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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02727

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Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus

¹ Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
² MIT Center for Cancer Research, Building E17-51, 40 Ames Street, Cambridge, MA 02139, USA
³ Mathematical & Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, 12 South Drive, Bethesda, MD 20892, USA
⁴ Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Building 41, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA

View larger version (24K): [in a new window]   Fig. 1. Transcription analysis of Mash1 in ES and neural cells. (A) RT-PCR of Mash1 and control genes from OS25 ES and neural cell RNA. After 30 cycles of PCR Mash1 is detected in the neural but not the ES sample. Markers for ES to neural differentiation, Oct4, Sox2 and Sox1, show expected expression patterns. ß-actin was used as a template input control. -RT, reverse-transcriptase-free negative control. (B) Semi-quantitative RT-PCR shows that transcription of Mash1 occurs at a negligible level in ES cells but is dramatically upregulated (at least 125-fold) in neural cells. Dilutions were: 1, 1:5, 1:25, 1:125, left to right. S26, small ribosomal protein 26 template-input control. E15 head, positive control for Mash1 expression. dH2O, negative control.

View larger version (28K): [in a new window]   Fig. 2. Spatial relationship of the Mash1 locus to centromeric heterochromatin and the nuclear periphery. (A) By 3D FISH we observed that the Mash1 locus (green) is rarely associated with centromeric heterochromatin (-satellite probe is red) in either ES or neural cells. Numbers in brackets are FISH signal scores. (B) Frequency (mean ± s.d.) of FISH signals scored as peripheral (80% of nuclear radius) in undifferentiated OS25 ES cells and differentiated OS25 neural cells. Numbers in bars are total fish signals scored. (C) Representative 2D FISH result showing Mash1 locus (green) in OS25 ES and neural cells. Nuclei were counterstained with DAPI (blue). (D) Representative 3D FISH result showing Mash1 locus (green) in individual z slices of DAPI-stained ES and neural nuclei. Frequency of perinuclear Mash1 signals scored in 3D analysis is indicated. Numbers in brackets represent the total FISH signals scored. Bar, 5 µm.

View larger version (60K): [in a new window]   Fig. 3. Dissociation of Mash1 from nuclear periphery is specific to neural fate. (A) RT-PCR shows Mash1 is upregulated upon neural differentiation of MR-7 ES cells by PA6 stromal cell layer method. Oct4 and Sox1 controls are also shown. (B) Frequency (mean ± s.d.) of FISH signals scored as peripheral in MR-7 ES cells, PA6-differentiated MR-7 neural cells, ex vivo Sox1-positive neural cells, mesoderm-differentiated OS25 cells, ex vivo wild-type keratinocyte precursors and ex vivo wild-type T cells. Also shown are undifferentiated OS25 ES and neural cells from Fig. 2, for reference. Numbers in bars represent total FISH signals scored. (C) Representative 2D FISH results for each of these cell types. Mash1 locus is green. Nuclei are counterstained with DAPI (blue). Bar, 5 µm.

View larger version (25K): [in a new window]   Fig. 4. The genomic environment of Mash1. (A) A 2 Mb region surrounding the Mash1 gene on chromosome 10, with neighbouring genes annotated. (B) RT-PCR analysis of genes surrounding Mash1 in OS25 ES and NE cells. (C) Replication timing of genes surrounding Mash1 in ES and NE cells. Bar charts show the abundance of nascent DNA in each S-phase fraction (as a percentage of the total). (D) Positions of the 2 Mb region probes relative to nuclear periphery (probe positions shown annotated in A). Also shown are the positions of silent, late replicating genes, Neurod (NeuroD) and Sprr2 (SPRR2) and active, early replicating genes, Sox2 (Sox2) and Utf1 (UTF) as controls. Numbers beneath probes represent FISH signals scored in high-throughput analysis (blue, ES cells; pink, neural cells).

View larger version (33K): [in a new window]   Fig. 5. Chromatin organisation at Mash1. (A) Histone modifications of Mash1 and neighbouring genes in ES and neural cells. Bars show relative levels of real-time PCR products amplified from ES (pink) and NE (blue) genomic DNA following ChIP with antibodies to AcH3K9, pan-AcH4, Me3H3K9, Me3H3K20 and Me3H3K27. Results are normalized to histone H3 ChIP PCR levels. (B) Mash1 positioning in ES cells lacking chromatin-silencing factors. Bar chart shows number of Mash1 FISH signals scored as peripheral in Eed-/-, G9a-/-, SuV39h1-/-, Dnmt1-/- and Dnmt3ab-/- ES cells. Also shown are OS25 ES and neural cells from Fig. 2, for reference. Numbers in bars represent total FISH signals scored. (C) Representative 2D FISH results for each of these knockout ES cells. Mash1 locus, green. Nuclei counterstained with DAPI (blue). Bar, 5 µm.