Posterior transformation of the axis in Pcl2^GT/GT and Pcl2^Δ/Δ mutants. (A) Skeletal alterations in Pcl2^GT/GT (GT/GT) and Pcl2^Δ/Δ (Δ/Δ) mutants. (Frames a, b, and c) Lateral views of the upper part of the vertebral column are shown. Yellow arrowheads indicate the first cervical vertebra. The arrows and brackets in frames b and c indicate the ectopic ribs associated with C7 and the anteriorly shifted sternum, respectively. (Frames d, e, and f) Ventral views of the rib cages are shown. In frames e and f, the ectopic ribs associated with C7 are indicated by arrows. (Frames g, h, and i) Overviews of isolated C1 vertebrae. Odontoid processes fused with C1 are indicated by arrowheads in frames h and i. (Frames j, k, and l) Overviews of isolated C2 vertebrae. The odontoid process is indicated by an arrowhead in frame j. (Frames m, n, and o) Overviews of isolated C7 vertebrae. Ectopic ribs are indicated by arrows in frames n and o. (B) Schematic summary of the axial homeotic transformations in Pcl2^GT/GT and Pcl2^Δ/Δ mice. The following parameters, identified by letters on the figure, were scored, and the frequency of each alteration is indicated: C1 → C2, presence of the odontoid process on the C1 vertebra (a); C2 → C3, lack of the odontoid process from the C2 vertebra (b); C5 → C6, appearance of anterior tuberculum (c); C7 → T1, appearance of cervical ribs on C7 (d); T1 → T2, prominent spinous process on T1 (e); T7 → T8, dissociation of seventh rib from the sternum (f); T13 → L1, loss of the rib on 20th vertebra (g); lumbar vertebra 5 (L5) or L6 → sacral vertebra 1 (S1), formation of the sacroiliac joint in 25th or 26th vertebra (h). (C) Expression of Hox genes in 11.5-dpc wild-type and Pcl2^Δ/Δ embryos. The expression of Hoxb4 (a and b), Hoxb6 (c and d), Hoxb8 (e and f), and Hoxd4 (g and h) is shown. Prevertebrae are numbered starting from the proatlas, and dotted lines indicate the segment boundaries. (D) Derepression of Hox genes in Pcl2^Δ/Δ ES cells. Hoxa1, Hoxb4, Hoxb6, Hoxb8, and Tbx3 were derepressed in Pcl2^Δ/Δ ES cells compared to the wild-type controls by real-time PCR. Fold expression of respective genes in Pcl2^Δ/Δ ES cells compared to that of the wild type is shown by bars. Statistically significant differences are indicated by asterisks.

The expression and target binding of Ring1B and Pcl2 are regulated in a mutually independent manner in ES cells. (A) Pcl2 and Suz12 are expressed in a Ring1B-independent manner. The expression of Ring1B, Pcl2, and Suz12 in Ring1B^+/+ and Ring1B^−/− ES cells is shown. γ-Tubulin was used as a dose control. (B) Pcl2 binds to Hox promoters in a Ring1B-independent manner. Pcl2 association to the Hox promoter regions in Ring1B^+/+ and Ring1B^−/− ES cells is shown. The hprt gene was used as a negative control. (C) Global levels of H3K27 trimethylation (H3K27me3), M33, Ring1B, and H2A monoubiquitinylation (uH2A) were not significantly altered in Pcl2^Δ/Δ ES cells (Δ/Δ) compared to levels in the wild type (+/+). γ-Tubulin was used as a dose control. (D) Binding of Suz12, Eed, H3K27me3, and Ring1B at Hox promoter regions was not significantly changed in Pcl2^Δ/Δ ES cells (Δ/Δ). IgG and hprt genes were used as negative controls.

The role of Pcl2 in replicative senescence. (A) ChIP-seq binding patterns at Cdkn2a and Cdkn2b loci are shown for H3K4me3, H3K27me3, Ezh2, Ring1B, and Pcl2 in ES cells. CpG islands are shown as green bars (below) (B) Termination of replicative senescence in Pcl2^Δ/Δ MEFs as revealed by a modified 3T9 assay. Levels of replicative senescence were compared among the wild-type (+/+; +/+), Pcl2^Δ/Δ (Δ/Δ; +/+), Phc2^−/− (+/+; −/−) and Pcl2^Δ/Δ; Phc2^−/− (Δ/Δ; −/−) cells. (C) The role of Pcl2 in replicative senescence. Termination of replicative senescence in Pcl2^GT/GT MEFs as revealed by a modified 3T9 assay. Levels of replicative senescence were compared between the wild-type (+/+; +/+) and Pcl2^GT/GT MEFs. (D) Overexpression of Pcl2 induced senescence in Pcl2^GT/GT MEFs. (E) Morphology of wild-type (+/+) and Pcl2^Δ/Δ (Δ/Δ) MEFs at passage 12. Light microscopic views are shown. (F) Decreased expression of p19^arf and p16^ink4a in Pcl2^Δ/Δ (Δ/Δ) MEFs at passage 10 in comparison with that of the wild type (+/+). The expression of lamin B was examined as a dose control. (G) Quantitative analysis of p19^arf and p16^ink4a transcripts in Pcl2^Δ/Δ (Δ/Δ) MEFs at passage 10. Note the concomitant reduction of these transcripts in Pcl2^Δ/Δ MEFs in comparison with wild type (+/+). (H) Cdkn2a repression is involved in inhibiting cellular senescence in Pcl2^Δ/Δ MEFs. Overexpression of either p19^arf or p16^Ink4a in Pcl2^Δ/Δ MEFs considerably inhibited their proliferation. The total cells were counted on day 2 and day 4 after retroviral transduction.

The role of Pcl2 for PRC2 and PRC1. (A and B) Enrichment of H3K27me3, H3K4me3, and Ring1B at the p16^Ink4a promoter region was determined by ChIP and site-specific real-time PCR. The β-actin promoter was used as a control. (C) A model for Pcl2 functions during A-P specification of the axis and stress-induced cellular senescence. In developing embryos, Pcl2 is a functional component of PRC2 and also functionally cooperates with PRC1 to mediate repression of Hox cluster genes. Upon replicative stress, Pcl2 activates Cdkn2a expression and suppresses cellular senescence, likely by suppressing the catalytic activity of PRC2 at the Cdkn2a locus.

Primary structure and the expression of mouse Pcl2 gene products. (A) Schematic representation of the two predicted Pcl2 isoforms. The black box corresponding to aa 44 to 101 representing the Tudor domain. The two dark blue boxes represent the PHD fingers. Three red vertical bars delineate the putative nuclear localization signals (NLS). A light blue box at aa 569 to 589 shows the chromo-like domain region at the C terminus. Positions for siRNA are demonstrated by light blue bars. The cDNA fragment isolated by two-hybrid screening for Mel18 interacting factors is shown by a green line at the top. The peptide and the fragment used to generate anti-Pcl2 antibodies are represented by two black lines. All ATG codons in the correct reading frame are indicated by colored triangles. Putative translation initiation codons for the 67- and 55-kDa isoforms are demarcated by red triangles, and those for 52- and 46-kDa isoforms are indicated by green triangles. (B) Expression of multiple Pcl2 isoforms in ES cells. The 67-, 55-, 52-, and 46-kDa bands were detected on Western blots of whole-cell lysates from wild-type ES cells using the Pcl2 MAb. The expression of Pcl2 siRNA proportionally reduced the intensity of all four bands, whereas they were unaffected by the control siRNA. Lamin was used as a loading control. (C) Schematic representation of the genomic organization of Pcl2^GT and Pcl2^Δ alleles. The Pcl2^GT allele resulted from insertion of a gene trap vector (indicated by an open triangle) into exon 3. In the Pcl2^Δ allele, the genomic region encompassing the fourth intron to the middle of exon 5 is deleted. Genomic positions for putative translation initiation codons for the 67- and 55-kDa isoforms are indicated by red triangles, and those for 52- and 46-kDa isoforms are indicated by green triangles. (D) The 67-, 55-, 52-, and 46-kDa bands detected by the Pcl2 MAb were absent in Pcl2^GT/GT ES cells, whereas only the 67- and 55-kDa bands were affected in Pcl2^Δ/Δ ES cells. (E) Polyclonal antibodies against the N-terminal peptide of Pcl2 (anti-N) exclusively immunoprecipitated the 67-kDa isoform in the wild-type ES cells. WCE, whole-cell extract. The blot was probed with the Pcl2 MAb.

PRC2-dependent binding of Pcl2 to Hox genes. (A) Pcl2 association at Hox promoter regions revealed by conventional ChIP assay using anti-N polyclonal in Pcl2^+/+ (+/+) and Pcl2^Δ/Δ (Δ/Δ) ES cells. For the input, genomic DNA extracted from the original whole-cell lysate equivalent to 1/40 of the volume used for the ChIP analysis was subjected to PCR; the hprt gene was used as a negative control. (B) The distribution of Pcl2, H3K27me3, and Ring1B at the promoter regions of the genes in ES cells. Agilent mouse promoter ChIP-on-Chip Sets 244K were used for Hoxa10, Hoxb4, and Tcf4 promoter region analysis. (C) ChIP-seq binding patterns for Evx2 and Hoxd loci are shown for H3K4me3, H3K27me3, Ezh2, Ring1B, and Pcl2 in ES cells. CpG islands are shown as green bars (below). (D) Impact of Suz12 deficiency on the expression of Pcl2. The expression of Suz12, Pcl2, Rnf2, and γ-tubulin in Suz12^+/+ (+/+) and Suz12^−/− (−/−) ES cells as revealed by Western blotting. (E) Pcl2 association at Hox promoter regions in Suz12^+/+ and Suz12^−/− ES cells. For the input, genomic DNA extracted from the original whole-cell lysate equivalent to 1/40 of the volume used for the ChIP analysis was subjected to PCR; the hprt gene was used as a negative control. (F) Expression changes of Pcl2, Suz12, Eed, and Ezh2 in Suz12^−/− ES cells. Expression changes of Pcl2, Suz12, Eed, and Ezh2 in Suz12^−/− ES cells were revealed by microarray analysis using Affymetrix Mouse 430.2. Detected signals were normalized using a quantile normalization method, and only signals having significant intensity from background were counted. The average signal intensity of respective genes in Suz12^−/− and wild-type ES cells was calculated, and the log ratios of Suz12^−/− against wild type are shown by bars.

Synergistic activity of Pcl2 with PRC2 and PRC1 components during A-P specification of the axis. (A) Lateral views of the upper part of the vertebral column in Pcl2^Δ/Δ and Pcl2^Δ/Δ; Suz12^+/− newborn mice. Genotypes of Pcl2 and Suz12 loci are indicated at the top. Anterior tubercles, ectopic ribs, and prominent spinous processes are indicated by yellow arrowheads, black arrows, and yellow arrows, respectively. A bracket in Pcl2^Δ/Δ; Suz12^+/− indicates the cranially shifted sternum. (B) Schematic representation of the frequency of the axial homeotic transformations in respective mutants. The following parameters, identified by letters on the figure, were scored, and the frequency of each alteration is indicated: C1 → C2, presence of the odontoid process on the C1 vertebra (a); C2 → C3, lack of the odontoid process from the C2 vertebra (b); C5 → C6, appearance of the anterior tubercle of the transverse process on the C5 (c); C7 → T1, appearance of cervical ribs on C7 (d); T1 → T2, prominent spinous process on T1 (e); T13 → L1, loss of the rib on 20th vertebra (f); L5 or L6 → S1, formation of the sacroiliac joint in the 25th or 26th vertebra (g). (C) Skeletal alterations seen in Pcl2^Δ/Δ, Pcl2^Δ/Δ; Mel18^−/−, and Pcl2^Δ/Δ; Phc2^−/− mice. Only the mutant Pcl2, Mel18, and Phc2 alleles are depicted. (Upper panels show lateral views of the upper part of the vertebral column. Arrowheads indicate ectopic ribs associated with C7. For Pcl2^Δ/Δ; Mel18^−/− and Pcl2^Δ/Δ; Phc2^−/− mice, yellow arrows and brackets indicate ectopic arches of the occipital bone and cranially shifted sternum, respectively. Lower panels show overviews of the scapula. Acromion are indicated by arrows, which are rudimentary in the compound mutants. Holes (asterisks) were generated in the center of blades in Pcl2^Δ/Δ; Mel18^−/− and Pcl2^Δ/Δ; Phc2^−/− mice. (D) Schematic representation of the frequency of the axial homeotic transformations in respective mutants. The following parameters, identified by letters on the figure, were scored, and the frequency of each alteration is indicated: supraoccipital bone → C1, appearance of ectopic bones seen in the craniodorsal region of the C1 vertebra or ectopic arch of the occipital bones (a); C1 → C2, presence of the odontoid process on the C1 vertebra (b); C2 → C3, lack of the odontoid process from the C2 vertebra (c); C7 → T1, appearance of cervical ribs on C7 (d); T7 → T8, dissociation of seventh rib from the sternum (e); T13 → L1, loss of the rib on the 20th vertebra (f); L5 or L6 → S1, formation of the sacroiliac joint in 25th or 26th vertebra (g).

Pcl2 regulates cellular senescence through Cdkn2a. (A) Schematic representation of the transgene to overexpress Myc-tagged Pcl2 in a Cre recombinase-dependent manner. Briefly, the lacZ gene in CAG-CAT-Z (4) was replaced by Myc-tagged Pcl2. Pcl2 expression is therefore induced by deletion of chloramphenicol acetyltransferase (CAT) gene cassette placed between two loxP sites (shown by arrows) by Cre recombinase. A transgenic line harboring this inducible construct was bred with another transgenic line expressing ERT2-Cre to generate MEFs in which Pcl2 could be expressed upon 4-hydroxytamoxifen ([OHT] 0.5 μM) treatment. The location of primers used to assess deletion of the CAT gene cassette is shown. (B) Impact of Myc-tagged Pcl2 on replicative senescence as revealed by a modified 3T9 assay. Double transgenic MEFs were treated by 0.5 μM tamoxifen at passage 3 (OHT+), and growth rate was examined after the treatment by using untreated MEFs as a control (OHT−). Overexpression of Pcl2 (OHT+) clearly induced premature proliferation arrest. (C) Induction of p19^arf expression upon deletion of the CAT gene in two different MEF lines harboring double transgenes. The top panel is an assessment of tamoxifen-induced recombination of the transgene, the middle shows results of Western blotting for p19^arf expression, and the bottom shows results of Western blotting for lamin B expression as dose controls.