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First published online February 6, 2009
doi: 10.1242/10.1242/dev.028548

Development 136, 715-721 (2009)
Published by The Company of Biologists 2009
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REST selectively represses a subset of RE1-containing neuronal genes in mouse embryonic stem cells

Helle F. Jørgensen1,*, Anna Terry1, Chiara Beretta1, C. Filipe Pereira1, Marion Leleu1, Zhou-Feng Chen2, Claire Kelly1, Matthias Merkenschlager1 and Amanda G. Fisher1,*

1 MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
2 Departments of Anesthesiology, Psychiatry, Developmental Biology, Washington University School of Medicine Pain Center, St Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Repression of neural determinants is not compromised in REST-deficient mouse ES cells. (A) The top panel shows transcript levels in Rest+/- and Rest-/- relative to wild-type (WT) ES cells, as assessed by real-time quantitative PCR. The lower panel compares gene expression in wild-type ES cells transfected with siRNA targeting either a control sequence (siCtrl) or Rest (siREST), relative to mock-transfected cells. Values were normalised to house keeping genes (Ywhaz, Hmbs, Gapdh). Error bars indicate the s.d. from three to six experiments. Significant differences (two-tailed Student's t-test) between wild-type and Rest-/- (top) or between siREST and siCtrl (bottom) samples are indicated: *P<0.05, **P<0.005, ***P<0.0005. (B) Schematic representation of a 49 kb region flanking mouse Mash1 (top panel). Arrow, transcription start site; black boxes, exons; the putative REST binding site (REST bs) is indicated. The subnuclear location of Mash1 in wild-type ES cells (ESWT), Rest-/- ES cells (ESRest-/-), undifferentiated wild-type 46C ES cells (ES46C) and neural stem cells derived from 46C ES cells (NSC46C) is shown in the middle panel. The bar chart shows the percentage of cells with two peripheral Mash1 alleles (P.P.), one peripheral and one internal allele (P. I.) or two internal alleles (I.I.), as assessed in 3D FISH analysis. Representative confocal images of a single optical section are shown beneath for each cell type. Arrows mark Mash1 FISH signals. Scale bars: 2 µm. The bottom panel shows a replication timing analysis of Mash1 in wild-type and Rest-/- ES cells. The relative amount of newly synthesised (BrdU-labelled) locus-specific DNA in G1, four sequential S-phase fractions and G2-M is shown. Data for control genes are shown in Fig. S2 in the supplementary material. Error bars indicate s.d. from two experiments. For comparison, the replication profile of Mash1 in neural progenitor cells (NPES+RA) is included (Williams et al., 2006Go).

 

Figure 2
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  		Fig. 2. Loss of REST in mouse ES cells causes upregulation of RE1-containing neuronal genes. (A) Venn diagram showing the number and intersection of genes that are at least 1.4-fold up- or downregulated between Rest-/- and wild-type (WT) ES cells or between shREST- and shCtrl-transfected ES cells (P<0.05). (B) Overlap between the 87 genes that are misregulated in both Rest-/- and shREST cells as defined in A and the 511 genes that contain RE1 sites within -1 kb to +5 kb relative to the transcription start site (TSS) (Otto et al., 2007Go). (C) ChIP analysis of REST binding in wild-type and REST-deficient ES cells. IgG, control. The average and s.d. of three to five experiments is shown. Significant REST-enrichment in wild-type relative to Rest-/- ES cells (one-tailed Student's t-test) is indicated: *P<0.05, **P<0.005, ***P<0.0005. (D,E) Confirmation that six candidate REST target genes are upregulated in the absence of REST. Gene expression detected by RT-PCR in Rest+/- and Rest -/- ES cells is shown relative to that in the wild type (D). Gene expression in siREST- and siCtrl-transfected ES cells is presented relative to that in mock-transfected controls (E). The expression levels were normalised to house keeping genes (Ywhaz, Hmbs). Bars show the average of three to six experiments and error bars indicate s.d. Significantly higher expression (one-tailed Student's t-test) in Rest-/- relative to wild type (D) and in siREST relative to siCtrl samples (E) is indicated: *P<0.05, **P<0.005, ***P<0.0005. (F) Reconstitution of Rest-/- ES cells with full-length Flag-tagged REST protein. Expression analysis of REST-dependent genes after ectopic expression of Flag-tagged full-length REST in wild-type or Rest-/- ES cells as compared with cells transfected with the control construct. The expression levels relative to `WT, Vector' were normalised to house keeping controls. Bars show the average of two experiments and error bars indicate s.d. Significantly reduced expression (one-tailed Student's t-test) in Rest-/- ES cells transfected with Flag-REST relative to vector-transfected cells is indicated: *P<0.05, **P<0.005. A western blot verifying overexpression of REST is shown in Fig. S5 in the supplementary material.

 

Figure 3
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  		Fig. 3. REST-deficient ES cells retain multi-lineage potential and reprogramming ability. (A) The kinetics of induction of differentiation-associated transcripts (Sox1, Bry, Gata4) and loss of Oct4 expression in wild-type or Rest-/- undifferentiated ES cells following LIF withdrawal and embryoid body formation. Retinoic acid was added at day 4 of differentiation where indicated (RA). For comparison, gene expression levels in control tissues (Ctrl, grey), E15 heads (Sox1), ES cell-derived mesoderm (Bry) and E15 liver (Gata4) are provided. Transcript levels were normalised to house keeping controls (Hmbs, Ywhaz, Gapdh). The average and s.d. from two to three experiments are shown. (B) Reprogramming activity of wild-type and Rest-/- ES cells assessed by heterokaryon formation after fusion of mouse ES cells with human B cells (hB). The ability of mouse ES cell lines to reprogram human B cells is indicated by the kinetics of induction of human (h) OCT4, NANOG, CRIPTO and DNMT3B transcripts detected 0, 1, 2 and 3 days after fusion. Bars show the average of two experiments and error bars indicate s.d.

 

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