (column 2) at the mof locus using copia [43] and β-tubulin [44] probes. The results are consistent with RT PCR analyses (Figure 3a) and demonstrate higher levels of copia expression in flies carrying the mutant mof¹ allele. No significant difference in β-tubulin expression was detected.

Figure 4

Immunostaining of Drosophila polytene chromosomes with MOF-antibody. Shown are preparations from strains representing two Drosophila species- (a) D. melanogaster and (b) D. simulans. Reduced binding of MOF to D. simulans autosomes is consistent with the reduced number of LTR retrotransposons present in this species.

Figure 5

MOF is physically associated with the copia untranslated leader region (ULR) in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed to determine whether MOF is physically associated with copia DNA sequences in vivo. Precipitation reactions performed independently with the MOF, the MSL-1 and MSL-3 antibodies, rabbit IgG antibody (non-specific control) and no antibody were used as templates for quantitative PCR from Kc cells. The average and standard errors for triplicate reactions were plotted to reveal the average fold increase in precipitated copia DNA for each antibody relative to the no antibody reactions. Precipitated DNA was amplified using primers specific for the copia ULR, the roX-1 gene (positive control) and the Spt4 gene (negative control) (see Methods). MOF and other members of the MSL complex have previously been demonstrated to bind to the roX-1 gene [e.g., 23]. Spt-4 was chosen as a negative control because it is not a target site for the MSL complex [e.g., 22]. The results indicate that MOF, MSL-1 and MSL-3 are all associated with the copia ULR and roX-1 in vivo. There was no binding above background to the Spt-4 gene.