Figure 1.
LIN-61 specifically interacts with H3K9me2/3.
(A), (B) Affinity purification of the recombinant three MBT repeats of hL3MBTL1 or C. elegans extract using the indicated biotinylated histone tail peptides carrying different methyl-lysine marks immobilized on avidin agarose resin. (C) Affinity purification as in (A) and (B) using C. elegans extract from wild type or hpl-1 and hpl-2 mutant worms. (D) Affinity purification experiment using bacterially produced recombinant MBP-LIN-61 and the indicated immobilized peptides. (E) Affinity purification of bacterially produced recombinant MBP-LIN-61 using unmodified and H3K9me3 peptides in presence of the indicated competitor peptides (100-fold excess). Western blot analyses of the recovered material using the indicated antibodies are shown. Input, 4% (A) or 2% (B-E); mock, avidin agarose resin without peptide.
Figure 2.
The three C-terminal MBT repeats of LIN-61 are essential for H3K9me3 interaction.
(A) Schematic representation of the LIN-61 protein indicating the amino acid position of the four (1-4) MBT repeat domains on top (according to GenBank using RPS-BLAST). Bottom, boundaries of the deletion constructs used (amino acid positions). (B) Sequence alignment of the four LIN-61 MBT core domains with MBT core domains of other MBT factors implicated in methyl-lysine binding. Amino acids identical in at least three of the sequences are in black. Residues shown to be essential for methyl-lysine interaction of dScm, hL3MBTL1 (isoform I), hL3MBTL2, dSfmbt (isoform C) and hMBTD1 are boxed in red (aromatic cage residues mediating hydrophobic and π-cation interactions) or in blue (conserved aspartate residue mediating ion pairing and hydrogen bonding to mono- and dimethylammonium moiety of lysine ε-amino group). MBT repeat four of LIN-61 containing all residues determined to be essential in methyl-lysine binding of other MBT domain proteins is highlighted in red. Amino acid positions of point mutants generated in this study (red and blue) or corresponding to lin-61 alleles identified in genetic screens (green) are indicated. (C) Affinity purification experiments of bacterially produced recombinant GST-LIN-61 proteins corresponding to the indicated MBT regions using immobilized unmodified and H3K9me3 peptides. αGST Western blot analyses of the recovered material are shown. Input, 2%. (D) Affinity purification experiments of in vitro translated wild type or point mutant MYC-LIN-61 proteins using immobilized unmodified and H3K9me3 peptides. αMYC Western blot analyses of the recovered material are shown. Input, 7.5%.
Figure 3.
LIN-61–H3K9me3 binding is essential for C. elegans vulva development within the synMuvB pathway.
(A) Schematic representation of C. elegans vulva development pathways. The anchor cell (AC) secretes the EGF factor LIN-3, thereby inducing vulval cell fate determination in three out of six vulva precursor cells (VPC). Normally, LIN-3 expression and secretion in the hypodermis (hyp7) is repressed by the parallel synMuvA and synMuvB pathways. When components of each class of factors, synMuvA (e.g. lin-15A) and synMuvB (e.g. lin-61) are mutated, spurious LIN-3 signal results in induction of additional VPCs causing pseudovulvae formation (adapted from ref. [49]). (B) Representative images (DIC optics) of worms of the indicated genotypes. Arrowheads point to pseudovulvae. Scale bar represents 100 µm. (C) lin-61; lin-15A double mutant worms were injected with a genomic fragment of the lin-61 gene or the lin-61 gene encoding for a F452A/W455A/F459A triple mutant protein. rol-6(su1006) served as marker for transgenic worms. Animals with a minimum of one ectopic ventral protrusion (pseudovulva) were scored Muv. For each independently established worm line, the indicated number (n) of worms was analyzed. (D) Western blot analysis of worm extracts of the indicated transgenic worm lines using the indicated antibodies. (E) Affinity purification experiments of in vitro translated wild type or point mutant MYC-LIN-61 proteins using immobilized unmodified and H3K9me3 peptides. αMYC Western blot analyses of the recovered material are shown. Input, 7.5%.
Table 1.
Genetic interaction of lin-61 with other factors in causing Muv phenotype.
Figure 4.
lin-61 and hpl-2 act synergistically in C. elegans vulva development and fertility control.
(A) Representative images (DIC optics) of worms of the indicated mutant genotypes. Arrowheads point to pseudovulvae. Scale bar represents 100 µm. (B) Representative images of C. elegans of lin-61, hpl-2 and lin-61; hpl-2 mutant genotype raised at 24.5°C. DNA was stained with DAPI, Nomarski images were taken with DIC optics. The shape of gonad arms is outlined (solid lines). Dashed lines outline the embryos. White arrowheads mark nuclei of endomitotic cells implying failure in oocyte maturation and/or fertilization. Grey arrowheads mark oocytes. Asterisk marks spermatheca. Scale bar represents 50 µm. Mean brood size of non-sterile C. elegans of the indicated genotype at 20°C (C) and 24.5°C (D). Error bars reflect standard deviation. n, number of worms analyzed. One asterisk marks a significance interval of p<0.02 and three asterisks mark a significance interval of p<0.001 determined using a two-tailed Student's t-test.
Table 2.
Fertility analysis of lin-61 in conjunction with other factors.