Key master regulators of co-expressed genes

Using advantages of the design of the experiment allowing time-series style analysis, we identified master regulators and key-nodes (master regulators with positive feedback loop) from clusters of co-expressed genes and reconstructed diagrams of interactions between master regulators, transcription factors, and target genes for the top five master regulators [8].

We found that the main regulatory clusters were similar in both slow and fast muscles, but several unique patterns were discovered for each muscle type. In slow muscle, clusters were governed by regulators of proteasomal degradation and cell cycle regulators.

In particular, the most of regulators in slow muscle correspond to several E3 ubiquitin ligases (MAFbx/atrogin 1, Parkin) and components or regulators of SCF ubiquitin ligase complex (CUL1, SKP1, DDB1, ROC1, Ubs14) which are known signatures of disuse muscle atrophy in mammals as well as in humans [8,9]. In the fast muscle, proteasomal degradation and cell cycle regulators also played an important role, but the master regulators, transcription factors and target genes were different.

Unlike the slow muscle, calpain-2, for example, acts as one of the crucial master-regulators in fast muscles. Calpains are the members of calcium-dependent proteolytic system which along with ubiquitin-proteasomal system is an important player in muscle atrophy mechanism ((Taillandier et al. 1996; Murphy 2010; Huang and Forsberg 1998);

To summarize, using the atlas and time-series design of analysis we demonstrated that several genes that were known for their involvement in muscle biology act as master regulators in genetic response to atrophy.

Slow muscle (soleus)

Among key master-regulators that we identified from co-expressed genes clusters, genes encoded proteins involved in proteasomal degradation were overrepresented (Figure 6 ). In particular, the most of regulators in slow muscle correspond to several E3 ubiquitin ligases, among which there is a SCF family member Muscle atrophy F-box protein (MAFbx/atrogin 1). MAFbx together with another E3 ubiquitin ligase Muscle-specific RING finger protein 1 (MuRF1) are known signatures of disuse muscle atrophy in mammals as well as in humans [8,9]. In this cluster one of the most common key master-regulators is cullin-1 (CUL1). Cullin-1 is a major scaffold protein of SCF ubiquitin ligase complex, through SKP1 it binds to various F-box proteins, thus generating different types of muscle-specific ubiquitin ligases ( [36] [37]). In mammals CUL1 as well as other elements of SCF complex involve in ubiquitination of target substrates for subsequent degradation by 26S proteasome system ( [38,39];. Moreover, by ChIP-seq analysis it was shown that CUL1 indirectly interacts with DNA and can repress transcription of c-Myc-associated genes involved in RNA splicing and mitochondria regulation [40]. Among other master regulators SKP1 (S-phase kinase-associated protein 1) and DDB1 (DNA damage-binding protein 1) are adapter proteins providing binding cullin and F-box proteins thereby recognizing substrate for ubiquitination. ROC1 is also part of the SCF complex and appears as a regulator of cullin. In particular, ROC1 interacts with CUL1, indirectly promoting its nuclear accumulation by NEDD8 modification in HeLa cell culture [41]. Parkin is another ubiquitin-ligase which is involved in the elimination of defective mitochondria (mitophagy). Particularly, in skeletal muscles parkin plays an important role in maintenance of the mitochondrial function during endurance training [8,42].

Figure 6. Proteasomal cluster, soleus muscle. Network of top 5 master regulators. See SF for complete view with intermediate signal transducers shown

The second identified cluster (Figure 7) resembles mixing of some regulatory modules. One of them is fairly reasonable and comprises components of proteasomal degradation pathway like 26S proteasome as well as downstream PSMA7, PSMC2 and PSMD4 targets. In this context, an activation of the pathway in the course of disuse is obvious and consistent with the function of the above-described cluster. Other master-regulators relevant to the multiple signal transduction pathways via protein tyrosine kinase-phosphatase activities (CK1a, SHP-2 and Grb2), may have alternate and opposite roles in the process of the muscle disuse and recovery in the meantime. It is intriguing that increase of their expression in the process of disuse may trigger active programmed cell death leading to the loss of muscle fibers or promotes muscle growth via regulatory activation of cells division and proliferation [43–45]. We hypothesize that continuing increase of their expression during the first three days of recovery is apparently a residual phenomenon which switches to the significant decrease of regulators expression at the last day of the experiment.

Figure 7. Proteasomal degradation (regeneration) cluster, soleus muscle. Network of top 5 master regulators. See SF for complete view with intermediate transducers of proteasomal degradation signal shown

In alternative cell cycle-related third cluster in slow muscle (Figure 8) structural elements of an ubiquitin-ligase complex are still dominated. Notably, a deubiquitinating enzyme Ubs14, regulating proteasomal degradation and autophagy, is activated and raised up its expression all recovery period. Usp14 inhibits proteasomal activity, by removing ubiquitin chains [46]. MKP1 is a phosphatase that mediates dephosphorilization of MAP kinase 1, which is activated in skeletal muscle of obese humans and in high-fat diet-fed mice [47].

Figure 8. Alternative cell cycle related cluster, soleus muscle. Network of top 5 master regulators. See SF for complete view of intermediate transducers of cell cycle regulation signal

Fast muscle (EDL)

In EDL muscle, proteasomal degradation and cell cycle regulators also played an important role, but the master regulators, transcription factors and target genes were different

Unlike slow muscles, in fast muscles another regulatory program is undergoing. According to the first cluster (Figure 9), calpain-2 acts as one of the crucial master-regulators in slow muscles. Calpains are the members of calcium-dependent proteolytic system which along with ubiquitin-proteasomal system is an important player in muscle atrophy mechanism. It has been suggested that calpains induced myofibrillar disassembly, releasing actin and myosin from myofibrillar complex [48]. Data concerning the mRNA expression of calpain isoforms in disused skeletal muscles is fairly controversial. In several studies it has been reported that the mRNA expression of calpain-2 is upregulated in fast twitch muscles, such as m. EDL during sepsis [11] and m. tibialis anterior during hindlimb unloading [10] . Alternatively, in slow muscle, mRNA expression of calpain-2 did not significantly change in response to different types of disuse [49,50]. Moreover, it has been suggested that calpains can have a specific role in skeletal muscle which is the prevention of major degradation by reducing Ca2+ release from the sarcoplasmic reticulum [51]. This peculiarity of the protein family may complicate the functional role of calpains as master-regulators in the skeletal muscle.

Figure 9. Cell cycle cluster, EDL muscle.. Network of top 5 keynode master regulators. (See SF for complete view with intermediate nodes shown)

Overall, the master-regulators of proteasomal cluster in slow muscles (Figure 10) are similar to those in soleus muscle. However, Ubc12 is a distinctive element which relates to modification by tagging ubiquitin-like protein NEDD8. Neddylation is analogous to the ubiquitination process and involves controlling activity of cullin-RING-E3 ligases [52]. Meanwhile, Ubc12 provides neddylation of cullin proteins [53].

Figure 10. Proteasomal degradation cluster, EDL muscle. Top 5 master regulators and their effectors (intermediate nodes not shown). See SF for full top 5 diagram including intermediate nodes

SHP-2 is known protein tyrosine-phosphatase implicated in signal transduction and processes controlling cell proliferation, cell survival and cell adhesion. It’s proven that SHP-2 is necessary for skeletal muscle growth in mice postnatal development [54]. Moreover, SHP2 is involved in insulin signaling as a positive regulator and its protein expression increased during acute exercise and short-term training [55]

Figure 11. Alternative cell cycle regulators cluster, fast muscle. Top 5 master regulators and their effectors (intermediate nodes not shown). See SF for full top 5 diagram including intermediate

Cell cycle keynode regulators had only 19 features in common while 53 features were specific to each of the muscles (Figure 12, see ST14):

On the contrary, proteasomal clusters had more features in common (40) than specific to muscles (36 for EDL and 33 for soleus, Figure 13, see ST15): Surprisingly, clusters of alternative cell cycle regulation had only one feature in common between EDL and soleus - DUSP14. It is also notable that EDL cluster was significantly smaller and had only 4 unique features - 2 Ubc5A isoforms and 2 PP1β (Figure 14, see ST16)