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

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.Introduction The myogenesis of mammalian skeletal muscle cells is regulated by myogenic regulatory factors (MRFs), related in a hierarchical relationship [1], which are considered to be members of a superfamily of the highly conserved variant of the basic helix-loop-helix (bHLH) domain [2], that confers their peculiar myogenic potential [3]. The phylogenetic analysis of the sequences of these genes indicates that MRF genes have evolved from a single ancestral MRF gene progenitor, by gene duplication events followed by divergent mutations [4]. The four main basic helix-loop-helix myogenic regulatory factors, which exhibit a pivotal role in skeletal muscle development and which are responsible for coordinating muscle-specific gene expression in the developing embryo [5], are MyoD (Myf-3) [3], which coordinates an open chromatin structure at muscle-specific genes [1]; Myf-5 [6], which is required for the specification and proliferation of myoblasts [7-10], enhances myogenesis by promoting myoblast proliferation [11-13], because it is also the earliest to be expressed during myogenesis process, acting as a transcription factor in muscle progenitor cells (satellite cells) and myocytes [14]; Myogenin (Myf-1) [15], which is involved in the differentiation of myoblasts into multinucleated myotubes [16] and MRF4 [17], which is implicated in the latest phase of differentiation that includes myofiber maintenance [18]. The Myogenic regulatory factor (MRF) genes are expressed with a peculiar pattern [19,20] and they can auto- and cross-regulate the expression of each other and interact with the myocyte enhancer factor-2 (MEF2) family of transcription factors, to activate the transcription of muscle-specific genes [21]: currently, these MEF2 transcription factors are considered to drive the development of muscle, cardiac, skeletal, vascular, neural and blood cells, because of their pivotal effects on cell differentiation, proliferation, apoptosis, migration, shape and metabolism [22]. The activity of MRF proteins require heterodimerization with a member of the ubiquitously expressed E-protein family of bHLH proteins. This event leads the binding to the regulatory regions of muscle-specific genes on the E-box consensus sequence (CANNTG) [5]. Currently, only a small fraction of 14 million potential sites are available for MRF binding [23,24]. MyoD, the master regulator of the skeletal muscle gene expression program, activates genes which display the consensus E-box sequence VCASCTG (where V is A, C or G while S is C or G) within their promoter/enhancer regions [23,25-27] and it leads a gene expression program by heterodimerizations with E-proteins and giving rise to multinucleated myotubes [28]. The binding of MyoD on the E-box sequence (CANNTG) and the recruitment of factors, which are involved in remodelling the chromatin, are considered the crucial event for transcription [29,30] because MyoD activates pRb and p21 gene expression [31,32] to shoot down the cell cycle machinery [29]. The skeletal muscle differentiation is a strongly coupled event to the cell cycle exit [33] by an upregulation of cyclin-dependent kinase inhibitors (CDKIs), which inhibit cyclin-CDK complexes driving a downregulation of the activity of cyclin D1,E,A, and B-CDK complexes [34] and by an induction of the permanent cell cycle exit. This is a pivotal step because of the overexpression of cyclin/CDKs has been reported to inhibit the activity of MyoD by different mechanisms [29]. In particular, p21cip1/waf1, and p57/kip2, encoded respectively by Cdkn1a and Cdkn1c genes, control differentiation of skeletal muscle and their loss affects fiber formation: they have a key role both for cell cycle exit, both in triggering a muscle-specific transcriptional program [35]. The PRDM (Positive Regulatory Domain) gene family which consists of 17 orthologs in primates and 16 orthologs in rodents, birds and amphibians [36], encodes transcription factors with a PR domain and a variable number of zinc finger motifs [37], with the exception of PRDM11 [36,38]. The PR domain displays a 20-30% amino acid homology sequence to the catalytic SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain with hystone lysine methyltransferase (HMTs) activity [39]. In contrast to the SET domain proteins, currently only three PRDM proteins have been demonstrated to possess intrinsic HMTase activity [40] and a number of PRDMs have not endowed with catalytic activity towards histones/nucleosomes [41-44]. In particular the HMT’s activity has been found only in the PR domains of PRDM2/RIZ1, Prdm8 and Prdm9 [45-47]. In fact, the PR domain has diverged significantly from the SET domain [48-51] and most PR domains lack the H/RxxNHxC motif required for methyltransferase activity [52,53]. Generally, the PR domain is localized at the N-terminus of the protein, whereas the SET domain is often localized to the C-terminus [39]. A common characteristic of PRDM genes is the expression of molecular variants by alternative splicing or by alternative use of promoters. The PRDM1, PRDM2 and PRDM3, which is also called MECOM (MDS1-EVI1 complex locus) genes are expressed as two alternative forms, by intergenic splicing, which produce the PR plus and PR minus forms of these genes [54-57]. PR plus and PR minus forms are expressed at equimolar concentration and their ratio is maintained in a fine equilibrium [58]: an imbalance in the amounts of the two products, through either disruption or underexpression of the PR plus form or overexpression of the PR minus form commonly occurs in human cancers through genetic and epigenetic mechanisms [59-66]. PRDM2 gene gives two alternative products: RIZ1, the PR plus form, implicated in tumor suppressor function, and RIZ2, the PR minus form. This PR domain (PRDI-BF1 and RIZ homologous) [67] which is endowed with histone H3 K9 methyltransferase activity, is targeted by inactivating mutations in human cancers [68]. PRDM proteins mediate transcriptional activation or repression depending on the nature of their intrinsic HMT activity: PRDM proteins appear to function by modulating gene expression states either directly (via intrinsic HMTase activity), or indirectly (via recruitment of various cofactors), controlling critical aspects of cell integrity, spanning from cell differentiation to cell growth and apoptosis [58]. These genes also play a role in human cancer, where they mainly act as tumor suppressors: for example, PRDM1 is a tumor suppressor of diffuse large B cell lymphoma (DLBCL); PRDM3and PRDM16 show different isoforms with separate functions in leukemia [44,69]. PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16) controls a bidirectional cell fate switch between skeletal myoblasts and brown fat cells [70], the targeting of 3’UTR of Prdm16 is involved in the choise between myogenic and brown adipose determination of the adult skeletal muscle stem cells (satellite cells) [71], PRDM5 acts with a potential tumor suppressor role for gastrointestinal carcinogenesis [72-74]. The RIZ proteins regulate cell proliferation in a yin-yang manner [57,58,75]: gene silencing of the RIZ1 form, by genetic or epigenetic mechanisms, has been described in a variety of human tumors [14], whereas the RIZ2 form, lacking of PR domain, is always present or overexpressed [76]: this finding suggests a positive selection for RIZ2 in cancer progression. Further evidence indicates that forced expression of RIZ1 in tumor cell cultures induces growth arrest and apoptosis, possessing an anticancer activity in the PR domain [77], and the silencing of RIZ1 expression can stimulate breast cancer cell proliferation [78]. In addition, forced expression of the Zn-finger domain present in both RIZ forms increases the growth rate of breast cancer cells [79]. Based on these findings, RIZ1 could be considered as a crucial tumor suppressor gene candidate and the Zn-finger domain could be responsible for the putative oncogenic activity of the RIZ2 gene product. Such an effect might be more relevant in estrogen target tissues, where RIZ gene products are reported to directly interact with the estrogen receptor in a hormone-dependent manner through a LXXLL motif [80,81], promoting optimal estrogen response; conversely, in osteosarcoma cancer cell line (SAOS2), RIZ1 is expressed at high lev