Final Report Summary - SMYD3 AND MYOGENESIS (Potential role of the histonemethylase SMYD3 in myogenesis)
Final report PIRGES-224833
Role of the histonemethylase SMYD3 in myogenesis.
Giuseppina Caretti
SMYD3 is a histonemethylase that plays an important role in proliferation, cell adhesion and invasiveness in certain tumor types (Hamamoto et al., 2004, Van Aller et al., 2012).? It is expressed in the embryo but its expression levels are very low in adult tissues, with exception of skeletal muscle and few other tissues. Therefore, we proposed to analyse SMYD3 role in myoblasts, to elucidate the transcriptional pathways impacted by SMYD3. In C2C12 myoblasts and skeletal muscle stem cells, SMYD3 depletion led to a marked acceleration in the differentiation process and to a hypertrophic phenotype of the differentiated myotubes. Because of the hypertrophic phenotype of SMYD3 depleted myotubes we looked for a pathway that may be relevant in muscle size determination. Myostatin is a pivotal molecule in muscle mass determination and reduced levels of myostatin in myoblast cells cause a hypertrophic phenotype. Furthermore, inhibition of the myostatin pathway enhances terminal differentiation of satellite cells (McCroskery et al., 2003), further suggesting that defining the functional mechanism that accounts for SMYD3 involvement in regulating skeletal muscle differentiation and homeostasis may be very relevant for myoblast cells. We focused on the role of SMYD3 in regulating the myostatin pathway.? Employing the chromatin immunoprecipitation technique, we have shown that SMYD3 is recruited to the proximal promoter and the first intron of the myostatin gene. SMYD3 depletion by Sh-RNA interference dramatically reduced the recruitment of RNA Pol II phosphorylated at Serine 2, at the myostatin regulatory regions. Concurrently, we also observed a reduction in association regulators of transcription elongation, at myostatin chromatin regulatory regions. We revealed that the pattern of histone modifications, e.g. H3S10 phosphorylation and H4K16 acetylation, is not affected by SMYD3 depletion at myostatin regulatory regions. We therefore predict a model according to which SMYD3 interacts with elongation regulators and histones, possibly through binding to the SMYD3 DNA consensus site and to H3, phosphorylated at Serine 10 and bound by 14-3-3?/e (Li et al., 2011), at the myostatin gene. The link we revealed between SMYD3 and elongation factors would be indispensable for RNA Pol II phopshorylation on Serine 2, which triggers the elongation steps and confers to SMYD3 a special role in the elongation step of myostatin transcription process. SMYD3 may therefore fine-tune myostatin transcription at the elongation step both in embryonic muscle progenitors and satellite cells and it may contribute to expression of myostatin. Myostatin levels are extremely critical to inhibit skeletal muscle differentiation in C2C12 cells (McCroskery et al., 2003) and SMYD3 orchestrate a crucial checkpoint, after transcription initiation has occurred. Due to the prominent role of myostatin in muscle mass maintenance and in muscle related diseases, the link we disclosed between SMYD3 and myostatin may open novel avenues for pharmacological intervention to modulate myostatin abundance for instance during cancer-related cachexia.? SMYD3 mediated facilitation of transcription elongation may take place during transcription of other SMYD3-regulated targets, such as genes relevant for cancer cells proliferation. Therefore, SMYD3 abnormal over-expression in certain tumors may contribute to uncontrolled expression of genes that promote cancer progression and invasion through excessive elongation regulators recruitment to their chromatin regions. We proposed that SMYD3 target genes might need to be marked by H3S10 phosphorylation and H4K16 acetylation. Both chromatin regulatory regions within myostatin promoter and first intron were characterised by H3S10 phosphorylation and H4K16 acetylation. A molecular mechanism, analogous to the one we revealed for myostatin, may play a pivotal part in other sets of genes regulated by release of paused RNA polymerase II, such as other developmental genes and early response genes (Zippo et al., 2009).
Hamamoto, R., Furukawa, Y., Morita, M., Iimura, Y., Silva, F.P. Li, M., Yagyu, R., and Nakamura, Y. (2004). Nat Cell Biol 6, 731-740.?
Van Aller, G. S., N. Reynoird, O. Barbash, M. Huddleston, S. Liu, A. F. Zmoos, P. McDevitt, R. Sinnamon, B. Le, G. Mas, R. Annan, J. Sage, B. A. Garcia, P. J. Tummino, O. Gozani and R. G. Kruger (2012). Epigenetics 7(4): 340-343.
McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. (2003). J Cell Biol. Mc162(6):1135-47.
Li, Y., Sun, L., Zhang, Y., Wang, D., Wang, F., Liang, J., Gui, B., and Shang, Y. (2011). The histone modifications governing TFF1 transcription mediated by estrogen receptor. J Biol Chem 286, 13925-13936.
Zippo, A., Serafini, R., Rocchigiani, M., Pennacchini, S., Krepelova, A., and Oliviero, S. (2009). Cell 138, 1122-1136.?Levine, M. (2011). Cell 145, 502-511.
Role of the histonemethylase SMYD3 in myogenesis.
Giuseppina Caretti
SMYD3 is a histonemethylase that plays an important role in proliferation, cell adhesion and invasiveness in certain tumor types (Hamamoto et al., 2004, Van Aller et al., 2012).? It is expressed in the embryo but its expression levels are very low in adult tissues, with exception of skeletal muscle and few other tissues. Therefore, we proposed to analyse SMYD3 role in myoblasts, to elucidate the transcriptional pathways impacted by SMYD3. In C2C12 myoblasts and skeletal muscle stem cells, SMYD3 depletion led to a marked acceleration in the differentiation process and to a hypertrophic phenotype of the differentiated myotubes. Because of the hypertrophic phenotype of SMYD3 depleted myotubes we looked for a pathway that may be relevant in muscle size determination. Myostatin is a pivotal molecule in muscle mass determination and reduced levels of myostatin in myoblast cells cause a hypertrophic phenotype. Furthermore, inhibition of the myostatin pathway enhances terminal differentiation of satellite cells (McCroskery et al., 2003), further suggesting that defining the functional mechanism that accounts for SMYD3 involvement in regulating skeletal muscle differentiation and homeostasis may be very relevant for myoblast cells. We focused on the role of SMYD3 in regulating the myostatin pathway.? Employing the chromatin immunoprecipitation technique, we have shown that SMYD3 is recruited to the proximal promoter and the first intron of the myostatin gene. SMYD3 depletion by Sh-RNA interference dramatically reduced the recruitment of RNA Pol II phosphorylated at Serine 2, at the myostatin regulatory regions. Concurrently, we also observed a reduction in association regulators of transcription elongation, at myostatin chromatin regulatory regions. We revealed that the pattern of histone modifications, e.g. H3S10 phosphorylation and H4K16 acetylation, is not affected by SMYD3 depletion at myostatin regulatory regions. We therefore predict a model according to which SMYD3 interacts with elongation regulators and histones, possibly through binding to the SMYD3 DNA consensus site and to H3, phosphorylated at Serine 10 and bound by 14-3-3?/e (Li et al., 2011), at the myostatin gene. The link we revealed between SMYD3 and elongation factors would be indispensable for RNA Pol II phopshorylation on Serine 2, which triggers the elongation steps and confers to SMYD3 a special role in the elongation step of myostatin transcription process. SMYD3 may therefore fine-tune myostatin transcription at the elongation step both in embryonic muscle progenitors and satellite cells and it may contribute to expression of myostatin. Myostatin levels are extremely critical to inhibit skeletal muscle differentiation in C2C12 cells (McCroskery et al., 2003) and SMYD3 orchestrate a crucial checkpoint, after transcription initiation has occurred. Due to the prominent role of myostatin in muscle mass maintenance and in muscle related diseases, the link we disclosed between SMYD3 and myostatin may open novel avenues for pharmacological intervention to modulate myostatin abundance for instance during cancer-related cachexia.? SMYD3 mediated facilitation of transcription elongation may take place during transcription of other SMYD3-regulated targets, such as genes relevant for cancer cells proliferation. Therefore, SMYD3 abnormal over-expression in certain tumors may contribute to uncontrolled expression of genes that promote cancer progression and invasion through excessive elongation regulators recruitment to their chromatin regions. We proposed that SMYD3 target genes might need to be marked by H3S10 phosphorylation and H4K16 acetylation. Both chromatin regulatory regions within myostatin promoter and first intron were characterised by H3S10 phosphorylation and H4K16 acetylation. A molecular mechanism, analogous to the one we revealed for myostatin, may play a pivotal part in other sets of genes regulated by release of paused RNA polymerase II, such as other developmental genes and early response genes (Zippo et al., 2009).
Hamamoto, R., Furukawa, Y., Morita, M., Iimura, Y., Silva, F.P. Li, M., Yagyu, R., and Nakamura, Y. (2004). Nat Cell Biol 6, 731-740.?
Van Aller, G. S., N. Reynoird, O. Barbash, M. Huddleston, S. Liu, A. F. Zmoos, P. McDevitt, R. Sinnamon, B. Le, G. Mas, R. Annan, J. Sage, B. A. Garcia, P. J. Tummino, O. Gozani and R. G. Kruger (2012). Epigenetics 7(4): 340-343.
McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. (2003). J Cell Biol. Mc162(6):1135-47.
Li, Y., Sun, L., Zhang, Y., Wang, D., Wang, F., Liang, J., Gui, B., and Shang, Y. (2011). The histone modifications governing TFF1 transcription mediated by estrogen receptor. J Biol Chem 286, 13925-13936.
Zippo, A., Serafini, R., Rocchigiani, M., Pennacchini, S., Krepelova, A., and Oliviero, S. (2009). Cell 138, 1122-1136.?Levine, M. (2011). Cell 145, 502-511.