LIN-61, one of two Caenorhabditis elegans malignant-brain-tumor-repeat-containing proteins, acts with the DRM and NuRD-like protein complexes in vulval development but not in certain other biological processes

Abstract

Vulval development in Caenorhabiditis elegans is inhibited by the redundant functions of the synthetic multivulva (synMuv) genes. At least 26 synMuv genes have been identified, many of which appear to act via transcriptional repression. Here we report the molecular identification of the class B synMuv gene lin-61, which encodes a protein composed of four malignant brain tumor (MBT) repeats. MBT repeats, domains of approximately 100 amino acids, have been found in multiple copies in a number of transcriptional repressors, including Polycomb-group proteins. MBT repeats are important for the transcriptional repression mediated by these proteins and in some cases have been shown to bind modified histones. C. elegans contains one other MBT-repeat-containing protein, MBTR-1. We demonstrate that a deletion allele of mbtr-1 does not cause a synMuv phenotype nor does mbtr-1 appear to act redundantly with or in opposition to lin-61. We further show that lin-61 is phenotypically and biochemically distinct from other class B synMuv genes. Our data indicate that while the class B synMuv genes act together to regulate vulval development, lin-61 functions separately from some class B synMuv proteins in other biological processes.

Figure 1.—

A synMuv protein interaction map. Class A synMuv proteins are shown in yellow, Class B synMuv proteins in shades of blue, and Class C synMuv proteins in green. This assignment of proteins to specific classes is based on published classifications. The synMuv protein complexes indicated have been demonstrated directly in co-immunoprecipitation experiments, have been suggested by studies of protein stability, or are based on homology to complexes identified in other organisms. Interactions that have been suggested by yeast two-hybrid and GST pull-down experiments but not demonstrated in co-immunoprecipitation experiments are shown by double-headed arrows. References for the interactions shown are as follows: LIN-15A–LIN-56 (E. Davison and H. R. Horvitz, unpublished observations); LIN-8–LIN-35 (Davison et al. 2005); DRM complex (Harrison et al. 2006); NuRD-like complex (Unhavaithaya et al. 2002; Harrison et al. 2006); HDA-1–LIN-35 (Lu and Horvitz 1998); LIN-13—HPL-2 (Coustham et al. 2006); Tip60-like complex (Ceol and Horvitz 2004).

Figure 2.—

Molecular cloning of lin-61. (A) lin-61 maps between unc-14 and unc-15 on LGI. Part of cosmid R06C7 is shown below as a shaded bar. The rescuing StuI–SacII fragment of R06C7 is shown below the cosmid. Open boxes represent the exons of the predicted genes within the subclone. Arrows indicate the direction of transcription. (B) lin-61 gene structure as determined from cDNA and genomic sequences. Solid boxes indicate coding sequence. Open box indicates the 3′ untranslated region. Predicted translation initiation (ATG) and termination (TAA) codons are shown along with the site of polyadenylation [poly(A)] and of SL1 trans-splicing (SL1). The positions of the mutations found in the 10 lin-61 alleles are indicated above the gene structure. (C) Alignment of the MBT repeats from the C. elegans (Ce) proteins LIN-61 and MBTR-1 and the Homo sapiens (Hs) proteins L(3)MBT2 and L(3)MBT1, accession nos. Q969R5 and Q9Y468, respectively. Each repeat is shown separately with the repeat number indicated next to the protein name. The top portion corresponds to the N-terminal arm and the bottom portion corresponds to the contiguous β-core region of the MBT repeat, as defined by structural analysis (Sathyamurthy et al. 2003; Wang et al. 2003). Shaded residues indicate identities among >8 of the 15 MBT repeats. Circled residues indicate positions of missense mutations in LIN-61. The corresponding allele is indicated above the residue. The missense mutation n3624 is located between the first and second repeats. The boxed region indicates the 15 amino acids inserted in the second MBT repeats of LIN-61 and MBTR-1. (D) Schematic of C. elegans LIN-61, H. sapiens L(3)MBT2, and Mus musculus Mbtd1 proteins, accession nos. NP_492050, Q969R5, and AAH62907, respectively. Shaded boxes indicate the positions and relative sizes of the four MBT repeats.

Figure 3.—

MBTR-1 sequence and structure. (A) Alignment of MBTR-1 and LIN-61. Solid boxes indicate identities between LIN-61 and MBTR-1, and shaded boxes indicate similarities between the two proteins. Underlined regions correspond to the four MBT repeats. The solid box indicates the 15–16 amino acid insertions in the second MBT repeats of LIN-61 and MBTR-1. (B) mbtr-1 gene structure as determined from cDNA and genomic sequences. Solid boxes indicate coding sequence. Open boxes indicate 5′ and 3′ untranslated regions. Predicted translation initiation (ATG) and termination (TAA) codons are shown along with the site of polyadenylation [poly(A)]. The predicted gene within the first intron of mbtr-1, Y48G1A.2, is shown. The arrow depicts the direction of transcription. The genomic region deleted in n4775 is indicated by brackets. (C) Schematic of the MBTR-1 protein. Shaded boxes indicate the positions and relative sizes of the four MBT repeats.

Figure 4.—

LIN-61 is an ubiquitously expressed nuclear protein with punctate localization. (A) Affinity-purified antibodies raised against recombinant LIN-61 were used to blot extracts from both wild-type and lin-61(n3809) mutant animals. Asterisks denote nonspecific immunoreactivity. HM4077 antibodies were raised in a guinea pig. HM4078 antibodies were raised in a rabbit. (B, D, F, H, J, and L) Whole-mount staining with anti-LIN-61 antisera. HM4077 was used for whole-mount staining of embryos, and HM4078 was used for whole-mount staining of adults. (B) LIN-61 is expressed in discrete foci in the nuclei of the developing embryo. (C) 4′,6-diamidino-2-phenylindole (DAPI) staining of the embryo shown in (B). (D) Enlargement of the boxed portion of B. (E) Enlargement of the boxed portion of C. (F) LIN-61 was absent in lin-61(n3809) embryos. (G) DAPI staining of the embryo shown in F. (H) LIN-61 was broadly expressed in the adult hermaphrodite germline and was localized to condensed chromosomes. (I) DAPI staining of the germline shown in H. (J) Enlargement of the boxed portion of H. (K) Enlargement of the boxed portion of I. (L) LIN-61 staining was absent from the germline of lin-61(n3809) adult hermaphrodites. (M) DAPI staining of the germline shown in K. WT, wild type. Bars, 10 μm.

Figure 5.—

Residues within the β-core region MBT repeats of LIN-61 are likely to be important for protein folding and stability. LIN-61 levels are reduced in many lin-61 mutant animals. Equivalent amounts of protein from mixed-stage cultures of each of the genotypes indicated above the lanes were loaded in each lane. Proteins were separated by SDS–PAGE and immunoblotted with the antibodies indicated at the left. Antitubulin antibodies were used to assess protein loading and transfer. The asterisk denotes nonspecific immunoreactivity.

Figure 6.—

LIN-61 is not a core component of the DRM or NuRD-like complexes. (A) LIN-61 does not immunoprecipitate a number of synMuv proteins, including members of the DRM and NuRD-like complexes. Extracts from either wild-type or lin-61(n3809) mutant embryos (as indicated above the lanes) were precipitated using antibodies against LIN-61 (HM4077). Immunoprecipitates were analyzed using immunoblots with antibodies specific to the antigen indicated at the left. (B) DRM complex members LIN-37 and LIN-9 co-immunoprecipitate a number of class B synMuv proteins (Harrison et al. 2006), but fail to co-immunoprecipitate LIN-61. Extracts from wild-type embryos were precipitated using antibodies indicated above the lanes and immunoblotted with antibodies specific to the antigens indicated at the left. (C) DRM complex assembly and stability is not perturbed in animals lacking lin-61 function. Extracts from lin-61(n3809) mutant embryos were precipitated using antibodies against LIN-37 and immunoblotted with antibodies specific to the antigen indicated at the left. IN, 2% of the input; IP, 100% of the immunoprecipitate.