1. Volume 22, Issue 11, Pages 3032-3043 (March 2018)
Combinatorial Control of Recruitment of a Variant PRC1.6 Complex in Embryonic Stem Cells 
Yikai Huang, Wukui Zhao, Congcong Wang, Yaru Zhu, Mengjie Liu, Huan Tong, Yin Xia, Qing Jiang, Jinzhong Qin 
Cell Reports 
Volume 22, Issue 11, Pages (March 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 3032-3043DOI: (10.1016/j.celrep.2018.02.072)
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3. Figure 1
L3mbtl2 Functions Independently of the Methyl-Lysine Caging or SUMO Modification
(A) Schematic representation of the mouse L3mbtl2 protein with known aromatic cage residues and putative sumoylation sites indicated, including K215, K433, K541, K673, and K698. Alanine mutation of four aromatic residues lining the cage is designated as Cage mutant. Sumoylation-deficient mutant with all five sumoylation site lysines substituted for arginines is designated as K5R.
(B) Quantification of FLAG-tagged wild-type or mutant forms of L3mbtl2 occupancy on the Taf7L, Tex101, and Rhox13 promoters by ChIP-qPCR. Input control represents 5% of total amount of sonicated chromatin added to each IP reaction. The data shown represent the mean (± SD) of three independent experiments.
(C) Real-time PCR of Taf7L, Tex101, and Rhox13 expression in wild-type, L3mbtl2−/− ESC, and L3mbtl2−/− cell reintroduction of wild-type and mutant forms of FLAG-tagged mouse L3mbtl2 (F-L3mbtl2). The y axis represents fold change after normalization of data from three independent experiments to control cells (presented as mean ± SD).
(D) The expression of different L3mbtl2 mutant proteins in L3mbtl2−/− ESCs. FLAG antibody was used for detecting wild-type or mutant L3mbtl2 proteins. β-actin served as the loading control.
(E) Representative phase-contrast micrographs of ESC colony of indicated genotypes grown on mitomycin-C-inactivated feeders. Shown is colony size 7 days after seeding single-cell suspensions onto feeder layers in the presence of LIF.
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4. Figure 2
Structure of L3mbtl2 Progressive Deletion Mutants and Their Ability to Rescue Phenotypic Defects Observed in L3mbtl2 Null ESCs
(A) The structures of various constructs generated by deletion of the indicated regions of L3mbtl2 and their ability to rescue L3mbtl2−/− ESC growth phenotype are summarized schematically. Shown at the top of the panel is a schematic diagram of known structural features of wild-type L3mbtl2. Numbers refer to the positions of amino acid residues in the deduced protein sequence of L3mbtl2. The systematic deletion mutants made in this study are diagrammed below the wild-type L3mbtl2, with the amino acids removed by each deletion listed to the right or top of the drawing. Deleted regions are indicated by thin bent lines. Results of growth rescue assays using L3mbtl2−/− ESCs reexpressing L3mbtl2 wild-type or mutant proteins are presented on the right.
(B) Association of wild-type and deletion mutant L3mbtl2 with select promoters, detected by ChIP-qPCR experiments. Data are presented as mean ± SD of three independent experiments in triplicates.
(C) Real-time PCR detecting Taf7L, Tex101, and Rhox13 expression levels in L3mbtl2−/− ESCs following reexpression of wild-type and deletion mutant forms of L3mbtl2. Data of relative mRNA levels (y axis) from three independent experiments were normalized to β-actin and are presented as mean ± SD.
(D) Protein levels of respective L3mbtl2 mutants were examined by western blotting with anti-FLAG antibody.
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5. Figure 3
Effect of Chimeric L3mbtl2 Constructs on L3mbtl2-Mediated Chromatin Association and Activity in ESCs
(A) Schematic diagram representing the chimeric constructs generated by exchange of the indicated regions between L3mbtl2 and Mbtd1. The names of the mutants begin with SW (swapping) followed by the range of amino acids in L2mbtl2 that were swapped with Mbtd1. Each domain of L3mbtl2 and Mbtd1 is represented by a different color (the amino acid [aa] residue where the junction between L3mbtl2 and Mbtd1 occurred).
(B) Chromatin immunoprecipitated from L3mbtl2−/− ESCs reexpressing FLAG-tagged wild-type or swap mutants of L3mbtl2 with anti-FLAG antibody and enrichment on target promoters (Taf7L, Tex101, and Rhox13) measured by qPCR. Data are presented as mean ± SD of three independent experiments in triplicates.
(C) Real-time PCR of Taf7L, Tex101, and Rhox13 expression in L3mbtl2−/− cell reintroduction of wild-type and swap mutants of FLAG-tagged mouse L3mbtl2. Data of relative mRNA levels (y axis) from three independent experiments were normalized to β-actin and are presented as mean ± SD.
(D) The expression of different L3mbtl2 swap mutants in L3mbtl2−/− ESCs. FLAG antibody was used for detecting wild-type or hybrid L3mbtl2 proteins. β-actin served as the loading control.
(E) Representative phase-contrast pictures of ESC colony of indicated genotypes.
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6. Figure 4 L3mbtl2 Is a Mouse Homolog of Drosophila Sfmbt
(A) FLAG-tagged wild-type L3mbtl2, Mbtd1, Sfmbt1, and Sfmbt2 were generated as diagrammed. Shown at the top of the panel is a schematic diagram of known structural features of a four-MBT repeat protein, Sfmbt, in Drosophila. The different conserved domains are color coded according to the color key.
(B) CoIP of FLAG-tagged L3mbtl2, Mbtd1, Sfmbt1, and Sfmbt2 with PRC1.6 core complex components. ESCs transfected with expression plasmids for FLAG-tagged L3mbtl2, Mbtd1, Sfmbt1, or Sfmbt2, as indicated, were immunoprecipitated with control IgG (lanes 5–8) or an antibody to FLAG (M2) (lanes 9–12), followed by immunoblotting with antibodies to Ring1b, Max, Pcgf6, Hdac1, Hdac2 and YY1 as indicated. 5% of total cell lysates used in IP are shown as input (lanes 1–4).
(C) L3mbtl2 forms a complex (PRC1.6) similar in composition to the Drosophila PhoRC complex. The models were based on findings obtained in this study as well as those reported in previous studies (Alfieri et al., 2013; Kloet et al., 2016; Trojer et al., 2011; Zhao et al., 2017).
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7. Figure 5
Characterization of the Pho Spacer-Binding Pocket of Mouse L3mbtl2
(A) The structures of Drosophila Sfmbt and mouse L3mbtl2 are schematically represented along with their domain organizations. Aligned amino acid sequences for the Pho spacer-binding pocket are also shown with the positions of alanine substitution mutations. The key residues in the Pho spacer-binding pocket from Sfmbt and mouse L3mbtl2 are indicated with star symbols and highlighted (red).
(B) Association of FLAG-tagged proteins in L3mbtl2−/− ESCs stably expressing mock, FLAG-tagged L3mbtl2 wild-type, or indicated mutants with promoter regions of their representative target genes as determined by ChIP and site-specific real-time PCR. Data are presented as mean ± SD of three independent experiments in triplicates.
(C) Expression levels of Taf7L, Tex101, and Rhox13 in L3mbtl2−/− ESCs expressing mock, FLAG-tagged L3mbtl2 wild-type, or indicated mutants. Expression levels were normalized to a β-actin control and are depicted as fold changes relative to the wild-type ESCs. Error bars are based on the SD, as determined (derived) from triplicate PCR reactions.
(D) The expressions of transfected plasmids were confirmed by immunoblotting. β-actin served as the loading control.
(E) Representative phase-contrast images of colonies resulting from feeder-cultured wild-type, L3mbtl2−/− ESCs and L3mbtl2−/− ESCs rescued with wild-type or the Pho spacer-binding pocket mutants of L3mbtl2.
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8. Figure 6
Dependency of PRC1.6 Recruitment on Overlapping Action between Pcgf6, L3mbtl2, and Max In Vivo
(A) Wild-type, Ring1b−/−, Pcgf6−/−, L3mbtl2−/−, Max−/−, Pcgf1−/−, and Pcgf6−/−;L3mbtl2−/− ESCs were analyzed by ChIP using the indicated antibodies. The HPRT1 gene, which is not thought to be a PRC1.6 target, was not significantly enriched and serves as a negative control in all subsequent ChIP experiments. Data are presented as mean values ± SD from three independent biological replicate experiments. #p < 0.05; ##p < 0.01; and ###p < 0.001, for Pcgf6/L3mbtl2 double knockout versus Pcgf6 or L3mbtl2 single knockout.
(B) Western blot demonstrating changes in the levels of L3mbtl2, Pcgf6, Max, Ring1b, and Pcgf1 in ESCs of indicated genotypes. β-actin was used as a loading control.
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9. Figure 7
Overlapping as well as Independent Action among PRC1.6 Components in Chromatin Tethering
(A) Real-time PCR of Taf7L, Tex101, Rhox13, Tcam1, and Tdrkh expression in ESCs of the indicated genotypes. Expression levels were normalized to a β-actin control and are represented as fold changes relative to the wild-type ESCs. Error bars are based on the SD, as determined from triplicate PCR reactions. #p < 0.05; ##p < 0.01; and ###p < 0.001, for Pcgf6/L3mbtl2 double knockout versus Pcgf6 or L3mbtl2 single knockout.
(B) Representative phase-contrast images of colonies resulting from feeder-cultured ESCs of the indicated genotypes. Shown is the colony size 7 days after seeding single-cell suspensions onto MEF-feeder layers in the presence of LIF.
(C) Schematic model of the recruitment of PRC1.6 complex to chromatin by collaborative action between L3mbtl2 and Max (see Discussion for a detailed description).
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