BACKGROUND
mouse tis <dig>  its rat pc <dig>  <cit> , and human interferon-related developmental regulator  <dig>  <cit>  homologues were originally proposed to act as immediate early genes  <cit> .
tis <dig> was found to be upregulated upon c-jun activation and translocated to the nucleus  <cit> , where it functions as a transcriptional co-regulator, activating  <cit>  or repressing gene expression via interaction with transcription factors and histone deacetylase  <cit>  complexes  <cit> .
tis <dig> protein has been shown to be involved in the regulation of differentiation processes in multiple cell types, e.g., neurons  <cit> , enterocytes  <cit> , and myocytes  <cit> .
we have previously shown that tis <dig> knockout  mice are viable and fertile but suffer from delayed muscle regeneration upon muscle crush damage, which becomes even more apparent with increasing age  <cit> .
at the cellular level, primary muscle satellite cells from tis <dig> ko mice displayed significant differentiation deficiency  <cit> .
this regeneration phenotype can be explained by reduced differentiation potential caused by decreased mrna and protein expression levels of key muscle regulatory factor myod  <cit>  in tis <dig> ko muscle satellite cells .
we, together with others, have previously shown that tpa induced sequence  <dig>  <cit>  is a positive regulator of myogenesis and of myod levels in particular.
although there is experimental evidence for tis7-dependent regulation of multiple proteins related to the control of myod levels and its activity, e.g., depletion of the inhibitory effect of hdac <dig>  <cit>  or downregulation of nf-κb  <cit> , an exact tis7-dependent pathway regulating myod levels and thereby myogenesis remained thus far unknown.
though there is indication that transcriptional regulation of myogenic regulatory factors  myod, myf <dig>  and myogenin depends on the enzymatic activity of several histone modifying enzymes, such as protein arginine n-methyltransferase  <dig> , the mechanism regulating the particular choice of prmt <dig> substrate in adult myogenesis is yet to be identified.
prmt <dig> was found in the methylosome complex with the protein icln which, depending on its interacting partners, is also involved in transcriptional regulation .
icln was shown to be responsible for the prmt <dig> substrate specificity  <cit> .
however, whether icln levels correlate with the prmt <dig> activity and mainly how they are regulated in the process of myogenesis, remains to be studied.
herein, we investigated, at the molecular level, how tis <dig> may affect skeletal muscle regeneration and the regulatory processes of myoblast differentiation, in particular, the exact mechanism by which tis <dig> regulates myod levels.
first of all, we identified that the tis <dig> protein binds directly to dna, which substantially clarifies its role in transcriptional regulation.
we confirmed by several independent techniques the physical interaction between the tis <dig> protein and regulatory elements of the icln gene and even quantitatively determined the relationship between tis <dig> binding and icln transcriptional regulation.
next, we clarified the link between prmt <dig> and regulation of myod expression by showing that icln levels determine binding of prmt <dig> and thereby its methyl transferase activity on histone h <dig> arg  <dig> on particular gene locus, in this case myod.
finally, through in vivo experiments, we documented that the ectopic expression of either tis <dig>  icln, or myod in mscs derived from tis <dig> ko mice rescued their diminished differentiation potential.
on the basis of our current data, we propose that tis <dig> participates in the mechanism of epigenetic regulation of myogenesis via a novel player icln, in particular by its transcriptional regulation.
RESULTS
tis <dig> controls myod levels through epigenetic regulation of gene expression
myo5d and mef <dig> families of transcription factors are key regulators of skeletal muscle differentiation  <cit> .
we and others have previously shown that myod levels in myoblasts depend on tis <dig> expression  <cit> .
although the levels of several proteins related to myogenesis regulation  have been shown to be affected by the presence or absence of tis <dig>  <cit> , the explicit mechanism by which tis <dig> regulates myod expression is yet to be identified.
therefore, we decided to first analyze the ability of ectopic tis <dig> expression to restore myod mrna and protein levels in proliferating tis <dig> ko mscs.
consistent with previous data  <cit> , the loss of tis <dig> caused a significant downregulation of myod mrna levels, which was restored upon ectopic expression of tis <dig> in ko mscs .
western blot analyses of myod showed that  myod d protein levels were significantly reduced in tis <dig> ko mscs  and  significant rescue was achieved by tis <dig> ectopic expression  .fig.
<dig> tis <dig> epigenetically regulates myod expression.
a tis <dig> ko mscs were transfected with indicated plasmids.
quantitative pcr analysis of myod was normalized to gapdh expression .
values represent the mean ± sem and resulting data were analyzed via two-tailed type  <dig> student’s t-test, *** p <  <dig>  and * p <  <dig> .
b proliferating tis <dig> ko mscs were transfected with indicated plasmids and total cell lysates were probed with myod and control antibodies.
quantification  was performed by normalization to the tubulin signal.
values represent the mean ± sd and resulting data were analyzed via two-tailed type  <dig> student’s t-test, *** p <  <dig>  and ** p <  <dig> .
representative western blots are shown.
chip analyses using chromatin from tis <dig> wt or ko mscs transfected with either gfp or gfp-tis <dig> plasmid dna.
c schematic outline of mouse myod gene locus.
primer pairs used for chip assays are indicated by arrows.
the alignment of human and mouse sequences amplified by the pcr is shown.
antisera directed against symmetrically di-methylated histone h <dig> at arginine  <dig>  or prmt <dig>  were used for immunoprecipitation.
immunoprecipitated dna was analyzed by qpcr in triplicates using primers specific for the mouse myod regulatory region.
chip with control rabbit igg or  <dig>  % of total chromatin  were used as controls.
signals were normalized to input chromatin and shown as percentage of input, ** p ≤  <dig>  and *** p <  <dig>
tis <dig> regulates myod expression by an epigenetic mechanism
transcriptional co-regulator arginine methyltransferase prmt <dig> was identified to be necessary for proper myogenesis in zebrafish embryonic development, where it specifically regulates myod expression  <cit> .
in adult mice, prmt <dig> inactivation abrogated skeletal muscle regeneration  <cit> , which resembled the tis <dig> ko mouse phenotype reported by us  <cit> .
therefore, we decided to investigate whether the prmt5-dependent epigenetic control mechanism of myod expression is affected by tis <dig> ko.
we first tested the presence of arginine  <dig> dimethylated histone h <dig> on the myod gene.
for this purpose, we performed chromatin immunoprecipitation  using specific anti-histone h <dig>  antibodies followed by quantitative pcr detection of the myod regulatory region .
our data showed that there was a significant increase in symmetrical dimethylation of histone h <dig> on myod in tis <dig> ko when compared to wildtype  mscs , although the total amounts of histone h <dig> were comparable .
the increase in this modification, which is generally related to transcriptional inhibition, explained reduced myod expression in tis <dig> ko mscs.
importantly, the ectopic expression of tis <dig> in ko mscs significantly  reduced symmetrical dimethylation of histone h <dig> on myod to levels comparable with the wt mscs.
next, we analyzed by chip whether this difference was directly dependent on prmt <dig> recruitment to the myod regulatory region.
as expected, prmt <dig> occupancy on the myod gene was significantly higher in tis <dig> ko when compared to wt mscs.
as in the case of h <dig> methylation, we found that the ectopic expression of tis <dig> significantly reduced prmt <dig> binding to myod gene in ko mscs .
icln expression is downregulated in tis <dig> ko mscs
to understand how tis <dig> may affect prmt5-specific function in myogenesis we analyzed the effect of ectopic expression of a known regulator of prmt <dig>  namely icln   <cit>  in tis <dig> wt and ko mscs.
icln protein forms a complex with prmt <dig>  <cit>  that regulates its substrate specificity  <cit> .
therefore, we analyzed whether prmt <dig> epigenetic regulation of myod transcription was related to icln levels.
under an identical experimental setup as described above , we analyzed chromatin isolated from tis <dig> wt and ko mscs, transiently transfected either with control yfp or with a yfp-icln construct.
immunoprecipitation with both anti-histone h <dig>  and anti-prmt <dig> antibodies revealed an increase in arg <dig> dimethylated histone h <dig> and prmt <dig> bound to myod regulatory elements in tis <dig> ko mscs, respectively .
constitutive expression of icln significantly  decreased the amount of symmetrically dimethylated h <dig> arg <dig>  on myod regulatory elements; this effect could be explained either by increased prmt <dig> enzymatic activity or changes in its binding to chromatin.
two different independent methylation assays unveiled that the presence of icln had no effect on total prmt <dig> enzymatic activity both in vitro and in vivo .
we also could not detect any difference in subcellular distribution of prmt <dig> .
however, our chip experiments revealed that the ectopic expression of icln substantially decreased the symmetrically dimethylated arginine  <dig> histone h <dig> and prmt <dig> recruitment to myod regulatory elements .
the level of chromatin-associated prmt <dig> in icln-complemented tis <dig> ko mscs was similar to that observed in wt cells.
next, we checked whether icln ectopic expression in tis <dig> ko mscs rescued the myod expression.
as shown in fig.
2c, the ectopic icln expression rescued myod protein levels due to increased myod transcription, as documented by elevated myod mrna .
thereafter, we asked ourselves whether prmt5-mediated methylation of the myod regulatory region is solely responsible for tis7/icln-mediated regulation of myod transcription.
therefore, we generated, by lentiviral transduction and antibiotic selection, a stable and specific prmt <dig> knockdown in tis <dig> ko mscs .
qpcr analysis of myod levels in proliferating tis <dig> ko mscs did not show any difference between control sh gfp and sh prmt <dig> expressing cells .
however, after 7 days in differentiation medium, at a time point when wt tis <dig> mscs fully differentiate, we identified an 80 % increase in myod rna levels of sh prmt5-expressing tis <dig> ko mscs .
based on these results we concluded that prmt5-dependent methylation of histone h <dig> bound to myod regulatory elements is not the main regulatory mechanism in proliferating tis <dig> deficient cells, suggesting that other tis7-dependent mechanisms may contribute to this regulation.
however, prmt5-regulated methylation significantly affected myod expression during the differentiation of mscs.fig.
<dig> icln affects in vivo methylation of histone h <dig> and binding of prmt <dig> to myod locus, resulting in increased myod rna and protein levels.
chip analysis using chromatin from tis <dig> wt or ko mscs transfected with either yfp or yfp-icln plasmids.
antisera directed against symmetrically dimethylated histone h <dig> at arginine  <dig>  or prmt <dig>  were used for immunoprecipitation.
immunoprecipitated dna was analyzed in triplicate by qpcr using primers to amplify the mouse myod regulatory region.
chip with control rabbit igg or  <dig>  % of total chromatin  were used as controls.
signals were normalized to input chromatin and shown as percentage of input.
** p ≤  <dig>  and *** p <  <dig> .
c tis <dig> ko mscs were transfected with indicated plasmids and total cell lysates were probed with anti-myod and anti-yfp antibodies.
quantification  was performed by normalization to the tubulin signal.
representative immunoblots are shown.
d tis <dig> ko mscs were transfected with the indicated plasmids.
quantitative pcr analysis of myod was normalized to gapdh expression .
e prmt <dig> knockdown significantly increased myod rna levels in tis <dig> ko mscs transduced with sh prmt5-expressing lentivirus.
myod rna levels were measured by qpcr and prmt <dig> protein levels were documented by immunoblots with indicated antibodies
we thus conclude that icln regulates myod expression in mscs by changing the epigenetic status of chromatin through functional interference with prmt <dig>
the next question raised was whether there were significant differences in icln levels between tis <dig> ko and wt mscs.
western blot analyses of several independent biological experiments  revealed that icln protein levels were significantly reduced in both proliferating  and differentiating  ko mscs   when compared to tis <dig> wt mscs.
ectopic expression of tis <dig> restored the icln protein levels up to 90 % of the wt values, suggesting the role of tis <dig> in its regulation .fig.
<dig> tis <dig> regulates icln protein and rna levels.
a quantitative analyses of icln protein expression in proliferating  and differentiating  mscs.
equal amounts of total cell lysates were probed by immunoblotting with anti-icln antibodies and normalized by tubulin.
b tis <dig> ectopic expression rescued icln protein levels in tis <dig> ko mscs.
proliferating tis <dig> ko mscs were transfected with gfp plasmid as a control or with gfp-tis <dig> plasmid constructs and total cell lysates were probed with the indicated antibodies.
quantification  was based on tubulin signal.
representative western blots are shown.
c quantitative pcr analysis of icln was normalized to gapdh expression .
d tis <dig> overexpression rescued icln rna levels in tis <dig> ko mscs.
proliferating tis <dig> ko mscs were transfected with indicated plasmids and icln quantitative pcr was performed as in panel c. e tis <dig> transcriptionally regulates icln levels.
proliferating wt mscs were transiently co-transfected with full length icln promoter-luciferase reporter construct, β-galactosidase expression plasmid, and gfp plasmid as a control or gfp-tis <dig> plasmid constructs.
luciferase values were normalized to β-galactosidase values.
in all graphs, values represent the mean ± sd and resulting data were analyzed via two-tailed type  <dig> student’s t-test, *** p <  <dig> , ** p <  <dig> , and * p <  <dig>
we observed the same trend also in mrna levels where icln expression was approximately 50 % lower in both pm and dm tis <dig> ko mscs .
similarly to protein, the level of icln mrna was significantly upregulated by tis <dig> ectopic expression , indicating possible tis <dig> involvement in icln transcriptional regulation.
to test this, we transiently transfected wt and tis <dig> ko mscs with an icln promoter-driven reporter construct and measured the luciferase activities.
these were reduced in tis <dig> ko mscs and efficiently rescued by ectopic expression of tis <dig>  showing that the activity of icln promoter is dependent on tis <dig>  since little is known about icln gene regulation  <cit> , we applied luciferase assays using reporter constructs possessing various promoter truncations.
these experiments revealed a 41 nt sequence  representing a minimal essential region required for icln transcriptional activity in wt mscs .fig.
<dig> a defined  <dig> nucleotide stretch within the icln promoter represents the minimal region essential for tis7-regulated icln transcription.
a proliferating tis <dig> wt mscs were transiently co-transfected with indicated icln promoter-luciferase reporter constructs and with β-galactosidase expression plasmid for transfection control.
the activity of the wt full length icln promoter-driven luciferase reporter was set as 100 %.
values represent the mean ± sem of at least three biological replicates.
b activities of luciferase reporter constructs driven by two or four  repeats from icln promoter.
bars marked + tis <dig> represent samples co-transfected with gfp-tis <dig> plasmid vector.
control samples were co-transfected with gfp plasmid.
c tis <dig> binds to the minimal essential region necessary for the tis7-regulated transcription of icln.
gst  or gst-tis <dig>  recombinant proteins were incubated with the fluorescently labeled oligonucleotide probe containing the –154/– <dig> sequence of the icln promoter.
d specific tis <dig> binding to icln minimal essential region  determined by surface plasmon resonance measurements .
oligonucleotides representing either the icln mer or its scrambled version were incubated with increasing concentrations of gst-tis <dig> or gst alone as indicated.
spr steady-state response values  are presented as a function of protein concentration .
kd = dissociation constant.
e chip analysis of tis <dig> binding to the icln promoter region.
chromatin from tis <dig> wt and ko mscs was immunoprecipitated as above using either specific antibody against tis <dig> or control rabbit igg
tis <dig> directly binds to and activates icln promoter
next, we tested whether this 41 nt sequence was sufficient to regulate icln expression in a tis7-dependent manner.
we generated a luciferase reporter construct, which contained two repeats of the 41 nt sequence  of the icln promoter upstream of the luciferase gene.
wt mscs transfected with this construct displayed increased luciferase activity when compared to the empty pgl3b luciferase vector .
this basal activity was significantly induced  by co-transfection with tis <dig> .
even stronger luciferase activity was found when four repeats of the 41 nt sequence were cloned upstream of the luciferase gene .
these data demonstrated the functional relevance of this minimal essential region for tis7-mediated activation of icln transcription.
using bioinformatics prediction software , bindn+ , dp-bind , and dnabindr ; additional file 2: figure s2) we identified, with 70–80 % probability, three distinct dna-binding domains in the tis <dig> protein.
this prediction suggested that the tis <dig> protein may directly bind dna.
we tested this possibility by an electrophoretic mobility shift assay using an oligonucleotide probe containing the  sequence of the icln promoter.
as shown in fig.
4c, we detected a specific band representing a complex of gst-tis <dig> with a bound fluorescently labeled oligonucleotide probe .
the specificity of this complex was confirmed by the fact that this complex was not formed when the probe was incubated with gst alone .
additionally, tis <dig> binding to the labeled probe was specifically blocked by pre-incubation with a 20-fold access of a non-labeled “cold” oligonucleotide .
the presence of tis <dig> in this dna complex was further confirmed by pre-incubation with a tis7-specific antibody, which caused a “supershift”, i.e., further slowed the mobility of the complex on the native polyacrylamide gel .
in addition, we have quantified the specific binding of tis <dig> to this dna oligonucleotide using surface plasmon resonance.
the measurements showed that  the protein binding moiety is specific  and  the tis7-binding was sequence specific because there was a 10-fold difference in kd values between the native  icln promoter  and “scrambled” sequences.
using chip analysis, we investigated whether tis <dig> binds a native icln promoter in mscs.
we found that tis <dig> was bound to the icln promoter in tis <dig> wt but not ko mscs .
the specificity of tis <dig> binding was confirmed using control rabbit igg.
based on the results of these analyses we conclude that tis <dig> directly binds icln promoter dna and thereby positively regulates icln transcription in msc cells.
tis <dig> regulates myotube formation through icln and myod expression
to understand how tis <dig> regulates skeletal muscle differentiation, we characterized in detail the morphological differences between primary wt and tis <dig> ko mscs.
in general, proliferating mscs could be induced to differentiate into mature myotubes within 5– <dig> days.
although plated at the same density, while 70 % of wt mscs differentiated into myotubes, only 30 % of tis <dig> ko mscs formed myotubes .
this defect was completely restored by ectopic tis <dig> expression.
importantly, the ectopic expression of myod in tis <dig> ko mscs led to full recovery of the differentiation deficit and restored myotubes formation in these cells .
because of tis7-dependent regulation of icln we investigated whether icln can act downstream of tis <dig> to regulate myogenesis.
ectopic icln expression in tis <dig> ko cells rescued myotube formation in tis <dig> ko mscs, similar to the effects of tis <dig> or myod overexpression .fig.
<dig> msc differentiation is regulated by myod, icln, and tis <dig>  a tis <dig> wt and ko mscs were plated at the same cell density.
proliferating tis <dig> ko mscs were transfected with indicated plasmids and 6 h later the differentiation was induced.
after  <dig> d of differentiation, cells were fixed, stained with anti-myosin heavy chain antibody mf  <dig>  and the nuclei were visualized using hoechst.
scale bar = 100 μm.
b percentage of msc fusion was calculated as the number of nuclei localized in multinucleated  myotubes defined by mf <dig> staining compared to the total number of nuclei.
vertical bars denote the mean ± sd and were calculated from independent biological replicates .
the indicated number of nuclei was evaluated.
resulting data were analyzed via two-tailed type  <dig> student’s t-test, *** p <  <dig> .
c quantitative analysis of cells with the indicated nuclei numbers per cell defined by myosin staining.
this analysis showed a significant decrease in the number of multinucleated cells and a completely missing category of cells with more than  <dig> nuclei in tis <dig> ko mscs when compared to tis <dig> wt mscs.
ectopic expression of tis <dig>  myod, or icln significantly increased the number of multinucleated cells, mainly those with more than  <dig> nuclei in tis <dig> ko mscs when compared to tis <dig> wt mscs
a more detailed quantitative analysis revealed that the majority of tis <dig> ko mscs  remained mononucleated and the very large myotubes containing more than  <dig> nuclei were completely missing in tis <dig> ko mscs .
ectopic tis <dig> expression rescued the appearance of multinucleated cells, mainly the category of myotubes with more than  <dig> nuclei.
even more interestingly, the ectopic expression of myod reverted the distribution of different groups of multinucleated myotubes similar to that of tis <dig> wt mscs and recovered in tis <dig> ko mscs the missing group of very large myotubes with more than  <dig> nuclei .
similarly, the increased number of multinucleated cells and, specifically, the formation of very large myotubes with more than  <dig> nuclei  were observed in icln-transfected cells.
myod as a crucial myogenic regulatory factor is responsible for the myoblast differentiation leading to myotube formation and, based on data shown here, we concluded that tis <dig> regulates icln transcription and thereby myod levels responsible for reversion of the tis <dig> ko phenotype in mscs.
discussion
in this study, we show that icln, a protein with multiple physiological functions that depend on its subcellular localization and mainly on its protein interacting partners  <cit> , represents a novel regulator of skeletal myoblast differentiation.
we identified that tis <dig> transcriptionally regulated icln via direct binding to its promoter and, secondly, that ectopic expression of icln rescued the differentiation deficiency of tis <dig> ko mscs.
icln controlled muscle differentiation through the myod pathway since icln upregulated myod levels.
our further results presented here provide evidence for the epigenetic regulation of myod transcription by icln.
adult muscle regeneration depends on de novo myogenesis, which requires activation and commitment of quiescent satellite cells.
muscle regeneration ability is regulated by and depends on the differentiation potential of mscs .
this is assured by the interplay of mrfs myod, myogenin, myf <dig>  and myf <dig>   <cit> .
myod and myf <dig> are required for myogenic determination  <cit> , whereas myogenin and myf <dig> are markers of terminal differentiation  <cit> .
muscle damage induces msc proliferation followed by differentiation resulting in the formation of new muscle fibers.
myod is required in both processes; however, myoblast differentiation initiation and progression is governed by an interplay of myod with other muscle-specific factors, e.g., myf <dig> and myogenin  <cit> .
myod ko mice indicate a unique requirement of myod during adult muscle regeneration rather than during embryonic development  <cit> .
we found that the myod regulation at this stage of myogenesis is tightly regulated by the tis7/icln/prmt <dig> axis.
depletion of tis <dig> or reduced level of icln caused the epigenetic inhibition of myod leading to the general differentiation deficiency phenotype characterized by diminished myoblast fusion.
these findings indicate that the maintenance of specific myod levels by the tis7/icln/prmt <dig> axis is critical for the induction and early progression of myoblast differentiation.
it remains to be clarified whether this pathway is responsible for the regulation of other mrf genes.
myogenic gene expression is affected by chromatin remodeling enzymes such as histone acetylases and deacetylases, histone lysine methyltransferases, swi/snf family of atp-dependent enzymes, and protein arginine methyltransferases  <cit> .
among them, prmt <dig> was identified in several independent studies as one of the critical epigenetic regulators of myogenesis.
prmt <dig> was shown to control the expression of the mrfs myogenin, myod, and myf <dig> in zebra fish embryos  <cit> .
it was also shown that prmt <dig> is involved in transcriptional regulation of various other myogenic genes via symmetric arginine dimethylation of histones and components of the transcriptional machinery  <cit> .
prmt <dig> facilitates the recruitment of brg1-atpase, associated with swi/snf chromatin remodeling enzymes, and myod to the promoter of myogenin via h <dig> arg <dig> dimethylation resulting in upregulation of myogenin transcription  <cit> .
the same h <dig> arg <dig> modification by prmt <dig> exerts transcriptional repression effect on the p <dig> gene that facilitates msc proliferation and expansion, which has a positive impact on the process of muscle regeneration.
as a part of the methylosome, prmt <dig> also methylates and facilitates the recruitment of sm proteins to the survival motor neuron complex leading to its activation  <cit> .
interestingly, the loss of survival motor neuron-specific activity is observed in primary myoblasts from patients with the autosomal recessive disorder spinal muscular atrophy.
these myoblasts display a strong fusion deficiency, implying a primary muscle defect along with the well-described neuronal degeneration  <cit> .
we present here new evidence for a contrasting, but not conflicting, function of prmt <dig> in the regulation of myogenesis that is associated with inhibition of myod expression.
prmt <dig> represses myod expression using a similar epigenetic mechanism described for p <dig> gene that is mediated through h3arg <dig> symmetrical dimethylation at the myod locus.
this inhibits induction and early progression of msc differentiation into myotubes, which could represent the mechanism that together with p <dig> inhibition  <cit>  contributes to the maintenance of the proliferative potential of adult mscs.
in this scenario, prmt <dig> would allow efficient accumulation of a critical msc mass that could be committed to differentiation upon receiving specific stimuli.
the open question, however, remains how this prmtr <dig> function is controlled in mscs.
it has been shown before that prmt5-specific cellular functions are regulated by its interacting partners  <cit> .
icln, the component of the methylosome complex, was shown to directly interact with prmt <dig> and change its substrate specificity.
in the presence of icln, prmt <dig> methylation of sm proteins is stimulated, but methylation of histones is inhibited  <cit> .
consistent with these studies, we also observed that icln ectopic expression inhibited methylation of histone h <dig> in mscs.
this was achieved via inhibition of prmt <dig> binding to the myod gene  rather than changing prmt <dig> enzymatic activity .
the recruitment of prmt <dig> to the regulatory elements of genes needs complex formation with dna-binding or chromatin-associated adaptor proteins  <cit> .
we propose that formation of such a complex on the myod gene is prevented by the interaction of prmt <dig> with icln.
similarly, formation of an alternative prmt <dig> complex has been described for protein riok <dig>  which competes with icln for binding to prmt <dig>  <cit> .
this would lead to diminished h <dig> arg <dig> methylation thereby de-repressing myod expression.
icln has been found to stimulate prmt5-mediated sm protein methylation and at the same time to inhibit the nuclear function of prmt <dig>  namely methylation of histones  <cit> .
silencing of icln was shown to cause α-motor axon degeneration  <cit> , as seen in patients suffering from spinal muscular atrophy.
there is a substantial body of knowledge on icln functions, its interaction with other proteins and subcellular localization.
however, the regulation of icln gene expression remains to be solved.
here, we found a mechanism related to myogenesis where tis <dig> transcriptionally activates icln through binding to the newly identified regulatory element in the icln gene.
thus, we propose that myod expression in mscs is epigenetically regulated via icln/prmt <dig> downstream of tis <dig>  this notion is supported by the following facts:  deletion of prmt <dig> prevented msc differentiation  <cit> , which was a phenotype strikingly reminiscent of tis <dig> ko mscs;  prmt5-specific symmetrical dimethylation of h <dig> arg <dig> at the myod gene was enhanced in tis <dig> ko mscs; and  tis <dig> or icln ectopic expression reduced h <dig> arg <dig> methylation at the myod gene and rescued the muscle differentiation deficiency in tis <dig> ko mscs.
furthermore, overexpression of prmt <dig> alone in tis <dig> ko mscs did not rescue their fusion , which was consistent with the inhibitory role of prmt <dig> in msc-specific myod regulation.
CONCLUSIONS
as summarized in fig.
<dig>  we propose here a novel model for tis <dig> function in myogenesis, where tis <dig> upregulates icln transcription in mscs via direct binding to the regulatory element in the promoter of this gene.
increase in icln transcription leads to accumulation of icln protein in the cytoplasm and nucleus.
the increased amount of icln protein inhibits prmt <dig> recruitment to the myod locus and prevents the methylation of histone h <dig> at arg  <dig>  decreased methylation de-represses myod expression, which is critical for msc differentiation.
it is likely that prmt <dig> controls myod levels to balance the proliferation and differentiation potential of mscs.
thus, the components of this pathway and mainly their regulation could be seen as targets for development of new therapeutic approaches for treatment of skeletal muscle diseases.fig.
<dig> model of myogenesis regulation by the tis7/icln/prmt <dig> pathway.
in skeletal muscles, tis <dig> transcriptionally regulates icln.
the accumulated icln interacts with prmt <dig> and prevents its recruitment to chromatin-bound complexes on the myod gene.
this regulates prmt5-mediated symmetrical dimethylation of histone h <dig> arg <dig> and myod expression.
the regulated myod level affects the balance between proliferation and differentiation of mscs

 mouse tis its rat pc, and human interferon-related developmental regulator homologues were originally proposed to act as immediate early genes. tis was found to be upregulated upon c-jun activation and translocated to the nucleus, where it functions as a transcriptional co-regulator, activating or repressing gene expression via interaction with transcription factors and histone deacetylase complexes. tis protein has been shown to be involved in the regulation of differentiation processes in multiple cell types, e.g., neurons, enterocytes, and myocytes. we have previously shown that tis knockout mice are viable and fertile but suffer from delayed muscle regeneration upon muscle crush damage, which becomes even more apparent with increasing age. at the cellular level, primary muscle satellite cells from tis ko mice displayed significant differentiation deficiency. this regeneration phenotype can be explained by reduced differentiation potential caused by decreased mrna and protein expression levels of key muscle regulatory factor myod in tis ko muscle satellite cells. we, together with others, have previously shown that tpa induced sequence is a positive regulator of myogenesis and of myod levels in particular. although there is experimental evidence for tis7-dependent regulation of multiple proteins related to the control of myod levels and its activity, e.g., depletion of the inhibitory effect of hdac or downregulation of nf-κb, an exact tis7-dependent pathway regulating myod levels and thereby myogenesis remained thus far unknown. though there is indication that transcriptional regulation of myogenic regulatory factors myod, myf and myogenin depends on the enzymatic activity of several histone modifying enzymes, such as protein arginine n-methyltransferase, the mechanism regulating the particular choice of prmt substrate in adult myogenesis is yet to be identified. prmt was found in the methylosome complex with the protein icln which, depending on its interacting partners, is also involved in transcriptional regulation. icln was shown to be responsible for the prmt substrate specificity. however, whether icln levels correlate with the prmt activity and mainly how they are regulated in the process of myogenesis, remains to be studied. herein, we investigated, at the molecular level, how tis may affect skeletal muscle regeneration and the regulatory processes of myoblast differentiation, in particular, the exact mechanism by which tis regulates myod levels. first of all, we identified that the tis protein binds directly to dna, which substantially clarifies its role in transcriptional regulation. we confirmed by several independent techniques the physical interaction between the tis protein and regulatory elements of the icln gene and even quantitatively determined the relationship between tis binding and icln transcriptional regulation. next, we clarified the link between prmt and regulation of myod expression by showing that icln levels determine binding of prmt and thereby its methyl transferase activity on histone h arg on particular gene locus, in this case myod. finally, through in vivo experiments, we documented that the ectopic expression of either tis icln, or myod in mscs derived from tis ko mice rescued their diminished differentiation potential. on the basis of our current data, we propose that tis participates in the mechanism of epigenetic regulation of myogenesis via a novel player icln, in particular by its transcriptional regulation.  tis controls myod levels through epigenetic regulation of gene expression myo5d and mef families of transcription factors are key regulators of skeletal muscle differentiation. we and others have previously shown that myod levels in myoblasts depend on tis expression. although the levels of several proteins related to myogenesis regulation have been shown to be affected by the presence or absence of tis, the explicit mechanism by which tis regulates myod expression is yet to be identified. therefore, we decided to first analyze the ability of ectopic tis expression to restore myod mrna and protein levels in proliferating tis ko mscs. consistent with previous data, the loss of tis caused a significant downregulation of myod mrna levels, which was restored upon ectopic expression of tis in ko mscs. western blot analyses of myod showed that myod d protein levels were significantly reduced in tis ko mscs and significant rescue was achieved by tis ectopic expression.fig. tis epigenetically regulates myod expression. a tis ko mscs were transfected with indicated plasmids. quantitative pcr analysis of myod was normalized to gapdh expression. values represent the mean ± sem and resulting data were analyzed via two-tailed type student’s t-test, *** p <  and * p < . b proliferating tis ko mscs were transfected with indicated plasmids and total cell lysates were probed with myod and control antibodies. quantification was performed by normalization to the tubulin signal. values represent the mean ± sd and resulting data were analyzed via two-tailed type student’s t-test, *** p <  and ** p < . representative western blots are shown. chip analyses using chromatin from tis wt or ko mscs transfected with either gfp or gfp-tis plasmid dna. c schematic outline of mouse myod gene locus. primer pairs used for chip assays are indicated by arrows. the alignment of human and mouse sequences amplified by the pcr is shown. antisera directed against symmetrically di-methylated histone h at arginine or prmt were used for immunoprecipitation. immunoprecipitated dna was analyzed by qpcr in triplicates using primers specific for the mouse myod regulatory region. chip with control rabbit igg or  % of total chromatin were used as controls. signals were normalized to input chromatin and shown as percentage of input, ** p ≤  and *** p <  tis regulates myod expression by an epigenetic mechanism transcriptional co-regulator arginine methyltransferase prmt was identified to be necessary for proper myogenesis in zebrafish embryonic development, where it specifically regulates myod expression. in adult mice, prmt inactivation abrogated skeletal muscle regeneration, which resembled the tis ko mouse phenotype reported by us. therefore, we decided to investigate whether the prmt5-dependent epigenetic control mechanism of myod expression is affected by tis ko. we first tested the presence of arginine dimethylated histone h on the myod gene. for this purpose, we performed chromatin immunoprecipitation using specific anti-histone h antibodies followed by quantitative pcr detection of the myod regulatory region. our data showed that there was a significant increase in symmetrical dimethylation of histone h on myod in tis ko when compared to wildtype mscs, although the total amounts of histone h were comparable. the increase in this modification, which is generally related to transcriptional inhibition, explained reduced myod expression in tis ko mscs. importantly, the ectopic expression of tis in ko mscs significantly reduced symmetrical dimethylation of histone h on myod to levels comparable with the wt mscs. next, we analyzed by chip whether this difference was directly dependent on prmt recruitment to the myod regulatory region. as expected, prmt occupancy on the myod gene was significantly higher in tis ko when compared to wt mscs. as in the case of h methylation, we found that the ectopic expression of tis significantly reduced prmt binding to myod gene in ko mscs. icln expression is downregulated in tis ko mscs to understand how tis may affect prmt5-specific function in myogenesis we analyzed the effect of ectopic expression of a known regulator of prmt namely icln in tis wt and ko mscs. icln protein forms a complex with prmt that regulates its substrate specificity. therefore, we analyzed whether prmt epigenetic regulation of myod transcription was related to icln levels. under an identical experimental setup as described above, we analyzed chromatin isolated from tis wt and ko mscs, transiently transfected either with control yfp or with a yfp-icln construct. immunoprecipitation with both anti-histone h and anti-prmt antibodies revealed an increase in arg dimethylated histone h and prmt bound to myod regulatory elements in tis ko mscs, respectively. constitutive expression of icln significantly decreased the amount of symmetrically dimethylated h arg on myod regulatory elements; this effect could be explained either by increased prmt enzymatic activity or changes in its binding to chromatin. two different independent methylation assays unveiled that the presence of icln had no effect on total prmt enzymatic activity both in vitro and in vivo. we also could not detect any difference in subcellular distribution of prmt. however, our chip experiments revealed that the ectopic expression of icln substantially decreased the symmetrically dimethylated arginine histone h and prmt recruitment to myod regulatory elements. the level of chromatin-associated prmt in icln-complemented tis ko mscs was similar to that observed in wt cells. next, we checked whether icln ectopic expression in tis ko mscs rescued the myod expression. as shown in fig. 2c, the ectopic icln expression rescued myod protein levels due to increased myod transcription, as documented by elevated myod mrna. thereafter, we asked ourselves whether prmt5-mediated methylation of the myod regulatory region is solely responsible for tis7/icln-mediated regulation of myod transcription. therefore, we generated, by lentiviral transduction and antibiotic selection, a stable and specific prmt knockdown in tis ko mscs. qpcr analysis of myod levels in proliferating tis ko mscs did not show any difference between control sh gfp and sh prmt expressing cells. however, after 7 days in differentiation medium, at a time point when wt tis mscs fully differentiate, we identified an 80 % increase in myod rna levels of sh prmt5-expressing tis ko mscs. based on these results we concluded that prmt5-dependent methylation of histone h bound to myod regulatory elements is not the main regulatory mechanism in proliferating tis deficient cells, suggesting that other tis7-dependent mechanisms may contribute to this regulation. however, prmt5-regulated methylation significantly affected myod expression during the differentiation of mscs.fig. icln affects in vivo methylation of histone h and binding of prmt to myod locus, resulting in increased myod rna and protein levels. chip analysis using chromatin from tis wt or ko mscs transfected with either yfp or yfp-icln plasmids. antisera directed against symmetrically dimethylated histone h at arginine or prmt were used for immunoprecipitation. immunoprecipitated dna was analyzed in triplicate by qpcr using primers to amplify the mouse myod regulatory region. chip with control rabbit igg or  % of total chromatin were used as controls. signals were normalized to input chromatin and shown as percentage of input. ** p ≤  and *** p < . c tis ko mscs were transfected with indicated plasmids and total cell lysates were probed with anti-myod and anti-yfp antibodies. quantification was performed by normalization to the tubulin signal. representative immunoblots are shown. d tis ko mscs were transfected with the indicated plasmids. quantitative pcr analysis of myod was normalized to gapdh expression. e prmt knockdown significantly increased myod rna levels in tis ko mscs transduced with sh prmt5-expressing lentivirus. myod rna levels were measured by qpcr and prmt protein levels were documented by immunoblots with indicated antibodies we thus conclude that icln regulates myod expression in mscs by changing the epigenetic status of chromatin through functional interference with prmt the next question raised was whether there were significant differences in icln levels between tis ko and wt mscs. western blot analyses of several independent biological experiments revealed that icln protein levels were significantly reduced in both proliferating and differentiating ko mscs when compared to tis wt mscs. ectopic expression of tis restored the icln protein levels up to 90 % of the wt values, suggesting the role of tis in its regulation.fig. tis regulates icln protein and rna levels. a quantitative analyses of icln protein expression in proliferating and differentiating mscs. equal amounts of total cell lysates were probed by immunoblotting with anti-icln antibodies and normalized by tubulin. b tis ectopic expression rescued icln protein levels in tis ko mscs. proliferating tis ko mscs were transfected with gfp plasmid as a control or with gfp-tis plasmid constructs and total cell lysates were probed with the indicated antibodies. quantification was based on tubulin signal. representative western blots are shown. c quantitative pcr analysis of icln was normalized to gapdh expression. d tis overexpression rescued icln rna levels in tis ko mscs. proliferating tis ko mscs were transfected with indicated plasmids and icln quantitative pcr was performed as in panel c. e tis transcriptionally regulates icln levels. proliferating wt mscs were transiently co-transfected with full length icln promoter-luciferase reporter construct, β-galactosidase expression plasmid, and gfp plasmid as a control or gfp-tis plasmid constructs. luciferase values were normalized to β-galactosidase values. in all graphs, values represent the mean ± sd and resulting data were analyzed via two-tailed type student’s t-test, *** p < , ** p < , and * p <  we observed the same trend also in mrna levels where icln expression was approximately 50 % lower in both pm and dm tis ko mscs. similarly to protein, the level of icln mrna was significantly upregulated by tis ectopic expression, indicating possible tis involvement in icln transcriptional regulation. to test this, we transiently transfected wt and tis ko mscs with an icln promoter-driven reporter construct and measured the luciferase activities. these were reduced in tis ko mscs and efficiently rescued by ectopic expression of tis showing that the activity of icln promoter is dependent on tis since little is known about icln gene regulation, we applied luciferase assays using reporter constructs possessing various promoter truncations. these experiments revealed a 41 nt sequence representing a minimal essential region required for icln transcriptional activity in wt mscs.fig. a defined nucleotide stretch within the icln promoter represents the minimal region essential for tis7-regulated icln transcription. a proliferating tis wt mscs were transiently co-transfected with indicated icln promoter-luciferase reporter constructs and with β-galactosidase expression plasmid for transfection control. the activity of the wt full length icln promoter-driven luciferase reporter was set as 100 %. values represent the mean ± sem of at least three biological replicates. b activities of luciferase reporter constructs driven by two or four repeats from icln promoter. bars marked + tis represent samples co-transfected with gfp-tis plasmid vector. control samples were co-transfected with gfp plasmid. c tis binds to the minimal essential region necessary for the tis7-regulated transcription of icln. gst or gst-tis recombinant proteins were incubated with the fluorescently labeled oligonucleotide probe containing the –154/– sequence of the icln promoter. d specific tis binding to icln minimal essential region determined by surface plasmon resonance measurements. oligonucleotides representing either the icln mer or its scrambled version were incubated with increasing concentrations of gst-tis or gst alone as indicated. spr steady-state response values are presented as a function of protein concentration. kd = dissociation constant. e chip analysis of tis binding to the icln promoter region. chromatin from tis wt and ko mscs was immunoprecipitated as above using either specific antibody against tis or control rabbit igg tis directly binds to and activates icln promoter next, we tested whether this 41 nt sequence was sufficient to regulate icln expression in a tis7-dependent manner. we generated a luciferase reporter construct, which contained two repeats of the 41 nt sequence of the icln promoter upstream of the luciferase gene. wt mscs transfected with this construct displayed increased luciferase activity when compared to the empty pgl3b luciferase vector. this basal activity was significantly induced by co-transfection with tis. even stronger luciferase activity was found when four repeats of the 41 nt sequence were cloned upstream of the luciferase gene. these data demonstrated the functional relevance of this minimal essential region for tis7-mediated activation of icln transcription. using bioinformatics prediction software, bindn+, dp-bind, and dnabindr; additional file 2: figure s2) we identified, with 70–80 % probability, three distinct dna-binding domains in the tis protein. this prediction suggested that the tis protein may directly bind dna. we tested this possibility by an electrophoretic mobility shift assay using an oligonucleotide probe containing the sequence of the icln promoter. as shown in fig. 4c, we detected a specific band representing a complex of gst-tis with a bound fluorescently labeled oligonucleotide probe. the specificity of this complex was confirmed by the fact that this complex was not formed when the probe was incubated with gst alone. additionally, tis binding to the labeled probe was specifically blocked by pre-incubation with a 20-fold access of a non-labeled “cold” oligonucleotide. the presence of tis in this dna complex was further confirmed by pre-incubation with a tis7-specific antibody, which caused a “supershift”, i.e., further slowed the mobility of the complex on the native polyacrylamide gel. in addition, we have quantified the specific binding of tis to this dna oligonucleotide using surface plasmon resonance. the measurements showed that the protein binding moiety is specific and the tis7-binding was sequence specific because there was a 10-fold difference in kd values between the native icln promoter and “scrambled” sequences. using chip analysis, we investigated whether tis binds a native icln promoter in mscs. we found that tis was bound to the icln promoter in tis wt but not ko mscs. the specificity of tis binding was confirmed using control rabbit igg. based on the results of these analyses we conclude that tis directly binds icln promoter dna and thereby positively regulates icln transcription in msc cells. tis regulates myotube formation through icln and myod expression to understand how tis regulates skeletal muscle differentiation, we characterized in detail the morphological differences between primary wt and tis ko mscs. in general, proliferating mscs could be induced to differentiate into mature myotubes within 5– days. although plated at the same density, while 70 % of wt mscs differentiated into myotubes, only 30 % of tis ko mscs formed myotubes. this defect was completely restored by ectopic tis expression. importantly, the ectopic expression of myod in tis ko mscs led to full recovery of the differentiation deficit and restored myotubes formation in these cells. because of tis7-dependent regulation of icln we investigated whether icln can act downstream of tis to regulate myogenesis. ectopic icln expression in tis ko cells rescued myotube formation in tis ko mscs, similar to the effects of tis or myod overexpression.fig. msc differentiation is regulated by myod, icln, and tis a tis wt and ko mscs were plated at the same cell density. proliferating tis ko mscs were transfected with indicated plasmids and 6 h later the differentiation was induced. after d of differentiation, cells were fixed, stained with anti-myosin heavy chain antibody mf and the nuclei were visualized using hoechst. scale bar = 100 μm. b percentage of msc fusion was calculated as the number of nuclei localized in multinucleated myotubes defined by mf staining compared to the total number of nuclei. vertical bars denote the mean ± sd and were calculated from independent biological replicates. the indicated number of nuclei was evaluated. resulting data were analyzed via two-tailed type student’s t-test, *** p < . c quantitative analysis of cells with the indicated nuclei numbers per cell defined by myosin staining. this analysis showed a significant decrease in the number of multinucleated cells and a completely missing category of cells with more than nuclei in tis ko mscs when compared to tis wt mscs. ectopic expression of tis myod, or icln significantly increased the number of multinucleated cells, mainly those with more than nuclei in tis ko mscs when compared to tis wt mscs a more detailed quantitative analysis revealed that the majority of tis ko mscs remained mononucleated and the very large myotubes containing more than nuclei were completely missing in tis ko mscs. ectopic tis expression rescued the appearance of multinucleated cells, mainly the category of myotubes with more than nuclei. even more interestingly, the ectopic expression of myod reverted the distribution of different groups of multinucleated myotubes similar to that of tis wt mscs and recovered in tis ko mscs the missing group of very large myotubes with more than nuclei. similarly, the increased number of multinucleated cells and, specifically, the formation of very large myotubes with more than nuclei were observed in icln-transfected cells. myod as a crucial myogenic regulatory factor is responsible for the myoblast differentiation leading to myotube formation and, based on data shown here, we concluded that tis regulates icln transcription and thereby myod levels responsible for reversion of the tis ko phenotype in mscs. discussion in this study, we show that icln, a protein with multiple physiological functions that depend on its subcellular localization and mainly on its protein interacting partners, represents a novel regulator of skeletal myoblast differentiation. we identified that tis transcriptionally regulated icln via direct binding to its promoter and, secondly, that ectopic expression of icln rescued the differentiation deficiency of tis ko mscs. icln controlled muscle differentiation through the myod pathway since icln upregulated myod levels. our further results presented here provide evidence for the epigenetic regulation of myod transcription by icln. adult muscle regeneration depends on de novo myogenesis, which requires activation and commitment of quiescent satellite cells. muscle regeneration ability is regulated by and depends on the differentiation potential of mscs. this is assured by the interplay of mrfs myod, myogenin, myf and myf. myod and myf are required for myogenic determination, whereas myogenin and myf are markers of terminal differentiation. muscle damage induces msc proliferation followed by differentiation resulting in the formation of new muscle fibers. myod is required in both processes; however, myoblast differentiation initiation and progression is governed by an interplay of myod with other muscle-specific factors, e.g., myf and myogenin. myod ko mice indicate a unique requirement of myod during adult muscle regeneration rather than during embryonic development. we found that the myod regulation at this stage of myogenesis is tightly regulated by the tis7/icln/prmt axis. depletion of tis or reduced level of icln caused the epigenetic inhibition of myod leading to the general differentiation deficiency phenotype characterized by diminished myoblast fusion. these findings indicate that the maintenance of specific myod levels by the tis7/icln/prmt axis is critical for the induction and early progression of myoblast differentiation. it remains to be clarified whether this pathway is responsible for the regulation of other mrf genes. myogenic gene expression is affected by chromatin remodeling enzymes such as histone acetylases and deacetylases, histone lysine methyltransferases, swi/snf family of atp-dependent enzymes, and protein arginine methyltransferases. among them, prmt was identified in several independent studies as one of the critical epigenetic regulators of myogenesis. prmt was shown to control the expression of the mrfs myogenin, myod, and myf in zebra fish embryos. it was also shown that prmt is involved in transcriptional regulation of various other myogenic genes via symmetric arginine dimethylation of histones and components of the transcriptional machinery. prmt facilitates the recruitment of brg1-atpase, associated with swi/snf chromatin remodeling enzymes, and myod to the promoter of myogenin via h arg dimethylation resulting in upregulation of myogenin transcription. the same h arg modification by prmt exerts transcriptional repression effect on the p gene that facilitates msc proliferation and expansion, which has a positive impact on the process of muscle regeneration. as a part of the methylosome, prmt also methylates and facilitates the recruitment of sm proteins to the survival motor neuron complex leading to its activation. interestingly, the loss of survival motor neuron-specific activity is observed in primary myoblasts from patients with the autosomal recessive disorder spinal muscular atrophy. these myoblasts display a strong fusion deficiency, implying a primary muscle defect along with the well-described neuronal degeneration. we present here new evidence for a contrasting, but not conflicting, function of prmt in the regulation of myogenesis that is associated with inhibition of myod expression. prmt represses myod expression using a similar epigenetic mechanism described for p gene that is mediated through h3arg symmetrical dimethylation at the myod locus. this inhibits induction and early progression of msc differentiation into myotubes, which could represent the mechanism that together with p inhibition contributes to the maintenance of the proliferative potential of adult mscs. in this scenario, prmt would allow efficient accumulation of a critical msc mass that could be committed to differentiation upon receiving specific stimuli. the open question, however, remains how this prmtr function is controlled in mscs. it has been shown before that prmt5-specific cellular functions are regulated by its interacting partners. icln, the component of the methylosome complex, was shown to directly interact with prmt and change its substrate specificity. in the presence of icln, prmt methylation of sm proteins is stimulated, but methylation of histones is inhibited. consistent with these studies, we also observed that icln ectopic expression inhibited methylation of histone h in mscs. this was achieved via inhibition of prmt binding to the myod gene rather than changing prmt enzymatic activity. the recruitment of prmt to the regulatory elements of genes needs complex formation with dna-binding or chromatin-associated adaptor proteins. we propose that formation of such a complex on the myod gene is prevented by the interaction of prmt with icln. similarly, formation of an alternative prmt complex has been described for protein riok which competes with icln for binding to prmt. this would lead to diminished h arg methylation thereby de-repressing myod expression. icln has been found to stimulate prmt5-mediated sm protein methylation and at the same time to inhibit the nuclear function of prmt namely methylation of histones. silencing of icln was shown to cause α-motor axon degeneration, as seen in patients suffering from spinal muscular atrophy. there is a substantial body of knowledge on icln functions, its interaction with other proteins and subcellular localization. however, the regulation of icln gene expression remains to be solved. here, we found a mechanism related to myogenesis where tis transcriptionally activates icln through binding to the newly identified regulatory element in the icln gene. thus, we propose that myod expression in mscs is epigenetically regulated via icln/prmt downstream of tis this notion is supported by the following facts: deletion of prmt prevented msc differentiation, which was a phenotype strikingly reminiscent of tis ko mscs; prmt5-specific symmetrical dimethylation of h arg at the myod gene was enhanced in tis ko mscs; and tis or icln ectopic expression reduced h arg methylation at the myod gene and rescued the muscle differentiation deficiency in tis ko mscs. furthermore, overexpression of prmt alone in tis ko mscs did not rescue their fusion, which was consistent with the inhibitory role of prmt in msc-specific myod regulation.  as summarized in fig.  we propose here a novel model for tis function in myogenesis, where tis upregulates icln transcription in mscs via direct binding to the regulatory element in the promoter of this gene. increase in icln transcription leads to accumulation of icln protein in the cytoplasm and nucleus. the increased amount of icln protein inhibits prmt recruitment to the myod locus and prevents the methylation of histone h at arg decreased methylation de-represses myod expression, which is critical for msc differentiation. it is likely that prmt controls myod levels to balance the proliferation and differentiation potential of mscs. thus, the components of this pathway and mainly their regulation could be seen as targets for development of new therapeutic approaches for treatment of skeletal muscle diseases.fig. model of myogenesis regulation by the tis7/icln/prmt pathway. in skeletal muscles, tis transcriptionally regulates icln. the accumulated icln interacts with prmt and prevents its recruitment to chromatin-bound complexes on the myod gene. this regulates prmt5-mediated symmetrical dimethylation of histone h arg and myod expression. the regulated myod level affects the balance between proliferation and differentiation of mscs 
in particular, icln regulates myod expression via its interaction with prmt by an epigenetic modification that utilizes symmetrical di-methylation of histone h on arginine we provide multiple evidences that tis directly binds dna, which is a functional feature necessary for its role in transcriptional regulation. tpa induced sequence acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. we show that tis7/icln epigenetically regulate myod expression controlling protein methyl transferase activity.
 tpa induced sequence acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. we and others have shown that tis regulates adult myogenesis through myod, one of the essential myogenic regulatory factors.  here, we present data identifying icln as the specific, novel protein downstream of tis controlling myogenesis. we show that tis7/icln epigenetically regulate myod expression controlling protein methyl transferase activity. in particular, icln regulates myod expression via its interaction with prmt by an epigenetic modification that utilizes symmetrical di-methylation of histone h on arginine we provide multiple evidences that tis directly binds dna, which is a functional feature necessary for its role in transcriptional regulation.  we present here a molecular insight into tis7-specific control of myod gene expression and thereby skeletal muscle differentiation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tis7dna-bindingmethyl transferaseepigenetic regulationiclnmyodmyogenesishttp://dx.doi.org/ /501100002428austrian science fundp18531-b p22350-b12p24251-b20valovka taras vietor ilja issue-copyright-statement© the author 2016 