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Expression profiling of FSHD-1 and FSHD-2 cells during myogenic differentiation evidences common and distinctive gene dysregulation patterns.

Cheli S, François S, Bodega B, Ferrari F, Tenedini E, Roncaglia E, Ferrari S, Ginelli E, Meneveri R - PLoS ONE (2011)

Bottom Line: Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation.Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy.

ABSTRACT

Background: Determine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis.

Methodology/principal findings: By in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.

Conclusions/significance: FSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

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Real-Time PCR validation of FSHD-1 microarray data.A: Table reports the genes analyzed in qRT-PCR with fold-change and p-value. The data obtained in the FSHD-1 myoblasts gene chip array and the biological processes identified by the DAVID program, are also reported. The analysis was performed on seven FSHD-1 and six CN myoblast cell lines. B) Bar diagrams show relative expression of PAX3, MYOD1, MYOG and MYH2 in control and FSHD-1 myoblasts (day 0) and myotubes (day 8) relative to GAPDH. *** p-value < ,001.
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pone-0020966-g004: Real-Time PCR validation of FSHD-1 microarray data.A: Table reports the genes analyzed in qRT-PCR with fold-change and p-value. The data obtained in the FSHD-1 myoblasts gene chip array and the biological processes identified by the DAVID program, are also reported. The analysis was performed on seven FSHD-1 and six CN myoblast cell lines. B) Bar diagrams show relative expression of PAX3, MYOD1, MYOG and MYH2 in control and FSHD-1 myoblasts (day 0) and myotubes (day 8) relative to GAPDH. *** p-value < ,001.

Mentions: To confirm the FSHD-1 microarray data we focused our attention on some genes contained in the most enriched categories evidenced by GO analysis. The chosen representative genes were validated by multiplex Real–Time assay performed with Taqman® probes on seven FSHD-1 in comparison to six healthy controls (for a description of the used cell lines see Table 1 in Materials and Methods). The analyzed genes comprised: E2F7 (negative regulator of cell cycle progression), CDC6 (DNA replication), KIF18A (chromosome segregation) and SUV39H1 (histone methylation), MSH2, DCLRE1B, SOD2 (DNA damage and repair), LAMA4 (extracellular matrix), SDR (membrane raft) and PTPRN (cell growth and differentiation). mir 23-b was also analyzed. As shown in Fig.4A, the results of the Real–Time assay confirmed the data of the array. In fact all genes tested were regulated in the same direction with both methods. The data relative to the FSHD-2 genechip analysis were not validated by qRT-PCR due to the unavailability of other FSHD-2 cell lines in addition to those used for microarray experiments.


Expression profiling of FSHD-1 and FSHD-2 cells during myogenic differentiation evidences common and distinctive gene dysregulation patterns.

Cheli S, François S, Bodega B, Ferrari F, Tenedini E, Roncaglia E, Ferrari S, Ginelli E, Meneveri R - PLoS ONE (2011)

Real-Time PCR validation of FSHD-1 microarray data.A: Table reports the genes analyzed in qRT-PCR with fold-change and p-value. The data obtained in the FSHD-1 myoblasts gene chip array and the biological processes identified by the DAVID program, are also reported. The analysis was performed on seven FSHD-1 and six CN myoblast cell lines. B) Bar diagrams show relative expression of PAX3, MYOD1, MYOG and MYH2 in control and FSHD-1 myoblasts (day 0) and myotubes (day 8) relative to GAPDH. *** p-value < ,001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113851&req=5

pone-0020966-g004: Real-Time PCR validation of FSHD-1 microarray data.A: Table reports the genes analyzed in qRT-PCR with fold-change and p-value. The data obtained in the FSHD-1 myoblasts gene chip array and the biological processes identified by the DAVID program, are also reported. The analysis was performed on seven FSHD-1 and six CN myoblast cell lines. B) Bar diagrams show relative expression of PAX3, MYOD1, MYOG and MYH2 in control and FSHD-1 myoblasts (day 0) and myotubes (day 8) relative to GAPDH. *** p-value < ,001.
Mentions: To confirm the FSHD-1 microarray data we focused our attention on some genes contained in the most enriched categories evidenced by GO analysis. The chosen representative genes were validated by multiplex Real–Time assay performed with Taqman® probes on seven FSHD-1 in comparison to six healthy controls (for a description of the used cell lines see Table 1 in Materials and Methods). The analyzed genes comprised: E2F7 (negative regulator of cell cycle progression), CDC6 (DNA replication), KIF18A (chromosome segregation) and SUV39H1 (histone methylation), MSH2, DCLRE1B, SOD2 (DNA damage and repair), LAMA4 (extracellular matrix), SDR (membrane raft) and PTPRN (cell growth and differentiation). mir 23-b was also analyzed. As shown in Fig.4A, the results of the Real–Time assay confirmed the data of the array. In fact all genes tested were regulated in the same direction with both methods. The data relative to the FSHD-2 genechip analysis were not validated by qRT-PCR due to the unavailability of other FSHD-2 cell lines in addition to those used for microarray experiments.

Bottom Line: Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation.Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy.

ABSTRACT

Background: Determine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis.

Methodology/principal findings: By in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome.

Conclusions/significance: FSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role.

Show MeSH
Related in: MedlinePlus