Cooperation between myogenic regulatory factors and SIX family transcription factors is important for myoblast differentiation

Precise regulation of gene expression is crucial to myogenesis and is thought to require the cooperation of various transcription factors. On the basis of a bioinformatic analysis of gene regulatory sequences, we hypothesized that myogenic regulatory factors (MRFs), key regulators of skeletal myogenesis, cooperate with members of the SIX family of transcription factors, known to play important roles during embryonic skeletal myogenesis. To this day little is known regarding the exact molecular mechanism by which SIX factors regulate muscle development. We have conducted a functional genomic study of the role played by SIX1 and SIX4 during the differentiation of skeletal myoblasts, a model of adult muscle regeneration. We report that SIX factors cooperate with the members of the MRF family to activate gene expression during myogenic differentiation, and that their function is essential to this process. Our findings also support a model where SIX factors function not only ‘upstream’ of the MRFs during embryogenesis, but also ‘in parallel’ to them during myoblast differentiation. We have identified new essential nodes that depend on SIX factor function, in the myogenesis regulatory network, and have uncovered a novel way by which MRF function is modulated during differentiation

Cooperation between myogenic regulatory factors and SIX family transcription factors is important for myoblast differentiation

Precise regulation of gene expression is crucial to myogenesis and is thought to require the cooperation of various transcription factors. On the basis of a bioinformatic analysis of gene regulatory sequences, we hypothesized that myogenic regulatory factors (MRFs), key regulators of skeletal myogenesis, cooperate with members of the SIX family of transcription factors, known to play important roles during embryonic skeletal myogenesis. To this day little is known regarding the exact molecular mechanism by which SIX factors regulate muscle development. We have conducted a functional genomic study of the role played by SIX1 and SIX4 during the differentiation of skeletal myoblasts, a model of adult muscle regeneration. We report that SIX factors cooperate with the members of the MRF family to activate gene expression during myogenic differentiation, and that their function is essential to this process. Our findings also support a model where SIX factors function not only ‘upstream’ of the MRFs during embryogenesis, but also ‘in parallel’ to them during myoblast differentiation. We have identified new essential nodes that depend on SIX factor function, in the myogenesis regulatory network, and have uncovered a novel way by which MRF function is modulated during differentiation

Six1 Regulates MyoD Expression in Adult Muscle Progenitor Cells

<div><p>Quiescent satellite cells are myogenic progenitors that enable regeneration of skeletal muscle. One of the early events of satellite cell activation following myotrauma is the induction of the myogenic regulatory factor MyoD, which eventually induces terminal differentiation and muscle function gene expression. The purpose of this study was to elucidate the mechanism by which MyoD is induced during activation of satellite cells in mouse muscle undergoing regeneration. We show that Six1, a transcription factor essential for embryonic myogenesis, also regulates MyoD expression in muscle progenitor cells. Six1 knock-down by RNA interference leads to decreased expression of MyoD in myoblasts. Chromatin immunoprecipitation assays reveal that Six1 binds the Core Enhancer Region of <i>MyoD</i>. Further, transcriptional reporter assays demonstrate that Core Enhancer Region reporter gene activity in myoblasts and in regenerating muscle depends on the expression of Six1 and on Six1 binding sites. Finally, we provide evidence indicating that Six1 is required for the proper chromatin structure at the Core Enhancer Region, as well as for MyoD binding at its own enhancer. Together, our results reveal that MyoD expression in satellite cells depends on Six1, supporting the idea that Six1 plays an important role in adult myogenesis, in addition to its role in embryonic muscle formation.</p></div

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

peer reviewedLong-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions

Chromatin structure and MyoD binding at the CER depend on Six1 function.

<p><b>A)</b> Genomic binding profiles of Six1 in primary myoblasts (this study), and of MyoD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067762#pone.0067762-Cao1" target="_blank">[²²]</a>, H3K4me1 and mononucleosomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067762#pone.0067762-Asp1" target="_blank">[⁴⁹]</a> in C2C12 myoblasts. The position of the MEF3 sites and of three E-boxes (CANNTG) is also shown. <b>B)</b> Over-expression of MyoD in HEK293T cells leads to activation of the CER+PRR luciferase reporter gene. The results are reported over the luciferase activity obtained with an empty expression plasmid, and reflect the average of three independent replicates. Asterisk, p<0.05 by one-tailed paired t test. <b>C)</b> Schematic representation of the PCR strategy used to distinguish the endogenous CER and the exogenous CER+PRR transgene. Primers a and b together can only amplify the endogenous CER sequences, while primers c and d will only give an amplification product on the CER+PRR reporter gene, since the endogenous CER and PRR are separated by more than 20 kb of sequence. The white bars in the CER represent the two Six1 binding sites. <b>D) and E)</b> ChIP assays performed on chromatin from stable polyclones of the CER+PRR reporters (wild-type or double MEF3 sites mutant). <b>D)</b> The real-time PCR quantities of the endogenous CER (primers a+b) were expressed as percentage of input chromatin. The level of binding of Six1, MyoD, H3K4me1 and H3 on the endogenous CER is not significantly different in wild type and mutant polyclones. Because the H3K4me1 signal is very high, we divided the values plotted for this mark by 100. <b>E)</b> The real-time PCR quantities of the transgene (primers a+c) were expressed as percentage of input chromatin and normalized over the quantities of the endogenous CER locus (primers a+b, used as internal control). Enrichment at a control locus (not targeted by Six1 or MyoD) is also given, in each set of polyclones. The results are reported as a fraction of the enrichment obtained on the wild-type CER+PRR construct. The inset shows the enrichment of Six1, MyoD, H3K4me1 and H3 as percentage of input on the wild type transgene. n = 3 replicates for each reporter gene construct; bars: S.E.M. Asterisks, p<0.05 by unpaired two-tailed t test. <b>F)</b> Western blot showing the levels of Six1 and MyoD in C2iFRT-FL-MyoD cells transfected with the control (siNS) or Six1 knock-down (siSix1) siRNA duplexes, and treated or not with doxycycline to induce MyoD expression. Beta-tubulin is shown as loading control. <b>G)</b> ChIP assays performed on chromatin isolated from C2iFRT-FL-MyoD cells treated as in F. The enrichment is shown as percent of input chromatin. Because the H3K4me1 signal is very high, we divided the values plotted for this mark by 100. The Lrp5 locus serves as a control locus not targeted by Six1 or MyoD. Asterisks indicate p<0.05 by one-tailed paired T test.</p

Six1 directly binds to the core enhancer region of MyoD at two conserved MEF3 sites.

<p><b>A)</b> Profile of genomic binding of Six1 in primary myoblasts in growth phase, showing binding at the CER. The read density is expressed as reads per million mappable reads in bins of 25 base pairs above the read density in the input sample. The signal obtained with a non-specific antibody (non-immune rabbit IgG) is shown for comparison. <b>B)</b> Conventional gene-specific ChIP assays followed by real-time PCR were used to confirm the binding of Six1 to the CER, in proliferating myoblasts. n = 3 biological replicates (independent chromatin preparations). By one-tailed paired t test, the signal for anti-Six1 at the CER is significantly above that obtained on the negative control locus, and above that obtained at the CER with normal rabbit IgG, with p<0.05. Error bars, S.E.M. <b>C)</b> Sequences of the two MEF3 sites identified within the mouse core enhancer. The murine MyoD gene is on the+strand; the reverse-complement of site #1 is shown. Conservation across mammalian species is shown, along with the mutations created in the EMSA probes and reporter constructs. Positions in small script indicate divergent sequences using mouse as reference. Dots indicate sequence not shown; dashes indicate missing sequences in certain species. <b>D)</b> Direct binding of Six1 to the CER, shown by EMSA experiments using recombinant Six1 protein incubated with a wild-type CER probe, or with versions mutated at either or both MEF3 sites identified. Specificity of binding was assessed by competition with a 50-fold molar excess of unlabelled myogenin MEF3 site oligonucleotides, either wild-type sequence or mutated.</p

CER reporter constructs depend on Six1 expression and on Six1 binding sites for maximal activity.

<p><b>A)</b> Schematic representation of the reporter constructs used for these experiments. The backbone for luciferase is pGL3-Basic, while that for LacZ is p1230. <b>B)</b> The murine CER+PRR LacZ construct drives reporter gene expression at the expected locations, in E11.5 transgenic founder mouse embryos. ii) The white arrowhead points to the dorsal part of the somites, with enhanced reporter activity. The black arrowhead points to the ventral part of the myotomes. fb, forelimb bud. ii) Forelimb bud signal on a different embryo. Signal is often seen in the hindlimb bud as well, on other embryos (not shown). iii) A cross-section at the inter-limb level reveals that the ventral signal seen in i) comes from the myotome (mt). Non-specific transgene expression is also detected in the neural tube (nt). <b>C)</b> CER enhancer activity depends on Six1 expression, as shown by promoter reporter assays performed in C2C12 myoblasts transfected first with the indicated luciferase reporter plasmids, and 24 hours later with the indicated siRNAs: control non-silencing or targeting Six1. The normalized luciferase activity readings are reported as fold over the numbers obtained with the empty pGL3-Basic plasmid and non-silencing siRNA. Bars represent the average of 3 biological replicates; error bars, S.E.M. Asterisks indicate significance (p<0.05) by two-tailed paired t test. <b>D)</b> CER enhancer activity depends on Six1 binding sites, as shown by reporter assays performed in primary myoblasts and myotubes. Myoblasts were transfected with the indicated reporter constructs, and either harvested as myoblasts or induced to differentiate for 48 hours prior to harvest. For comparison, the effect of MEF3 site mutation is also shown for the myogenin promoter. In each case, reporter activity is reported as fraction of the activity of the wild-type reporter. n = 3, asterisks indicate significance by two-tailed paired t test (p<0.05). Error bars, S.E.M. <b>E)</b> CER enhancer activity increases in regenerating muscle and this depends on the Six1 binding sites. Wild-type or MEF3-mutated reporter constructs were injected and electroporated in uninjured TA muscles, or in TA muscles 3 days post-injury by cardiotoxin injection. The transfected tissues were harvested 4 days later for luciferase assays. Values reported are normalized luciferase readings for each individual mouse leg harvested (n = 8, each depicted by a different symbol). Significance of reporter activity differences was assessed by Wilcoxon rank-sum test, with p<0.05 as threshold.</p

Six1 is expressed in primary myoblasts and is necessary for MyoD expression.

<p><b>A)</b> Western blot on total protein lysates of primary myoblasts in growth phase (Mb), at confluence (T0), differentiated for 24 (T24) or 48 hours (T48). The antibodies used were anti-Six1, anti-MyoD and anti-myogenin. Anti-β-tubulin was used as a loading control. <b>B)</b> Western blot showing the expression of Six1, MyoD and GAPDH on total protein lysates of primary myoblasts in growth phase, 48 hours after their transfection with siRNA duplexes targeting Six1 (siSix1), or with a non-silencing siRNA (siNS). A low and a high film exposure are shown for the anti-MyoD western blot. Comparable results were obtained in three independent experiments.</p

Six1 is expressed along with Pax7 and MyoD in activated satellite cells of regenerating muscles.

<p><b>A)</b> Immunostaining of sections from paraffin-embedded resting TA muscles, using antibodies against Pax7 (green) and Six1 (red). Pax7-positive satellite cells are marked by blue arrowheads, while Six1-positive cells are marked by yellow arrows. Gamma settings were adjusted to 1.00 or 0.35 to increase the signal-to-noise ratio. DAPI was used as a counterstain to label nuclei. <b>B)</b> Immunostaining of frozen sections of resting TA, or muscles after 3 or 7 days following cardiotoxin injection, with antibodies against Six1 (red signal) and Pax7 (green signal). DAPI was used as a counterstain to label nuclei. <b>C)</b> Quantification of the anti-Six1 and anti-Pax7 staining signal in resting muscles and at 2, 3, 4 or 7 days post-injury, as shown in panel A. Bars indicate the average number of positively stained nuclei counted in 0.6 mm² fields of view, using 5 mice per condition. Error bars, S.E.M. <b>D)</b> Immunostaining performed in samples identical as in A, but using antibodies against Six1 and MyoD. Magnification as shown in A. <b>E)</b> Quantification of the anti-Six1 and anti-MyoD staining as shown in C.</p