Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb

Abstract

Background: Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1.

Results: We show that Corto and DSP1 proteins co-localize at 91 sites on polytene chromosomes and co-immunoprecipitate in embryos. They interact directly through the DSP1 HMG-boxes and the amino-part of Corto, which contains a chromodomain. In order to search for a common target, we performed a genetic interaction analysis. We observed that corto mutants suppressed dsp11 sex comb phenotypes and enhanced AntpScx phenotypes, suggesting that corto and dsp1 are simultaneously involved in the regulation of Scr. Using chromatin immunoprecipitation of the Scr ME, we found that Corto was present on this ME both in Drosophila S2 cells and in embryos, whereas DSP1 was present only in S2 cells.

Conclusion: Our results reveal that the proteins Corto and DSP1 are differently recruited to a Scr ME depending on whether the ME is active, as seen in S2 cells, or inactive, as in most embryonic cells. The presence of a given combination of ETPs on an ME would control the recruitment of either PcG or TrxG complexes, propagating the silenced or active state.

Figure 1

Immunofluorescence detection of Corto and DSP1 on polytene chromosomes. (A-C) Simultaneous detection of Corto (green) and DSP1 (red) on Oregon-R polytene chromosomes stained with DAPI (blue). (D, E) Magnification of chromosome 2L end of Oregon-R (D) or dsp1¹ (E) labeled with anti-Corto (green) and anti-DSP1 (red). Sites shared by DSP1 and Corto in Oregon-R are yellow.

Figure 2

Comparison of Corto, DSP1 and PC localizations on polytene chromosomes of Oregon-R wild-type strain.

Figure 3

Corto and DSP1 interact in vivo. Co-immunoprecipitation of Corto with DSP1. Protein extracts from 0–14 hour-old w¹¹¹⁸ embryos were incubated either with rabbit serum (mock IP) or with anti-DSP1 antibodies (DSP1 IP). Western blotting was performed using rabbit anti-DSP1 antibodies (left) or rat anti-Corto antibodies (right). The arrows indicate the full-length DSP1 protein (50 kDa) and Corto (68 kDa), respectively. I : Input ; S : supernatant; IP : immunoprecipitated material; *: rabbit IgG. Note that full-length DSP1 and degradation products were retained on the protein A-agarose/anti-DSP1 beads.

Figure 4

Corto and DSP1 interact in vitro. (A) Far-western assays. Left, top: Coomassie-stained SDS-PAGE of the MBP-DSP1 fusion proteins. Left, bottom: Phosphorimager scan of the membrane after transfer of the proteins and incubation with radiolabeled Corto. Right: Schematic representation of DSP1 and DSP1 truncated forms (orange: HMG-A and HMG-B boxes; yellow: polyglutamine series and acidic tail). Corto is retained on MBP-DSP1 and on B22, C8, D16, E5, F33, J11 and M2 MBP-DSP1 truncated forms but not on G81, L5 and N4 MBP-DSP1 truncated forms. (B) GST pull-down assays Left, top: Coomassie blue staining of GST and GST-Corto fusion proteins (labeled with asteriks). Left, bottom: Autoradiography. ³⁵S-labeled DSP1 was retained on GST-Corto and GST-C1/324 proteins and not on GST-C325/550, GST-C127/203 or GST-C440/550. Input: 1/5 of the total radioactivity was loaded. Note that the full-length DSP1 protein as well as the truncated forms (degradation products or abortive translations) are retained on GST-Corto and GST-C1/324. Right : Schematic representation of Corto (blue : chromodomain).

Figure 5

Binding of Corto on the Scr 10-kb XbaI fragment. (A) The 10-kb XbaI Scr fragment is located about 37-kb upstream from the transcription start site. Numbers refer to NT_033777, i.e. the access number of the complete sequence of the Drosophila melanogaster 3R chromosome. The 7 internal sequences (S1 to S7) and the two distal sequences (SL and SR) were amplified with specific primers as described in Methods. The PvuII and XmnI fragments, where DSP1 has been previously shown to bind, are in purple [41]. (B) To determine the linear range of amplification, PCR samples were taken at the 29^th, 31^st, 33^rd and 35^th PCR cycles. (C-D) Chromatin immunoprecipitation analysis of 0–14 hour-old embryos (C) or S2 cells (D) using rabbit anti-Corto, rabbit anti-DSP1 or rabbit serum as a control (mock). The 31^st cycle samples were loaded on a 1% agarose gel. The PCR products were quantified using Image J software and expressed as a percentage of the total input DNA. The diagrams represent the mean of 3 independent experiments.