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. 2001 Apr 24;98(9):5061-6.
doi: 10.1073/pnas.081016198.

The Caenorhabditis elegans maternal-effect sterile proteins, MES-2, MES-3, and MES-6, are associated in a complex in embryos

L Xu  1 , Y Fong, S Strome
Affiliations

The Caenorhabditis elegans maternal-effect sterile proteins, MES-2, MES-3, and MES-6, are associated in a complex in embryos

L Xu et al. Proc Natl Acad Sci U S A. .

Abstract

The Caenorhabditis elegans maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, encode nuclear proteins that are essential for germ-line development. They are thought to be involved in a common process because their mutant phenotypes are similar. MES-2 and MES-6 are homologs of Enhancer of zeste and extra sex combs, both members of the Polycomb group of chromatin regulators in insects and vertebrates. MES-3 is a novel protein, and MES-4 is a SET-domain protein. To investigate whether the MES proteins interact and likely function as a complex, we performed biochemical analyses on C. elegans embryo extracts. Results of immunoprecipitation experiments indicate that MES-2, MES-3, and MES-6 are associated in a complex and that MES-4 is not associated with this complex. Based on in vitro binding assays, MES-2 and MES-6 interact directly, via the amino terminal portion of MES-2. Sucrose density gradient fractionation and gel filtration chromatography were performed to determine the Stokes radius and sedimentation coefficient of the MES-2/MES-3/MES-6 complex. Based on those two values, we estimate that the molecular mass of the complex is approximately 255 kDa, close to the sum of the three known components. Our results suggest that the two C. elegans Polycomb group homologs (MES-2 and MES-6) associate with a novel partner (MES-3) to regulate germ-line development in C. elegans.

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Figures

Figure 1
Figure 1
Coimmunoprecipitation of MES-2, MES-3, and MES-6 from C. elegans embryo extracts. Proteins were immunoprecipitated from embryo extracts with either rabbit anti-MES-2 (A and B) or rabbit anti-MES-6 (C). (A) The rabbit anti-MES-2 antibodies were crosslinked to Protein A-agarose beads. (B and C) Equal amounts of immunoprecipitates (P) and supernatants (S) were analyzed by SDS/PAGE and Western blot analysis by using rabbit anti-MES-2, rat anti-MES-3, rat anti-MES-4, or rabbit anti-MES-6 as indicated (see Materials and Methods). Signals indicated by * are due to cross-reactivity between the secondary antibodies (goat anti-rabbit or goat anti-rat) and the heavy chain of the antibodies used for immunoprecipitations. Numbers to the left are kDa.
Figure 2
Figure 2
MES-2 and MES-6 directly interact in vitro. 35S-labeled MES-2, MES-3, MES-4, and MES-6 proteins were synthesized in vitro (see Materials and Methods). Equal amounts of MES proteins were mixed and incubated together and then with anti-MES antibodies coupled to Protein A-agarose or Protein G-agarose beads. After extensive washing, proteins in the pellets were analyzed by SDS/PAGE and autoradiography. Tests for interactions were of radiolabeled MES-2 and MES-6 (A), nonradiolabeled (cold) MES-2 and radiolabeled MES-3 and MES-6 (B), and different combinations of radiolabeled MES-4 and MES-2, MES-3, or MES-6 (C). Numbers to the right are kDa.
Figure 3
Figure 3
The N terminus of MES-2 binds MES-6 in vitro. (A) Alignment of C. elegans MES-21–194 with the N termini of Drosophila E(Z), its mouse homolog (mENX-1), and its human homolog (hEZH1). The domain in E(Z) that interacts directly with ESC in vitro is flanked by narrow arrows (amino acids 34–66), and the domain in mENX-1 that interacts with the mammalian ESC homolog is flanked by wide arrows (amino acids 132–160). The alignment was done by San Diego Supercomputer Center biology workbench software (http://workbench.sdsc.edu). Residues identical to those in E(Z) are highlighted in black. (B) Radiolabeled N-terminal 194 aa (Upper) and C-terminal 579 aa (Lower) of MES-2 were tested for their ability to bind MES-6. Numbers to the right are kDa.
Figure 4
Figure 4
The MES-2/MES-6 complex is resistant to high KCl concentrations. Proteins were immunoprecipitated from embryo extracts with anti-MES-2 antibodies. The immunoprecipitates were washed with increasing concentrations of KCl. Equivalent amounts of the initial supernatant (S) and the precipitate after each wash were analyzed by SDS/PAGE and Western blot analysis by using a mixture of rabbit anti-MES-2 and rabbit anti-MES-6 antibodies.
Figure 5
Figure 5
Analysis of the MES-2/MES-3/MES-6 complex by sucrose density gradient centrifugation and gel filtration. Embryo extract was layered on a 7–47% linear sucrose gradient and centrifuged (A) or fractionated by Sephacryl S-300 chromatography (B). Fractions were analyzed by SDS/PAGE and Western blot analysis using anti-MES-2 and anti-MES-3. The fraction numbers are indicated at the top. The peaks of migration of various protein standards are indicated by arrows. The calibration curve was constructed by plotting Stokes radii of reference proteins vs. (−logKav)1/2. The Stokes radius of the MES-2/MES-3/MES-6 complex was determined by interpolation using this curve. For details see Materials and Methods. (C) Summary of the hydrodynamic properties of the MES-2/MES-3/MES-6 complex. The complex comigrated with β-amylase (8.9S) in two separate sucrose gradient experiments. The results of two different gel filtration experiments are shown, along with the resulting molecular mass estimates.
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