Fig 1.
Evolutionary conservation of mammalian PEG3 proteins.
The exon structure of mouse Peg3 is presented with boxes indicating individual exons. The conservation level of each exon at the amino acid sequence level was calculated with Clustal Omega or Cobalt using the 10 representative PEG3 sequences, which were further summarized with a scheme involving boxes with different colors. The sequence of Exon9 was further divided into protein subdomains, including 12 zinc finger motifs and two proline-rich motifs (P1 and P2).
Fig 2.
Identification of KRAB-A domain within PEG3.
The amino acid sequences of Exon7 of mammalian PEG3 were aligned using Clustal Omega (upper panel). The amino acid sequences of the KRAB-A exon of mammalian ZIM1 were also similarly analyzed (lower panel). The 9-amino-acid-long region at the C-terminus of Exon7 of PEG3 shows sequence similarity to that of the KRAB-A region as indicated with red. The sequence on bottom indicates the consensus sequence of the KRAB-A domain.
Fig 3.
Identification of the potential interacting partners of PEG3.
(A) The proteins extracts derived from a set of WT (W1-3) and KO (K1-3) MEF cells were immunoprecipitated with anti-PEG3 antibody. The quality of the isolated precipitates was first monitored through SDS-PAGE. The cell lysates (WT, KO) before the immunoprecipitation were also included along with size markers (M). (B) The isolated precipitates were further analyzed using a series of tandem mass spectrometry. A total of 1,274 proteins were successfully identified from each of the 6 tested samples. The relative abundance (RA) of each protein was summarized using a heat map showing different levels of abundance. (C) The relative abundance of three proteins in the WT and KO samples were summarized with graphs with statistical significances.
Fig 4.
KAP1 as an interacting partner of PEG3.
(A) A schematic representation indicates the exon structure and the ORF of mouse PEG3. The current study used three constructs expressing mouse PEG3 and human KAP1: PEG3-FL, the full-length PEG3 with the FLAG tag at the C-terminus; PEG3-Del(Ex7), the mutant version of mouse PEG3 lacking Exon7 with the FLAG tag at the C-terminus; KAP1, the full-length KAP1 with the HA tag at the N-terminus. (B) Co-localization experiments. As a control experiment, the cells were co-transfected with two constructs, PEG3-FL-FLAG and KAP1-HA constructs. The expression and subsequent localization within the nuclei were individually visualized with anti-FLAG (left) and anti-HA (middle) antibodies. These two images were later merged together (right). (C) The protein extracts derived from MEF cells were individually immunoprecipitated with anti-PEG3 antibodies, and further analyzed with western blotting using anti-KAP1 antibody (upper). A similar series of Co-IP experiments with anti-PEG3 antibody were repeated using the protein extracts from WT and KO MEFs, and later analyzed with western blotting using anti-LSD1 antibody (lower). (D) Neuro2a cells were co-transfected with the constructs expressing the full-length PEG3 and KAP1, immunoprecipitated with anti-FLAG and HA antibodies, and finally analyzed with western blotting using anti-KAP1 antibody (upper). A similar series of Co-IP experiments were repeated with two constructs expressing the full-length and mutant versions of PEG3. In this case, the immunoprecipitates were analyzed with western blotting using anti-HA antibody (lower).
Fig 5.
PEG3 binding to its targets with KAP1.
The potential interaction of PEG3 and KAP1 was further tested using ChIP experiments. PEG3 is known to bind to the zinc finger exon of Zim1, thus the 4 regions covering the Zim1 locus were used for this analysis (A). The chromatin prepared from WT and KO MEFs were precipitated with anti-KAP1 antibody, and the eluted DNA was subsequently analyzed using qPCR. The results were summarized with graphs with statistical significance (B).
Fig 6.
Exon7 for the repression role of PEG3.
A series of reporter assays were performed to test potential roles played by Exon7 for the repression function of PEG3. This series of assays used two reporters: promoterless basic (Empty) and H19-promoter-containing reporters (H19-prom). The H19-prom construct was also co-transfected individually with the full-length and mutant versions of PEG3 (A). The graph summarizes the results derived from this series of reporter assays. The obtained values of the luciferase activity were first normalized with the amount of total proteins, and later compared to the value of the sample transfected with the H19-prom construct only without the PEG3 expression construct. This series of experiments were repeated with two independent trials (B).