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Linkages between changes in the 3D organization of the genome and transcription during myotube differentiation in vitro

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ABSTRACT

Background: The spatial organization of eukaryotic genomes facilitates and reflects the underlying nuclear processes that are occurring in the cell. As such, the spatial organization of a genome represents a window on the genome biology that enables analysis of the nuclear regulatory processes that contribute to mammalian development.

Methods: In this study, Hi-C and RNA-seq were used to capture the genome organization and transcriptome in mouse muscle progenitor cells (C2C12 myoblasts) before and after differentiation to myotubes, in the presence or absence of the cytidine analogue AraC.

Results: We observed significant local and global developmental changes despite high levels of correlation between the myotubes and myoblast genomes. Notably, the genes that exhibited the greatest variation in transcript levels between the different developmental stages were predominately within the euchromatic compartment. There was significant re-structuring and changes in the expression of replication-dependent histone variants within the HIST1 locus. Finally, treating terminally differentiated myotubes with AraC resulted in additional changes to the transcriptome and 3D genome organization of sets of genes that were all involved in pyroptosis.

Conclusions: Collectively, our results provide evidence for muscle cell-specific responses to developmental and environmental stimuli mediated through a chromatin structure mechanism.

Electronic supplementary material: The online version of this article (doi:10.1186/s13395-017-0122-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Myoblasts were successfully differentiated into myotubes. a Muscle progenitor cells (myoblasts) were differentiated into myotubes in presence of differentiation media. This cell population was predominately comprised of myotubes although there were some myoblasts present. AraC treatment of the myotubes was undertaken to remove undifferentiated myoblasts. b Double immunostained images of cells harvested at different stages of myogenic progression. The AraC treatment resulted in the apparent removal of the myoblasts from the terminally differentiated myotubes as evidenced by the presence of cell-free patches in the AraC-treated cultures. Cells were immunostained for myosin (green), nuclei (blue) and mitochondria (red). cMyog and other skeletal muscle-specific genes (Acta1, Myh1, Myh2, Myh4, Tnnt1) are markedly upregulated in the myotubes with and without AraC treatment when compared to myoblasts. This indicates successful differentiation at the level of muscle-specific molecular markers. Transcript levels are shown as the mean of the log FPKM for the biological repeats ± SE
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Fig1: Myoblasts were successfully differentiated into myotubes. a Muscle progenitor cells (myoblasts) were differentiated into myotubes in presence of differentiation media. This cell population was predominately comprised of myotubes although there were some myoblasts present. AraC treatment of the myotubes was undertaken to remove undifferentiated myoblasts. b Double immunostained images of cells harvested at different stages of myogenic progression. The AraC treatment resulted in the apparent removal of the myoblasts from the terminally differentiated myotubes as evidenced by the presence of cell-free patches in the AraC-treated cultures. Cells were immunostained for myosin (green), nuclei (blue) and mitochondria (red). cMyog and other skeletal muscle-specific genes (Acta1, Myh1, Myh2, Myh4, Tnnt1) are markedly upregulated in the myotubes with and without AraC treatment when compared to myoblasts. This indicates successful differentiation at the level of muscle-specific molecular markers. Transcript levels are shown as the mean of the log FPKM for the biological repeats ± SE

Mentions: C2C12 cells were differentiated into myotubes (Fig. 1a) as evidenced by (1) the presence of sarcomeric myosin and (2) mitochondrial networks on days 3 and 7 following the medium switch (Fig. 1b). The percentage of differentiation was calculated by counting the number of DAPI-stained nuclei located within the myosin positive cell body, from nine randomly pre-selected fields of view (Additional file 3: Figure S1). The total number of DAPI staining nuclei counted within each field of view decreased from that observed at confluence (i.e. 2704). Following the addition of AraC, the total nuclei per field (i.e. 1303; Additional file 3: Figure S1A) decreased further with the loss of residual myoblasts. In contrast, the percentage of nuclei located within the myosin positive cell body (i.e. the percentage of differentiation) showed a statistically significant increase (p < 0.05, one-way ANOVA) to 76.2% over the 7-day culture period (Additional file 3: Figure S1). Notably, there was no significant difference in the percentage of differentiation between day 5 (81.7%), where myotubes reached their maximum percentage of differentiation, and day 7 (76.2%) cultures. Thus, we concluded that there was little morphological difference between myotubes on day 5 or day 7 differentiation.Fig. 1


Linkages between changes in the 3D organization of the genome and transcription during myotube differentiation in vitro
Myoblasts were successfully differentiated into myotubes. a Muscle progenitor cells (myoblasts) were differentiated into myotubes in presence of differentiation media. This cell population was predominately comprised of myotubes although there were some myoblasts present. AraC treatment of the myotubes was undertaken to remove undifferentiated myoblasts. b Double immunostained images of cells harvested at different stages of myogenic progression. The AraC treatment resulted in the apparent removal of the myoblasts from the terminally differentiated myotubes as evidenced by the presence of cell-free patches in the AraC-treated cultures. Cells were immunostained for myosin (green), nuclei (blue) and mitochondria (red). cMyog and other skeletal muscle-specific genes (Acta1, Myh1, Myh2, Myh4, Tnnt1) are markedly upregulated in the myotubes with and without AraC treatment when compared to myoblasts. This indicates successful differentiation at the level of muscle-specific molecular markers. Transcript levels are shown as the mean of the log FPKM for the biological repeats ± SE
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5382473&req=5

Fig1: Myoblasts were successfully differentiated into myotubes. a Muscle progenitor cells (myoblasts) were differentiated into myotubes in presence of differentiation media. This cell population was predominately comprised of myotubes although there were some myoblasts present. AraC treatment of the myotubes was undertaken to remove undifferentiated myoblasts. b Double immunostained images of cells harvested at different stages of myogenic progression. The AraC treatment resulted in the apparent removal of the myoblasts from the terminally differentiated myotubes as evidenced by the presence of cell-free patches in the AraC-treated cultures. Cells were immunostained for myosin (green), nuclei (blue) and mitochondria (red). cMyog and other skeletal muscle-specific genes (Acta1, Myh1, Myh2, Myh4, Tnnt1) are markedly upregulated in the myotubes with and without AraC treatment when compared to myoblasts. This indicates successful differentiation at the level of muscle-specific molecular markers. Transcript levels are shown as the mean of the log FPKM for the biological repeats ± SE
Mentions: C2C12 cells were differentiated into myotubes (Fig. 1a) as evidenced by (1) the presence of sarcomeric myosin and (2) mitochondrial networks on days 3 and 7 following the medium switch (Fig. 1b). The percentage of differentiation was calculated by counting the number of DAPI-stained nuclei located within the myosin positive cell body, from nine randomly pre-selected fields of view (Additional file 3: Figure S1). The total number of DAPI staining nuclei counted within each field of view decreased from that observed at confluence (i.e. 2704). Following the addition of AraC, the total nuclei per field (i.e. 1303; Additional file 3: Figure S1A) decreased further with the loss of residual myoblasts. In contrast, the percentage of nuclei located within the myosin positive cell body (i.e. the percentage of differentiation) showed a statistically significant increase (p < 0.05, one-way ANOVA) to 76.2% over the 7-day culture period (Additional file 3: Figure S1). Notably, there was no significant difference in the percentage of differentiation between day 5 (81.7%), where myotubes reached their maximum percentage of differentiation, and day 7 (76.2%) cultures. Thus, we concluded that there was little morphological difference between myotubes on day 5 or day 7 differentiation.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The spatial organization of eukaryotic genomes facilitates and reflects the underlying nuclear processes that are occurring in the cell. As such, the spatial organization of a genome represents a window on the genome biology that enables analysis of the nuclear regulatory processes that contribute to mammalian development.

Methods: In this study, Hi-C and RNA-seq were used to capture the genome organization and transcriptome in mouse muscle progenitor cells (C2C12 myoblasts) before and after differentiation to myotubes, in the presence or absence of the cytidine analogue AraC.

Results: We observed significant local and global developmental changes despite high levels of correlation between the myotubes and myoblast genomes. Notably, the genes that exhibited the greatest variation in transcript levels between the different developmental stages were predominately within the euchromatic compartment. There was significant re-structuring and changes in the expression of replication-dependent histone variants within the HIST1 locus. Finally, treating terminally differentiated myotubes with AraC resulted in additional changes to the transcriptome and 3D genome organization of sets of genes that were all involved in pyroptosis.

Conclusions: Collectively, our results provide evidence for muscle cell-specific responses to developmental and environmental stimuli mediated through a chromatin structure mechanism.

Electronic supplementary material: The online version of this article (doi:10.1186/s13395-017-0122-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus