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1.
Fig. 8.

Fig. 8. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Model for the effects of REST on PRC1 binding at distal versus proximal RE1 elements. The opposite rotational orientations of the multicolored wheel represent different configurations of PRC1 binding at distal versus proximal RE1 elements. We hypothesize that the opposite effects of REST on PRC1 binding at distal versus proximal RE1 elements are due to the distinct configurations of PRC1 binding. The data do not allow a determination of whether PRC1 interacts with REST at proximal RE1 elements.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
2.
Fig. 4.

Fig. 4. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Effects of RestΔN knockout on PRC1 binding at sites occupied by REST versus sites not occupied by REST in ES cells. The effects of the RestΔN knockout on PRC1 binding at distal RE1 elements (left column) and at sites not occupied by REST (right column) were compared in ES cells, as described in the legend of Fig. 3. The efficiencies of precipitation of the regions indicated above each graph by antibodies directed against the proteins indicated below the bars are shown for Rest+/+ and RestΔN knockout cells. Monoclonal anti-Ring1b antibody was used. The bars show the amounts of input chromatin precipitated on a logarithmic scale (*, P < 0.05; **, P < 0.01).

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
3.
Fig. 6.

Fig. 6. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Selectivity of REST, Rcor1, and PRC1 subunit knockdown and RestΔN knockout. (A) Effects of RestΔN knockout and of the shRNA constructs indicated below the bars on the REST and PRC1 subunit transcripts indicated above the bars. The bars show the ratios between the transcript levels in RestΔN knockout cells and those in Rest+/+control cells or between the transcript levels in cells expressing the knockdown shRNA and those in cells expressing the control shRNA. Two different shRNAs directed against different sequences in Cbx2, Cbx7, and Mel18 were used (solid bars and striped bars). The primers used to detect REST transcripts also detect transcripts from the RestΔN knockout locus. The results shown represent the means of data from two or more independent experiments. (B) Effects of the RestΔN knockout and of the shRNA constructs indicated below the bars on the levels of the pluripotency- and differentiation-associated transcripts indicated above the bars. The transcript levels were measured in ES cells (ESC) and in embryoid bodies (EB) and were normalized relative to Gapdh levels.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
4.
Fig. 5.

Fig. 5. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Coregulation of neuronal genes by REST and PRC1 subunits in ES cells. (A) Derepression of the transcripts indicated below the bars in knockout ES cells containing the mutations indicated above the bars. Transcripts in Ring1bfl/fl Ring1a−/− and Ring1bfl/fl ES cells were analyzed 24 and 48 h after the addition of 4-hydroxytamoxifen (OHT). The bars show the ratio between the transcript levels in the mutant cells relative to untreated cells or cells isolated from wild-type control mice. The transcript levels were normalized relative to the levels of Gapdh transcripts in the same cells. The results shown are representative of data from two experiments using independently cultured ES cells (*, P < 0.05; **, P < 0.01). (B) Derepression of the transcripts indicated below the bars in ES cells expressing the shRNAs indicated above the bars. Two different shRNAs directed against different sequences in Cbx2, Cbx7, and Mel18 were used. The bars show the ratios between the transcript levels in cells expressing the knockdown shRNA and cells expressing the control shRNA. The transcript levels were normalized relative to the levels of Gapdh transcripts in the same cells. The results shown are representative of data from two experiments using independently cultured ES cells (*, P < 0.05; **, P < 0.01).

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
5.
Fig. 7.

Fig. 7. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Effects of RestΔN knockout on PRC1 binding and neuron-specific gene transcription during neuronal differentiation. (A) Embryoid bodies were differentiated into neuronal stem cells (NSCs), stimulated to proliferate (NSC-P), and induced to form differentiating neurons (Neuron) (39). NSC-P (top) and differentiating neurons (bottom) derived from Rest+/+ control (left) and RestΔN knockout (right) ES cells were stained using Tuj1, antinestin, and antineurofilament (NF) antibodies (green) and Hoechst dye (blue). The images are representative of all cells in each population. The numbers indicate the percentages of cells stained by the antibodies. (B to D) Neuronal gene transcription in Rest+/+ control (solid) and RestΔN knockout (striped) cells at each stage of differentiation. (E and F) Precipitation of the RE1 elements indicated above each graph from differentiating Rest+/+ control and RestΔN knockout neurons by antibodies directed against the proteins indicated below the bars. The results shown are representative of data from two independent experiments with differentiating neurons and one experiment with neuronal stem cells (*, P < 0.05; **, P < 0.01). ChIP analysis of the Cdh2 RE1 site could not be performed with differentiating neurons due to the limited number of cells and the low sensitivity of detection at this site.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
6.
Fig. 3.

Fig. 3. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Effects of RestΔN knockout and REST knockdown on PRC1 subunit, Rcor1, Sin3a, and REST binding at distal versus proximal RE1 elements. The precipitation of the RE1 elements indicated above each graph by antibodies directed against the proteins and histone modifications indicated below the bars was measured by ChIP analysis. The efficiencies of precipitation were compared between Rest+/+ control and RestΔN knockout ES cells as well as between shControl and shREST knockdown ES cells. The data were plotted using a logarithmic scale to accommodate the data using different antibodies in the same graph (Pre, preimmune serum). Cbx2 and Cbx7 were precipitated using rabbit sera, whereas Ring1b and Mel18 were precipitated using purified IgG, which produced lower background signal. The left column corresponds to distal RE1 sites (>5 kb from transcription start), and the right column corresponds to proximal RE1 sites (<5 kb from transcription start). Proteins that are associated with PRC1 or with REST are indicated by the brackets below the graphs. The results shown are representative of data from two experiments using independently cultured ES cells (*, P < 0.05; **, P < 0.01). The difference between the effects of the RestΔN knockout as well as shREST knockdown on PRC1 occupancy at each distal versus each proximal RE1 element was statistically significant (P < 0.05 by a pairwise-difference test). The diagrams to the right of the graphs show the effects of REST on PRC1 (multicolored wheel) binding at distal versus proximal RE1 elements. The opposite rotational orientations of PRC1 represent distinct configurations of PRC1 binding.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
7.
Fig. 1.

Fig. 1. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

Purification of proteins associated with Cbx family members from mouse ES cells. (A) Flow diagram for immunoaffinity purification and identification of proteins associated with Cbx family members from mouse ES cells that expressed Cbx fusion proteins (43). (B) The enrichments of Cbx fusion proteins and of Ring1b during purification from the cell lines indicated to the left of each blot were evaluated. An equal proportion of each fraction (indicated above the lanes) was analyzed by immunoblotting using anti-GFP (left) and anti-Ring1b (right) antibodies. Multiple exposures of the blots were scanned, and the total protein concentrations were measured to calculate the fold enrichment of each Cbx protein and of Ring1b at each stage of purification. The final fold enrichments of each Cbx protein and of Ring1b were 310,000 and 240 (Cbx2), 1,000 and 1.5 (Cbx6), 12,000 and 1,000 (Cbx7), and 6,600 and 390 (Cbx8), respectively, relative to the cell lysates. ESC, parental ES cell line used to generate the ES cell lines expressing Cbx fusions. (C) SDS-PAGE analysis of the fractions purified from cells that expressed the Cbx fusion proteins indicated above the lanes or the parental cells (ESC). The mobilities of PRC1- and REST-associated proteins identified by mass spectrometry are indicated by the labels at the right of the images. The number of unique peptides corresponding to each protein is indicated in parentheses after the name of each protein. (D) Fractions purified from cells that expressed the Cbx fusions indicated above the lanes were analyzed by immunoblotting using antibodies directed against the proteins connected to the lines on the left of the images. The Cbx fusions were detected by using anti-GFP antibodies (bottom). Equal proportions of the fractions purified from cells that expressed Cbx2, Cbx7, and Cbx8 fusions and from the parental cells (ESC) were analyzed. A 10-fold-larger proportion of the fraction purified from cells that expressed the Cbx6 fusion was analyzed to compensate for the loss of the Cbx6 fusion during the isolation of nuclei.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.
8.
Fig. 2.

Fig. 2. From: REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements.

REST interactions with Cbx proteins in living cells and with Ring1b in native cell extracts. (A) Principle for BiFC analysis of REST interactions with Cbx proteins. REST and Cbx proteins fused to fluorescent protein fragments were coexpressed. The interaction of REST with Cbx proteins was detected based on the facilitated association of the fluorescent protein fragments. (B) The subnuclear distributions of complexes formed by the Cbx family proteins and REST indicated at the left of the images were visualized by using BiFC analysis (green) in live HEK293T cells stained by Hoechst dye (red). Different exposure times were used to compensate for differences in fluorescence intensities of BiFC complexes formed by different Cbx proteins with REST. The images are representative of the majority of cells in each population. (C) Efficiencies of BiFC complex formation by REST with wild-type (WT) and mutant (I17F or I16F) Cbx proteins measured by flow cytometry. The bars show the mean fluorescence intensities of 20,000 cells in each of two independent experiments. The levels of expression of different Cbx fusion proteins were compared by analyzing equal amounts of the cell extracts by immunoblotting using anti-GFP antibodies (inset above bars) (*, cross-reactive proteins). The levels of wild-type and mutant Cbx fusion protein expression were similarly compared (below the graph). The levels of Gapdh were measured to determine the amount loaded into each lane. (D) The diagrams indicate the truncated REST variants used to map interactions with Cbx proteins. The stripes indicate the zinc finger DNA-binding domain. The region replaced by Pgk-Neo in RestΔN knockout cells is indicated by a gray bar (9). (E) Efficiencies of BiFC complex formation by the Cbx proteins indicated above the bars with the truncated REST proteins indicated below the bars measured by flow cytometry. The bars show the mean fluorescence intensities of 20,000 cells in each of two independent experiments. The levels of expression of full-length and truncated REST fusions were measured by immunoblotting using anti-REST antibody (below each graph) (*, cross-reactive proteins). Gapdh was measured to determine the amount loaded into each lane. (F) Analysis of endogenous REST and Ring1b interactions in ES cell extracts. Extracts from Rest+/+ and RestΔN ES cells were precipitated by using anti-REST antibodies or beads alone (mock). The precipitates were analyzed by immunoblotting using antibodies directed against the proteins indicated at the left of the image. The input lanes contained 5% of the extract.

Xiaojun Ren, et al. Mol Cell Biol. 2011 May;31(10):2100-2110.

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