The cooperation of cis -elements during M-cadherin promoter activation

: M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis -elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. that of muscle kinase of culture conditions differentiation We also that GC and CDE Using ChIP assay, significant reduction of when

Cadherins are trans-membrane adhering proteins that connect neighboring cells in a Ca +2 -dependent manner (4,5). Among them, M-and N-cadherin are the major members expressed in the developing SKM cells and they allow myoblasts to recognize, and adhere to one another before fusing into multinucleated myofibers. The homotypic binding of extracellular domain from M-and N-cadherins ensures that fusion only happens between myoblasts (6).
Inter-cellular Cadherin-binding transduces signals into the nucleus to regulate the expression and function of MRFs and to promote cell cycle exit (7). The importance of Cadherins in myogenesis can be demonstrated by the finding that the fusion of myoblasts can be blocked by antibodies targeting the extracellular domain of either M-cadherin or N-cadherin (8)(9)(10)(11), and similar effect can also been achieved by incubating with peptides blocking the binding between M-cadherins on neighboring cells (12).
Although SKM cells express M-, N-, and R-cadherin, M-cadherin is the only musclespecific one and its expression is highly induced during myogenesis (13,14). We have demonstrated the strong activation of M-cadherin promoter by MyoD and other MRFs and this pathway might be responsible for its muscle-specific expression as all myogenic regulatory factors are muscle-specific (15). M-cadherin is moderately expressed in proliferating satellite cells and myoblasts, and its highest expression is observed during early myogenic differentiation when myoblasts adhere to fuse together, then, the expression declines in maturing myocytes, and similar phenomenon is observed in developing somites in utero (16,17).
The proximal promoter of M-cadherin contains 5 E-boxes and some other cis-elements, including GC-boxes and a conserved downstream element (CDE). E-boxes 3 and 4 have been found to mediate most of the activation effects by MyoD and the conserved downstream element (CDE) also plays important role during this process (15). The sequence context of CDE is predicted to be the target site of the heterodimeric factor Pbx/Meis, and the involvement of this factor in M-cadherin transcriptional activation has been demonstrated before (18). Pbx/Meis belongs to the TALE (three amino acid loop extension) subfamily of the homeodomain DNA-binding transcription factors and they are important cofactors regulating/modulating DNA-binding and transactivation of homeodomain transcription factors (19,20). A subset of myogenic genes, including Myogenin, are targeted by Pbx/Meis for initial relaxation of chromatin structure before recruitment of the MyoD for their transcription (18). Direct interaction on target sites between MyoD and Pbx/Meis has been examined (21,22).
It is of interest to elucidate how the Meis/Pbx recruited to CDE might cross-talk with MyoD or other MRFs bound to E-box to determine the chromatin for transcriptional activation.
In adult skeletal muscle, M-cadherin expression is restricted to the apical side of the myogenic stem cells (called satellite cells) that are located within the basal lamina of each myocyte (23). The expression of M-cadherin is activated after damage of skeletal muscle and the highest level is found in the fusing myoblasts. However, its expression is not induced by denervation that leads to SKM atrophy, implying its participation in the stem cells mediated regeneration (8,17,23). As M-cadherin is expressed in the proliferating myoblasts and quiescent satellite cells where most myogenic genes are not expressed, which implies the existence of a unique mechanism by which M-cadherin is activated but without inducing other myogenic genes participating in terminal differentiation.
Elucidating this M-cadherin-specific activation mechanism in myogenic precursors will certainly gain insight into the mechanism by which MyoD activates gene transcription and the acquirement of myogenic stem cell characteristics.
Therefore, a detailed analysis of the cooperation between cis-elements in the proximal promoter of M-cadherin was performed here. Then, the mixture was transferred onto cells (70-80% confluence) and the transfection was allowed to proceed overnight before the medium was replaced by fresh medium and incubated for another 24 hr before harvested for determining their luciferase activity using the Bio-Tek Clarity 2 luminometer. All reactions were done in triplicate and repeated at least 3 times.

Plasmids
The cloning of M-cadherin promoter of full length and proximal regions into pGL3basic vector has been reported before (15)

Chromatin immunoprecipitation (ChIP) assay
The detailed procedure of CHIP assay has been described in our previous works (26)(27)(28)

Quantitative RT-PCR (qRT-PCR)
The detailed protocol of qRT-PCR has been described in our previous works (29,30).
Briefly, cells and embryos were solubilized in Solution D and then, total RNA was extracted with series of phenol/chloroform mixture. Their cDNA was synthesized by the Superscript III kit (Invitrogen) according to the manufacturer's protocol. The

Western blot
The detailed protocol of Western blot has been described in our previous papers (29,31). Briefly, C2C12 cells were lysed in RIPA buffer containing protease inhibitors.
Total protein (50 μg) was resolved on SDS-PAGE gel, and then transferred onto PVDF membrane. After blocking with blocking buffer (PBS containing 0.5% Tween 20 and 5% milk), the membrane was hybridized by primary antibody at 4℃ overnight. HRPconjugated secondary antibodies were added into the membrane and incubated at room temperature for 1 hr. After washed with PBS, the HRP signal was detected by an enhanced chemiluminescence kit and viewed with X-ray film. Here is the list of  Results: Factors regulating M-cadherin are induced during myogenesis The proximal promoter of M-cadherin contains several predicted cis-elements, including 5 E-boxes, 2 GC-boxes, and a conserved downstream element (CDE; Fig. 1A and Supplementary fig. 1A). Among them, cis-elements around transcription initiation site (TIS) are highly conserved between mammals ( Fig. 1A & B). As Mcadherin expression is SKM-specific and highly induced during myogenesis, it is of interest to examine the expression of factors targeting its cis-elements during this process. This process can be recapitulated precisely in vitro by myoblast lines, such as C2C12 and Sol8, that are widely used for studying gene expression and other changes. was further induced in MT. SP1 is a ubiquitously expressed transcription factor and its expression was not much affected during myogenesis.
The expression of these factors was further examined in myogenesis of embryos of 11.5-12.5 dpc. As somitogenesis proceeds in the direction of rostral to caudal, tissues in the rostral part contain mature somites and those in the caudal part contain early somites and presomitic mesoderm; therefore, somites at various developing stages can be seen in the trunk of a single embryo. Here the embryos were separated into rostral, interlimb, and tail parts to represent somites from mature to early stages (Fig. 1D). We The cooperation of companion E-boxes with E4 was dissected with mutation of multiple E-boxes, and we found that double E-box mutants Mt1/2, Mt1/5, or Mt2/5 did not affect its activation but mutations of E-boxes 1/3, 2/3, 3/4, or 3/5 significantly  2D) than that in 10T1/2 fibroblasts, indicating a better working environment for MyoD in myoblasts. However, the requirement of multiple E-boxes for full activation and the importance of E3 and E4 boxes were conserved in both cell types. cells kept in growth medium but is gradually down-regulated during differentiation (33,34). We found that MyoD could highly activate the activity of both M-cadherin and Myogenin, but not MCK, promoters in cells kept in growth medium (Fig. 3A), demonstrating their lower sensitivity to the presence of Id. Consequently, cotransfection of E47 or expression of MyoD-E47 fusion protein did not further potentiate this activation. As expected, the activation of Mt1/2/5 was significantly lower than that of wildtype in GM (Fig. 3B).
In cells kept in differentiation medium we found that supplementation of E47 significantly enhanced MyoD mediated activation of M-cadherin, but not MCK and Myogenin, promoter (Fig. 3C); however, the activation of Mt1/2/5 was still significantly lower than that of wildtype. Fusion of MyoD and E47 into a single chimeric protein significantly enhanced the activation of MCK promoter, but only achieved an activation level lower than that of M-cadherin promoter triggered by MyoD and E47 cotransfection. These observations suggest that M-cadherin promoter has different requirement/sensitivity for E47 or other E-proteins before and after the initiation of terminal differentiation: before differentiation, M-cadherin promoter has low sensitivity to E47, but after the initiation of differentiation, it becomes highly sensitive to the presence of E47, and MyoD/E47 heterodimer efficiently formed and activates Mcadherin promoter with efficiency similar to that of MyoD-E47 chimeric protein.
Since supplementation of either E47 or MyoD-E47 failed to rescue the activation of Mt1/2/5 to the level of wildtype promoter (Fig. 3B), it confirms again that binding of MyoD/E47 heterodimer to only E3/4 boxes is not sufficient for full activation of Mcadhrin promoter and multiple E-boxes might cooperate to recruit multiple MyoD/E47 heterodimers to achieve higher activation.
E3-and E4-boxes play differential role during promoter activation Our previous study has shown that the E3 box in the M-cadherin promoter is a perfect target site of the Bhlhe40 protein (15), and studies from other groups also indicate that Bhlhe40 interferes with the dimerization of MyoD and E47, resulting in the downregulation of Myogenin, Mef2c, and myosin heavy chain (MHC) and prevention of myogenic differentiation (35). Therefore, it is of interest to test whether Bhlhe40 can still repress MyoD transactivation on mutant M-cadherin promoters. We found that Bhlhe40 mediated repression was highly dependent on the E3 as its mutation abolished this repression (Fig. 4A). On the contrary, E4 is essential to MyoD transactivation as it is abolished by E4 mutation. The differential dependence of MyoD and Bhlhe40 on E3 and E4 prompted us to ask whether their reciprocal mutation will enhance the activity of either transcription factor. When the sequence of E4 was changed to that of E3 in the reporter called double E3 (DB3), MyoD transactivation was significantly reduced and could be totally repressed by Bhlhe40 (Fig. 4B). Surprisingly, mutation of E3 to E4 (DB4) did not enhance but reduced MyoD transactivation, and at the same time the repression by Bhlhe40 was still reserved. Furthermore, the binding of both MyoD and Bhlhe40 to the promoter was increased in the DB3 but the binding of Bhlhe40 to DB4 probe was totally lost (Fig. 4C). These observations suggest that E4 is essential for MyoD transactivation but the presence of E3 is helpful, which is

CDE element is important for MyoD recruitment
Although Pbx1/Mes1 heterodimer does not seem to participate in the activation of transiently transfected M-cadherin promoter, its role in the epigenetic regulation of this promoter needs to be examined. The wildtype and ΔCDE promoter were cloned into the pStable-luc vector (Supple. Fig. 1B) that allowed integration of the reporter by G418 selection (27). Stable clones of C2C12 myoblasts carrying wildtype and ΔCDE reporters were generated and the recruitment of MyoD to reporters was examined by ChIP assay. Using a primer set specific to ectopic M-cadherin promoter, we found the recruitment of MyoD to chromatin packed ΔCDE reporter was sharply reduced as compared to that of wildtype reporter (Fig. 7C). This observation confirmed the importance of CDE element in the recruitment of MyoD to M-cadherin promoter, and it also suggests that the chromatin modifying function of Pbx1/Meis1 is critical to Mcadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.
To explore the involvement of CDE in M-cadherin expression in vivo, a CRISPER/dCcas9 approach was adopted. C2C12 myoblasts were over-expressed Over-expression of a single guide RNA (sgRNA) targeting sequence (+18~+36) immediately downstream of CDE can recruit KRAB-dCas9 to CDE to block its function (Fig. 8A). We found that M-cadherin was significantly repressed by KRAB-dCas9 in confluent myoblasts (Fig. 8B & C), demonstrating the importance of CDE element in M-cadherin transcriptional activation. Catenin accumulation under plasma membrane, but decreases Rac1-GTPase activity (11,37,38). Therefore, Rac1-and RhoA-GTPase might be the regulatory core mediating the compensative expression of these two genes. It will be interesting to examine if the promoters of both genes are affected directly by the signaling of Rac1and RhoA-GTPase.
Intriguingly, M-cadherin promoter activity was not affected in the C2-shPbx cells in transient transfection assays, probably due to lacks of proper chromatin structure.
However, stable clones of M-cadherin-ΔCDE-luc could not recruit MyoD efficiently to the ectopic M-cadherin promoter (Fig. 7), confirming that CDE recruited Mes1/Pbx1 plays key role in opening up chromatin structure for subsequent binding of MyoD/Myf5 in satellite cells and myoblasts. The CDE function in vivo was also demonstrated by the sgRNA recruited Krab-dCAS9, in which M-cadherin was highly repressed (Fig. 8). It will be interesting to know whether Krab-dCAS9 recruited to other cis-elements, such as E3 and E4, can have the same effects.

Collaboration and substitution between E-boxes
The myogenic bHLH domain targets MRFs to the class II (tissue-specific) E-boxes (consensus sequence: CANNTG) in the control regions of most, but not all, musclespecific genes (39,40). Multiple E-boxes are found in the promoters of many myogenic genes and their collaboration and substitution during transcriptional activation is an issue that has not been resolved.

The E-boxes 3 and 4 in M-cadherin promoter have been identified as the key ciselements mediating its activation by MyoD in our studies. Mutation of E3 and E4
reduced the activation to 40% and 20%, respectively (Fig.2), indicating differential roles played by either E-box and can not be substituted by each other. E3 box is targeted by the repressor Bhlhe40 and its repressing effect was indeed relieved when E3 was mutated. As E4 was targeted by MyoD, it prompted us to change the sequence of E3 to that of E4 in the hope that it will increase the activation by MyoD. In contrary to our expectation, the activation of DB4 was lower than that of wildtype promoter and it was still repressible by Bhlhe40 (Fig. 4B). The lower activation might be explained by the weaker binding affinity by MyoD and it suggests a novel sequence requirement for MyoD binding to target site, in which a Bhhle40 site and a MyoD site form an optimal activation/binding site for MyoD. Although DB3 increased the binding of both Bhlhe40 and MyoD, its activation by MyoD was very poor, indicating the irreplaceable role of E4 (Fig. 4C) and an optimal binding affinity is requirement for higher activation. .

Participation of E-proteins and other bHLH factors
As the expression of Id is high in proliferating myoblasts but is low in differentiating myoblasts (33,34), the formation of functional heterodimers between MyoD and E proteins in cells kept in GM should be difficult than those kept in DM. Therefore, it is expectable that the activation of M-cadherin promoter by MyoD was much higher in DM than in GM and supplement of E47 only showed significant effect in DM (Fig. 3).
Fusion of MyoD and E47 bypassed the inhibiting effect of Id, so higher activation could be seen in GM. However, in DM, MyoD~E47 fusion protein achieved similar activation as MyoD/E47 co-transfection, indicating that heterodimerization is as efficient as both factors are fused.
It was a surprise to find that, even with MyoD~E47, the activation of M-cadherin promoter in GM was still much lower than that in DM (Fig. 3 in myogenic determination and that should be the next priority in this field. There are 4 Pbx isoforms (Pbx1~4) in mammals and the expression of Pbx1~3 can be found in most embryonic tissues but with that of Pbx4 found majorly in testes. The Pbx1 -/homozygous mutant embryos die in utero with dramatic abnormality in multiple organs, while no detectable phenotype can be found in Pbx2 -/homozygous mutant mice.
These Pbx genes knockout studies suggest that distinct roles might be played by different Pbx isoforms during embryogenesis and their functional redundancy is limited. Actually, in C2C12 myoblasts, Pbx1 showed the strongest expression and followed by that of Pbx2 and -3, and much weaker level of Pbx4 was found (Supple. fig. 2B), indicating that Pbx1 is the major isoform in C2C12 myoblasts.
However, the involvement of other isoforms in myogenesis remains to be determined.
In the near future, isoform-specific shRNA or KRAB-dCas9 mediated gene knockdown approach should be employed to specifically reduce the expression of each isoform so their function in myogenic gene activation can be revealed.