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First published online 25 April 2006
doi: 10.1242/jcs.02945

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Acquisition and extinction of gene expression programs are separable events in heterokaryon reprogramming

Rémi Terranova^*, Carlos Filipe Pereira, Camille Du Roure^*, Matthias Merkenschlager and Amanda G. Fisher

Lymphocyte Development Group, MRC, Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK

View larger version (44K): [in a new window]   Fig. 1. Reprogramming human B-lymphocytes in mouse C2C12 heterokaryons. (A) Protocol used to generate interspecies (hBxC2) heterokaryons. Mouse C2C12 myoblasts were differentiated into myotubes and fused with human B lymphocytes (hB). Mouse and human nuclei were distinguished by FISH with probes specific for mouse -satellite DNA (red) or human -satellite DNA (green), or by DAPI staining (blue). (B) Confocal image of hB nuclei before fusion. (C) Confocal images of a myotube containing a human (arrow) and a mouse nucleus. (D) Confocal section of a reprogrammed heterokaryon identified by hNCAM expression (5.1H11 antibody, red). One mouse (DAPI intense foci) and one human (arrows) nucleus are seen. Bars, 10 µm. (E) Expression-kinetics of human NCAM (detected by 5.1H11 antibody) by lymphocyte-derived nuclei (mean ± s.d., n=3, 50 nuclei per experiment).

View larger version (45K): [in a new window]   Fig. 2. Nuclear reorganisation is an early event in lymphocyte reprogramming. (A) Single optical section showing lamin A/C immunofluorescence-labelling of human B lymphocyte nuclei (hB) before day 0 (d0) and three days (d3) after fusion with C2C12 myotubes. (B) Volume of hB nuclei before (d0), and 2 and 4 days after heterokaryon formation (d2 and d4, respectively) compared with C2C12 myotubes 2 and 4 days after serum withdrawal and differentiation (d2 and d4, respectively). Nuclear volume was estimated as described in Materials and Methods (mean ± s.d., n=3 with 50 nuclei per experiment). (C) CREST antisera (green) reveal the centromere distribution in hB nuclei (d0) and two days after heterokaryon formation (d2), where individual chromocentres are indicated (arrowed). Confocal images are maximal projection of multiple optical z-sections. (D) Distribution of constitutive heterochromatin in hB nuclei before (d0, open bars) and 2 days after fusion in hNCAM expressing (d2, black bars) and hNCAM-negative (not yet reprogrammed) heterokaryons (grey bars) was compared by assessing the number of discrete CREST signals and number of chromocentres per nucleus (n=100). (E) Myogenin protein (green) in a human nucleus (arrowheads) 1 day after fusion. Images show single optical sections. Bars, 10 µm.

View larger version (37K): [in a new window]   Fig. 3. Ordered expression of human myogenic genes by lymphocyte-derived nuclei. (A) Expression of human muscle-specific (hMYF5, hMYOG, hMRF4) and human lymphocyte-specific (hCD45, hPAX5) genes detected by RT-PCR. Prior to fusion, human B cells expressed hGAPDH, hCD45 and hPAX5, but not muscle-specific genes. Following heterokaryon formation, human MYF5, MYOG and MRF4 expression was initiated and the expression of human lymphocyte-specific genes CD45 and PAX5 declined. Mouse C2C12 samples (C2) were used as negative controls to confirm the specificity of primers to human transcripts and hGAPDH was used to standardise input. (B) Expression kinetics of human NCAM in hBxC2 heterokaryons in the presence (solid line) or absence (broken line) of TSA (20 nM). For each time point, 100 heterokaryons were analysed in two separate experiments and values shown are the mean ± s.d. (C) hMYOG, hMRF4, hMYF5 and hGAPDH gene expression in human B lymphocytes (hB) and in day 4 (d4) and day 7 (d7) hBxC2 heterokaryons cultured in the presence (+) or absence (–) of 20 nM TSA. As positive controls for PCR analysis (hC+) RNA was isolated from differentiating human muscle. Mouse C2C12 cells (C2) were used as negative control. hGAPDH was used to standardise input.

View larger version (56K): [in a new window]   Fig. 4. HDAC inhibition prevents the shutdown of lymphocyte-specific genes during reprogramming. (A) Impact of 20 nM TSA treatment on the expression of hCD45, hPAX5, hCD20 and hCD37 in interspecies hBxC2 heterokaryons. Green boxes highlight that lymphocyte-associated genes are not extinguished following TSA treatment. (B) Expression of hPAX6, hREX1, hTERT, hNANOG and hOCT4 in human B lymphocytes (hB) and in day 4 (d4) and day 7 (d7) hBxC2 heterokaryons cultured in the presence (+) or absence (–) of 20 nM TSA. As positive controls for PCR analysis (hC+), RNA was isolated from human embryonic stem cells (for hOCT4, hNANOG, hREX1, hTERT) and human neuronal progenitor cells (for hPAX6), and hGAPDH was used to standardise input. Mouse ES cells (C-mES) and mouse C2C12 cells (C-mC2), used as negative controls to confirm the specificity of primers to human transcripts, are shown on the right. (C) RT-PCR analysis of mouse transcripts for mMash1, mNeuroD, mNurr1, mPax2, mEngrailed1, mShh (neural-associated) and mCD45, mRag1, mCD4, mLambda-5, mCD19 (lymphocyte-associated) in mouse myoblasts and myotubes cultured in absence or in presence of 20 nM TSA. Positive controls in this analysis include RNA isolated from mouse brain and foetal liver (mC+); mHPRT was used to standardise input. The sequence of human- or mouse-specific pairs of primers used for PCR amplification is available upon request. (D) Increased levels of histone H3 Lys9 acetylation (H3K9ac) in human nuclei in response to treatment of heterokaryons with 20 nM TSA (+). Confocal microscope settings and laser power were kept constant so that the relative abundance of H3K9 acetylation could be directly compared (and quantified, right hand panel) to untreated control (mean ± s.d., 50 nuclei per experiment). Bar, 10 µm.

View larger version (89K): [in a new window]   Fig. 5. Coexpression of lymphocyte- and muscle-specific genes in individual nuclei treated with HDAC inhibitors TSA and VPA. (A) Human B lymphocytes were analysed for hCD20 (green) and hNCAM (red) transcripts using RNA FISH. Two foci of hCD20 signal are evident in hB cell nuclei that were removed by pre-treatment of samples with RNAse A (+RNase), but not DNAse (+DNAse). Application of 20 nM TSA did not impair hCD20 signal detection (+TSA). (B) Human NCAM transcripts (red) were detected in human myoblast nuclei but not in lymphocytes. (C) RNA FISH showing examples of nuclei that express hCD20 transcripts alone (green) and hNCAM transcripts alone (red) or both in lymphocyte-derived nuclei within day 5 hBxC2 heterokaryons. Confocal images are maximal projection of multiple optical z-sections (Bars, 10 µm). (D) the proportion of nuclei expressing hNCAM (red), hCD20 (green), or both hNCAM and hCD20 (red/green) signals in heterokaryons cultured in the absence (untreated) or presence of 20 nM TSA or 1 mM VPA is expressed as a proportion of human nuclei in which RNA transcripts were detected (see supplementary material Table S1).