Research Article

The master regulator gene PRDM2 controls C2C12 myoblasts proliferation and Differentiation switch and PRDM4 and PRDM10 expression

Di Zazzo Erika, Bartollino Silvia* and Moncharmont Bruno

Published: 09/25/2017 | Volume 1 - Issue 1 | Pages: 075-091

Abstract

The Positive Regulatory Domain (PRDM) protein family gene is involved in a spectrum variety of biological processes, including proliferation, differentiation and apoptosis: its member seem to be transcriptional regulators highly cell type and tissue peculiar, towards histones modifications or recruitment of specific interaction patters to modify the expression of target genes. In this study we analyzed the expression profile of different member of PRDM gene family focusing our attention on the role of PRDM2, PRDM4 and PRDM10 genes in mouse C2C12 cell line, during the differentiation of myoblasts into myotubes and speculate about the role of the protein Retinoblastoma protein-interacting zinc finger protein 1-RIZ1, coded by PRDM2 gene, as a regulator of the proliferation/differentiation switch.

Results showed a reduction of PRDM2, PRDM4 and PRDM10 expression level during the commitment of the differentiation of myoblasts into myotubes. The RIZ1 silencing stimulated myoblasts differentiation, similar to the effect of serum deprivation on these cells, associated with an increase of Myogenin expression level, which is considered to be involved in the differentiation of myoblasts into multinucleated myotubes. As demonstrated by chromatin immunoprecipitation experiments, RIZ1 is associated with Myogenin promoter in proliferation condition and after 24h from differentiation induction, negatively controlling therefore Myogenin expression. Moreover RIZ1 silencing induced a reduction in PRDM4 and PRDM10 expression levels leaving us to speculate that the PRDM genes have a redundant role and they are hierarchically organized.

Read Full Article HTMLDOI: 10.29328/journal.hjbm.1001007Cite this Article

References

  1. Singh K, Dilworth FJ. Differential modulation ofcell cycle progression distinguishes members of the myogenic regulatoryfactor family of transcription factors. FEBS J. 2013; 280: 3991-4003. Ref.: https://goo.gl/mw5Y9p
  2. Davis RL, Weintraub H. Acquisition of myogenicspecificity by replacement of three amino acid residues from MyoD intoE12. Science. 1992; 256: 1027-1030. Ref.: https://goo.gl/kD7f3S
  3. Tapscott SJ, Davis RL, Thayer MJ, Cheng PF,Weintraub H, et al. MyoD1: a nuclear phosphoprotein requiring a Mychomology region to convert fibroblasts to myoblasts. Science. 1988; 242:405-11. Ref.: https://goo.gl/6eXUeW
  4. Atchley WR, Fitch WM, Bronner-Fraser M. Molecularevolution of the MyoD family of transcription factors. Proc Natl AcadSci.1994; 91: 11522-11526.  Ref.: https://goo.gl/d2oZHf
  5. Berkes, CA, Tapscott SJ. MyoD and thetranscriptional control of myogenesis. Semin Cell Dev Biol. 2005; 16: 585-595.Ref.: https://goo.gl/cSHJLx
  6. Braun T, Buschhausen-Denker G, Bober E, TannichE, Arnold HH. A novel human muscle factor related to but distinct fromMyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO J. 1989; 8:701-709. Ref.: https://goo.gl/Vwy7uo
  7. Kablar B, Rudnicki MA. Development in the absenceof skeletal muscle results in the sequential ablation of motor neuronsfrom the spinal cord to the brain. Dev Biol. 1999; 208: 93-109. Ref.: https://goo.gl/z5nXSY
  8. Rudnicki, MA, Schnegelsberg PN, Stead RH, BraunT, Arnold HH., et al. MyoD or Myf-5 is required for the formation ofskeletal muscle. Cell 1993; 75: 1351-1359. Ref.: https://goo.gl/cWenqL
  9. Tajbakhsh S, Rocancourt D, Buckingham M. Muscleprogenitor cells failing to respond to positional cues adopt non-myogenicfates in myf-5 null mice. Nature. 1996; 384: 266-270. Ref.: https://goo.gl/FpK37B
  10. Reijntjes S, Francis-West P, Mankoo BS. Retinoicacid is both necessary for and inhibits myogenic commitment anddifferentiation in the chick limb. Int J Dev Biol. 2010; 54: 125-134. Ref.: https://goo.gl/pD5Xh2
  11. Ustanina S, Carvajal J, Rigby P, Braun T. Themyogenic factor Myf5 supports efficient skeletal muscle regeneration byenabling transient myoblast amplification. Stem Cells. 2007; 25: 2006-2016.Ref.: https://goo.gl/qJ7YU6
  12. Gayraud-Morel B, Chrétien F, Flamant P, Gomès D,Zammit PS, Tajbakhsh S. A role for the myogenic determination gene Myf5 inadult regenerative myogenesis. Dev Biol. 2007; 312: 13-28. Ref.: https://goo.gl/DR6jKq  
  13. Montarras D, Lindon C, Pinset C, Domeyne P.Cultured myf5 null and myoD null muscle precursor cells display distinctgrowth defects. Biol cell. 2000; 92: 565-572. Ref.: https://goo.gl/rQLzi9
  14. Panda AC, Abdelmohsen K, Martindale JL, DiGermanio C, Yang X, et al. Novel RNA-binding activity of MYF5 enhancesCcnd1/Cyclin D1 mRNA translation during myogenesis. Nucleic Acids Res.2016; 44: 2393-2408. Ref.: https://goo.gl/oKMM6P
  15. Wright WE, Sassoon DA, Lin VK. Myogenin, a factorregulating myogenesis, has a domain homologous to MyoD. Cell. 1989; 56:607-617. Ref.: https://goo.gl/DmD2qg
  16. Hashimoto N, Ogashiwa M, Iwashita S. Role oftyrosine kinase in the regulation of myogenin expression. Eur J Biochem 1995;227: 379-387. Ref: https://goo.gl/Lz6TQF
  17. Rhodes SJ, Konieczny SF. Identification of MRF4:a new member of the muscle regulatory factor gene family. Genes Dev. 1989;3: 2050-2061. Ref: https://goo.gl/uMgCbh
  18. Arnold HH, Braun T. Targeted inactivation ofmyogenic factor genes reveals their role during mouse myogenesis: areview. Int J Dev Biol. 1996; 40: 345-353. Ref: https://goo.gl/WJ5MPb
  19. Moncaut N, Rigby PWJ, Carvajal, JJ. Dial M (RF)for myogenesis. FEBS J. 2013; 280: 3980-3990. Ref: https://goo.gl/YSZk62
  20. Chandra S, Terragni J, Zhang G, Pradhan S,Haushka S, et al. Tissue-specific epigenetics in gene neighborhoods:myogenic transcription factor genes. Hum Mol Genet. 2015; 24: 4660-4673. Ref: https://goo.gl/4SPWMT
  21. Molkentin JD, Black BL, Martin JF, Olson EN.Cooperative activation of muscle gene expression by MEF2 and myogenic bHLHproteins. Cell. 1995; 83: 1125-1136. Ref: https://goo.gl/YLEyhp
  22. Pon JR, Marra MA. MEF2 transcription factors:developmental regulators and emerging cancer genes. Oncotarget. 2016; 7:22970-22312. Ref: https://goo.gl/kbzRUj
  23. Cao Y, Yao Z, Sarkar D, Lawrence M, Sanchez GJ, etal. Genome-wide MyoD binding in skeletal muscle cells: a potential forbroad cellular reprogramming. Dev Cell 2010; 18: 662-674. Ref: https://goo.gl/M39LeS
  24. Soleimani VD, Yin H, Jahani-Asl A, Ming H, KockxCEM, et al. Snail regulates MyoD binding-site occupancy to direct enhancerswitching and differentiation-specific transcription in myogenesis. MolCell. 2012; 47: 457-468. Ref: https://goo.gl/HTLeyZ
  25. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, etal. Interactions between heterologous helix-loophelix proteins generatecomplexes that bind specifically to a common DNA sequence. Cell 1989; 58:537-544. Ref: https://goo.gl/vcQBSt
  26. Blackwell TK, Weintraub H. Differences andsimilarities in DNA-binding preferences of MyoD and E2A protein complexesrevealed by binding site selection. Science. 1990; 250: 1104-1110. Ref: https://goo.gl/MEyBj2
  27. Blais A, Tsikitis M, Acosta-Alvear D, Sharan R,Kluger Y, et al. An initial blueprint for myogenic differentiation. GenesDev. 2005; 19: 553-569. Ref: https://goo.gl/FPp6dQ
  28. Aziz A, Liu QC, Dilworth FJ. Regulating a masterregulator: establishing tissuespecific gene expression in skeletal muscle.Epigenetics 2010; 5: 691-695. Ref: https://goo.gl/j6xEnj
  29. De Falco G, Comes F, Simone C. pRb: master ofdifferentiation. Coupling irreversible cell cycle withdrawal withinduction of muscle-specific transcription. Oncogene. 2006; 25: 5244-5249.Ref: https://goo.gl/ZPQ9W8
  30. Giacinti C, Bagella L, Puri PL, Giordano A,Simone C. MyoD recruits the cdk9/cyclin T2 complex on myogenic-genesregulatory regions. J Cell Physiol. 2006; 206: 807-813. Ref: https://goo.gl/mCAE7b
  31. Martelli F, Cenciarelli C, Santarelli G, PolikarB, Felsani A, et al. MyoD induces retinoblastoma gene expression duringmyogenic differentiation. Oncogene. 1994; 9: 3579-3590. Ref: https://goo.gl/LVfg3U
  32. Halevy O, Novitch BG, Spicer DB, Skapek SX, RheeJ, et al. Correlation of terminal cell cycle arrest of skeletal musclewith induction of p21 by MyoD. Science. 1995; 267: 1018-1021. Ref: https://goo.gl/aXwh2j
  33. Basile V, Baruffaldi F, Dolfini D, Belluti S,Benatti P, et al. NF-YA splice variants have different roles on muscledifferentiation. Biochim. Biophys Acta 2016; 1859: 627-638. Ref: https://goo.gl/RXTp41
  34. Besson A, Dowdy SF, Roberts JM. CDK Inhibitors:Cell Cycle Regulators and Beyond. Dev Cell 2008; 14: 159-169. Ref: https://goo.gl/LYEJ4R
  35. Zhang P, Wong C, Liu D, Finegold M, Harper JW, etal. p21(CIP1) and p57(KIP2) control muscle differentiation at the myogeninstep. Genes Dev. 1999; 13: 213-224. Ref: https://goo.gl/V2osBr
  36.  FumasoniI, Meani N, Rambaldi D, Scafetta G, Alcalay M, et al. Family expansion andgene rearrangements contributed to the functional specialization of PRDMgenes in vertebrates. BMC Evol Biol. 2007; 7: 187. Ref: https://goo.gl/w36vh7
  37. Vervoort M, Meulemeester D, Béhague J, Kerner P.Evolution of Prdm Genes in Animals: Insights from Comparative Genomics. MolBiol Evol. 2016; 33: 679-696. Ref: https://goo.gl/hQNY1L
  38. Kinameri E, InoueT, Aruga J, Imayoshi I, Kageyama R, et al. Prdm proto-oncogenetranscription factor family expression and interaction with the Notch-Hespathway in mouse neurogenesis. PLoS One. 2008; 3: e3859. Ref: https://goo.gl/26UZEg
  39. Huang S. Histone methyltransferases, dietnutrients and tumour suppressors. Nat Rev Cancer. 2002; 2: 469-476. Ref: https://goo.gl/b73pB9
  40. Fog CK, Galli GG, Lund AH. PRDM proteins:important players in differentiation and disease. Bioessays. 2012; 34: 50-60.Ref: https://goo.gl/G8bjFM
  41. Gyory I, Wu J, Fejér G, Seto E, Wright KL. PRDI-BF1recruits the histone H3 methyltransferase G9a in transcriptionalsilencing. Nat Immunol. 2004; 5: 299-308. Ref: https://goo.gl/z2qBqf
  42. Duan Z, Person RE, Lee HH, Huang S, Donadieu J,et al. Epigenetic regulation of protein-coding and microRNA genes by theGfi1-interacting tumor suppressor PRDM5. Mol Cell Biol. 2007; 27: 6889-6902.Ref: https://goo.gl/o9rsFs
  43. Davis CA, Haberland M, Arnold MA, Sutherland LB,McDonald OG, et al. PRISM/PRDM6, a transcriptional repressor that promotesthe proliferative gene program in smooth muscle cells. Mol Cell Biol. 2006;26: 2626-2636. Ref: https://goo.gl/F2qSto
  44. Zhang Y, Stehling-Sun S, Lezon-Geyda K, Juneja SC,Coillard L, et al. PR-domain-containing Mds1-Evi1 is critical for long-termhematopoietic stem cell function. Blood 2011; 118: 3853-3861. Ref: https://goo.gl/CAgCMP
  45. Derunes C, Briknarová K, Geng L, Li S, GessnerCR, et al. Characterization of the PR domain of RIZ1 histonemethyltransferase. Biochem Biophys Res Commun. 2005: 333: 925-934. Ref: https://goo.gl/6EFkQu
  46. Eom GH, Kim K, Kim SM, Kee H J, Kim JY, at al..Histone methyltransferase PRDM8 regulates mouse testis steroidogenesis. BiochemBiophys Res Commun 2009; 388: 131-136. Ref: https://goo.gl/F8KSX8
  47. Hayashi K, Yoshida K, Matsui YA. Histone H3methyltransferase controls epigenetic events required for meiotic prophase.Nature. 2005; 438: 374-378. Ref: https://goo.gl/eFVaU5
  48. Fears S, Mathieu C, Zeleznik-Le N, Huang S,Rowley JD, et al. Intergenic splicing of MDS1 and EVI1 occurs in normaltissues as well as in myeloid leukemia and produces a new member of the PRdomain family. Proc Natl Acad Sci USA. 1996; 93: 1642-1647. Ref: https://goo.gl/M8fJtd
  49. Sun X.J, Xu PF, Zhou T, Hu M, Fu CT, et al.Genome-wide survey and developmental expression mapping of zebrafish SETdomain-containing genes. PLoS One. 2008; 3: e1499. Ref: https://goo.gl/gcTckQ
  50. Wu H, Min J, Lunin VV, Antoshenko T, DombrovskiL, et al. Structural Biology of Human H3K9 Methyltransferases. PLoS One.2010; 5: e8570. Ref: https://goo.gl/Vy9nKX
  51. Zannino DA, Sagerström CG. An emerging role forprdm family genes in dorsoventral patterning of the vertebrate nervoussystem. Neural Dev. 2015; 10: 24. Ref: https://goo.gl/u2LDuH
  52. Rea S, Eisenhaber F, Carroll D, Strahl BD, Sun ZW,et al. Regulation of chromatin structure by site-specific histone H3methyltransferases. Nature. 2000; 406: 593-599. Ref: https://goo.gl/Pck8uA
  53. Hohenauer T, Moore AW. The Prdm family: expandingroles in stem cells and development. Development. 2012; 139: 2267-2282. Ref: https://goo.gl/jPCxk1
  54. Györy I, Fejér G, Ghosh N, Seto E, Wright KL.Identification of a functionally impaired positive regulatory domain Ibinding factor 1 transcription repressor in myeloma cell lines J Immunol.2003; 170: 3125-3133. Ref: https://goo.gl/rQW9ti
  55. Hirai H. The transcription factor Evi-1. Int JBiochem Cell Biol. 1999; 31: 1367-1371. Ref: https://goo.gl/3TKze6
  56. Liu L, Shao G, Steele-Perkins G, Huang S. Theretinoblastoma interacting zinc finger gene RIZ produces a PR domain-lackingproduct through an internal promoter. J Biol Chem. 1997; 272: 2984-2991. Ref: https://goo.gl/A3RY5D
  57. Zazzo E, De Rosa C, Abbondanza C, Moncharmont. BPRDM Proteins: Molecular Mechanisms in Signal Transduction andTranscriptional Regulation. Biology (Basel). 2013; 2: 107-141. Ref: https://goo.gl/GA4nXP
  58. Zazzo E, Porcile C, Bartollino S, Moncharmont B.Critical Function of PRDM2 in the Neoplastic Growth of Testicular Germ CellTumors. Biology (Basel). 2016; 5: 54. Ref: https://goo.gl/NAKVav
  59. Geli J, Kiss N, Kogner P, Larsson C. Suppressionof RIZ in biologically unfavourable neuroblastomas. Int J Oncol. 2010; 37:1323-1330. Ref: https://goo.gl/CGkbaM
  60. Geli J, Nord B, Frisk T, Edström Elder E, EkströmTJ, et al. Deletions and altered expression of the RIZ1 tumour suppressorgene in 1p36 in pheochromocytomas and abdominal paragangliomas. Int JOncol. 2005; 26: 1385-1391. Ref: https://goo.gl/GCX4Yd
  61. Piao Z, Fang W, Malkhosyan S, Kim H, Horii A, etal. Frequent frameshift mutations of RIZ in sporadic gastrointestinal andendometrial carcinomas with microsatellite instability. Cancer Res. 2000;60: 4701-4704. Ref: https://goo.gl/W7x2i6
  62. Poetsch M, Dittberner T, Woenckhaus C. Frameshiftmutations of RIZ, but no point mutations in RIZ1 exons in malignantmelanomas with deletions in 1p36. Oncogene. 2002; 21: 3038-3042. Ref: https://goo.gl/aeV6mN
  63. Sakurada K, Furukawa T, Kato Y, Kayama, T, Huang,S. et al. RIZ, the retinoblastoma protein interacting zinc finger gene, ismutated in genetically unstable cancers of the pancreas, stomach, andcolorectum. Genes Chromosomes Cancer. 2001; 30: 207-211. Ref: https://goo.gl/RPyP9V
  64. Sasaki O, Meguro K, Tohmiya Y, Funato T,Shibahara, S, et al. Altered expression of retinoblastoma protein-interactingzinc finger gene, RIZ, in human leukaemia. Br J Haematol. 2002; 119: 940-948.Ref: https://goo.gl/7BPA7K
  65. Steele-Perkins G, Fang W, Yang XH, Van Gele M,Carling T, et al. Tumor formation and inactivation of RIZ1, an Rb-bindingmember of a nuclear protein-methyltransferase superfamily. Genes Dev. 2001;15: 2250-2262. Ref: https://goo.gl/LDUqkS
  66. Mzoughi S, Tan YX, Low D, Guccione E. The role ofPRDMs in cancer: one family, two sides. Curr Opin Genet Dev. 2016; 36: 83-91.Ref: https://goo.gl/aLyPC2
  67. Marmorstein R. Structure of SET domain proteins:a new twist on histone methylation.
  68. Trends Biochem Sci. 2003; 28: 59-62. Ref: https://goo.gl/fC7vwY
  69. Kim KC, Geng L, Huang S. Inactivation of ahistone methyltransferase by mutations in human cancers. Cancer Res. 2003;63: 7619-7623. Ref: https://goo.gl/aTL3SA
  70. Matsuo H, Goyama S, Kamikubo Y, Adachi S. Thesubtype-specific features of EVI1 and PRDM16 in acute myeloidleukemia. Haematologica. 2015; 100: e116-e117. Ref: https://goo.gl/X2BvyD
  71. Seale P, Bjork B, Yang W, Kajimura S, Chin S, etal. PRDM16 controls a brown fat/skeletal muscle switch.Nature. 2008; 454: 961-967. Ref: https://goo.gl/emNeao
  72. Yin H, Pasut A, Soleimani VD, Bentzinger CF,Antoun G, et al. MicroRNA-133 Controls Brown Adipose Determination inSkeletal Muscle Satellite Cells by Targeting Prdm16. Cell Metab. 2013; 17: 210-224. Ref: https://goo.gl/xU9ohE
  73. Deng Q, Huang S. PRDM5 is silenced in humancancers and has growth suppressive activities. Oncogene. 2004; 23: 4903-4910.Ref: https://goo.gl/fxFuV8
  74. Watanabe Y, Toyota M, Kondo Y, Suzuki H, Imai T,et al. PRDM5 identified as a target of epigenetic silencing in colorectaland gastric cancer. Clin Cancer Res. 2007; 13: 4786-4794. Ref: https://goo.gl/heTiF3
  75. Cheng HY, Chen XW, Cheng L, Liu YD, Lou G. DNAmethylation and carcinogenesis of PRDM5 in cervical cancer. J Cancer ResClin Oncol. 2010; 136: 1821-1825. Ref: https://goo.gl/uJwpLx
  76. Huang S. The retinoblastoma protein-interactingzinc finger gene RIZ in 1p36-linked cancers. Front Biosci. 1999; 4: D528-32.Ref: https://goo.gl/MEhmBJ
  77. Jiang Gl, Liu L, Buyse IM, Simon D, Huang S.Decreased RIZ1 expression but not RIZ2 in hepatoma and suppression ofhepatoma tumorigenicity by RIZ1. Int J cancer 1999; 83: 541-546. Ref: https://goo.gl/6tBdTJ  
  78. Dong SW, Zhang YW, Chen Y, Wang S, Sun P, et al.PRDIBF1-RIZ domain of retinoblastoma protein-interacting zinc finger gene1 induces apoptosis and exerts anticancer activity in esophageal squamouscell carcinoma cells. Oncol Lett. 2015; 9: 341-346. Ref: https://goo.gl/Uv8Xc1  
  79. Gazzerro P, Abbondanza C, Arcangelo A, Rossi M,Medici N, et al. Modulation of RIZ gene expression is associated toestradiol control of MCF-7 breast cancer cell proliferation. Exp Cell Res.2006;  312: 340-9. Ref: https://goo.gl/GzBzCz
  80. Rossi M, Abbondanza C, Arcangelo A,  Gazzerro P, Medici N, Moncharmont B,
  81. Puca GA. The Zn-finger domain of RIZ protein promotes MCF-7 cell proliferation. Cancer
  82. Lett. 2004; 215: 229-37. Ref: https://goo.gl/Sj7TsU
  83. Medici N, Abbondanza C, Nigro V, Rossi V, PilusoG, et al. Identification of a DNA binding protein cooperating withestrogen receptor as RIZ (retinoblastoma interacting zinc finger protein).Biochem Biophys Res Commun. 1999; 264: 983-989. Ref: https://goo.gl/uN4qs9
  84. Abbondanza C, Medici N, Nigro V, Rossi V, GalloL, et al. The retinoblastoma-interacting zinc-finger protein RIZ is adownstream effector of estrogen action. Proc Natl Acad Sci. USA. 2000; 97:3130-3135. Ref: https://goo.gl/XoLXWf
  85. He L, Yu JX, Liu L, Buyse IM, Wang MS, et al.RIZ1, but not the alternative RIZ2 product of the same gene, isunderexpressed in breast cancer, and forced RIZ1 expression causes G2-Mcell cycle arrest and/or apoptosis. CancerRes. 1998; 58: 4238-4244.Ref: https://goo.gl/6xk6VN
  86. Koh-Stenta X, Joy J, Poulsen A, Li R, Tan Y, etal. Characterization of the histone methyltransferase PRDM9 usingbiochemical, biophysical and chemical biology techniques. Biochem J 2014; 461:323-334. Ref: https://goo.gl/M3WQge
  87. Li X, Wang J, Jiang Z, Guo F, Soloway PD, Zhao R.Role of PRDM16 and its PR domain in the epigenetic regulationof myogenic and adipogenic genes during transdifferentiation of C2C12cells. Gene 2015; 570: 191-198. Ref: https://goo.gl/ixp4Kr
  88. Harms MJ, Ishibashi J, Wang W, Lim HW, Goyama S, etal. Prdm16 is Required for the Maintenance of BrownAdipocyte Identity and Function in Adult Mice. Cell Metab. 2014; 19: 593-604. Ref: https://goo.gl/yTqTsM
  89. Burattini S, Ferri P, Battistelli M, Curci R,Luchetti F, et al. C2C12 murine myoblasts as a model of skeletal muscledevelopment: morpho-functional characterization. Eur J Histochem. 2004; 48:223-233. Ref: https://goo.gl/MwDgzE
  90. Cheedipudi S, Puri D, Saleh A, Gala HP, Rumman M,et al. A fine balance: epigenetic control of cellular quiescence by thetumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene.Nucleic Acids Res. 2015; 43:6236-56. Ref: https://goo.gl/B3UYF7
  91. Sabourin LA, Rudnicki M.A. The molecularregulation of myogenesis. Clin Genet. 2000; 57: 16-25. Ref: https://goo.gl/yHVw8p
  92. Morano A, Angrisano T, Russo G, Landi R, PezoneA, et al. Targeted DNA methylation by homology-directed repair inmammalian cells. Transcription reshapes methylation on the repaired gene.Nucleic Acids Res. 2014; 42: 804-821. Ref: https://goo.gl/NUDMA3
  93. Pfaffl MW. A new mathematical model for relativequantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: 45. Ref: https://goo.gl/ABUUGz
  94. Laemmli UK. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4. Nature. 1970; 227:680-685. Ref: https://goo.gl/jS8Gfa
  95. Crescenzi M, Crouch DH, Tatò F. Transformation bymyc prevents fusion but not biochemical differentiation of C2C12myoblasts: mechanisms of phenotypic correction in mixed culture withnormal cells. J Cell Biol. 1994;125: 1137-1145. Ref: https://goo.gl/bNkD5j
  96. Mal A, Harter ML. MyoD is functionally linked tothe silencing of a muscle-specific regulatory gene prior to skeletalmyogenesis. Proc Natl. Acad Sci. 2003; 100: 1735-1739. Ref: https://goo.gl/cdc72F
  97. Gazzerro P, Bontempo P, Schiavone EM, AbbondanzaC, Moncharmont B, et al. Differentiation of myeloid cell lines correlateswith a selective expression of RIZ protein. Mol Med. 2001; 7: 552-560. Ref: https://goo.gl/PJDGtc
  98. Abbondanza C, Nigris F, Rosa C, Rossiello R, PucaGA, et al. Silencing of YY1 downregulates RIZ1 promoter in humanosteosarcoma. Oncol Res. 2008;17: 33-41. Ref: https://goo.gl/Cuwz43
  99. Mal AK. Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic differentiation. EMBO J. 2006; 25: 3323-3334. Ref: https://goo.gl/jr1Sse