An Unbiased Proteomic Platform for Activity-based Arginylation Profiling.

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general activity-based arginylation profiling (ABAP) platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. ABAP has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 µg sample input, with 229 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma.

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.

SMAD2 promotes myogenin expression and terminal myogenic differentiation.

SMAD2 is a transcription factor, the activity of which is regulated by members of the transforming growth factor ß (TGFß) superfamily. Although activation of SMAD2 and SMAD3 downstream of TGFß or myostatin signaling is known to inhibit myogenesis, we found that SMAD2 in the absence of TGFß signaling promotes terminal myogenic differentiation. We found that, during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGFß receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog.

CCAAT/enhancer-binding protein beta promotes muscle stem cell quiescence through regulation of quiescence-associated genes.

Regeneration of skeletal muscle depends on resident muscle stem cells called satellite cells that in healthy, uninjured muscle remain quiescent (noncycling). After activation and expansion of satellite cells postinjury, satellite cell numbers return to uninjured levels and return to mitotic quiescence. Here, we show that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPß) is required to maintain quiescence of satellite cells in uninjured muscle. We show that C/EBPß is expressed in quiescent satellite cells in vivo and upregulated in noncycling myoblasts in vitro. Loss of C/EBPß in satellite cells promotes their premature exit from quiescence resulting in spontaneous activation and differentiation of the stem cell pool. Forced expression of C/EBPß in myoblasts inhibits proliferation by upregulation of 28 quiescence-associated genes. Furthermore, we find that caveolin-1 is a direct transcriptional target of C/EBPß and is required for cell cycle exit in muscle satellite cells expressing C/EBPß. The induction of mitotic quiescence is considered necessary for the long-term maintenance of adult stem cell populations with dysregulation driving increased differentiation of progenitors and depletion of the stem cell pool. Our findings place C/EBPß as an important transcriptional regulator of muscle satellite cell quiescence.

CCAAT/enhancer binding protein ß is required for satellite cell self-renewal.

BACKGROUND: Postnatal growth and repair of skeletal muscle relies upon a population of quiescent muscle precursor cells, called satellite cells that can be activated to proliferate and differentiate into new myofibers, as well as self-renew to replenish the satellite cell population. The balance between differentiation and self-renewal is critical to maintain muscle tissue homeostasis, and alterations in this equilibrium can lead to chronic muscle degeneration. The transcription factor CCAAT/enhancer binding protein beta (C/EBPß) is expressed in Pax7+ satellite cells of healthy muscle and is downregulated during myoblast differentiation. Persistent expression of C/EBPß upregulates Pax7, inhibits MyoD, and blocks myogenic differentiation. METHODS: Using genetic tools to conditionally abrogate C/EBPß expression in Pax7+ cells, we examined the role of C/EBPß in self-renewal of satellite cells during muscle regeneration. RESULTS: We find that loss of C/EBPß leads to precocious differentiation at the expense of self-renewal in primary myoblast and myofiber cultures. After a single muscle injury, C/EBPß-deficient satellite cells fail to self-renew resulting in a reduction of satellite cells available for future rounds of regeneration. After a second round of injury, muscle regeneration is impaired in C/EBPß conditional knockout mice compared to wild-type control mice. We find that C/EBPß can regulate Notch2 expression and that restoration of Notch activity in myoblasts lacking C/EBPß prevents precocious differentiation. CONCLUSIONS: These findings demonstrate that C/EBPß is a novel regulator of satellite cell self-renewal during muscle regeneration acting at least in part through Notch2.

Induction of CCAAT/Enhancer-Binding Protein ß Expression With the Phosphodiesterase Inhibitor Isobutylmethylxanthine Improves Myoblast Engraftment Into Dystrophic Muscle.

UNLABELLED: Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common muscular dystrophy. Characterized by rounds of muscle degeneration and regeneration, DMD features progressive muscle wasting and is fatal. One approach for treatment is transplantation of muscle progenitor cells to repair and restore dystrophin expression to damaged muscle. However, the success of this approach has been limited by difficulties in isolating large numbers of myogenic progenitors with strong regenerative potential, poor engraftment, poor survival of donor cells, and limited migration in the diseased muscle. We demonstrate that induction of the transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) using the cyclic adenosine monophosphate phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) results in enhanced myoblast expansion in culture and increased satellite cell marker expression. When equal numbers of IBMX-treated cells were transplanted into dystrophic muscle, they contributed to muscle repair more efficiently than did vehicle-treated cells and engrafted into the satellite cell niche in higher numbers, demonstrating improved cell migration from the site of injury and enhanced survival after transplantation. Thus, pharmacologic stimulation of C/EBPß expression reprograms myoblasts to a more stem cell-like state, promotes expansion in culture, and improves engraftment such that better transplantation outcomes are achieved. SIGNIFICANCE: Duchenne muscular dystrophy is a genetic disorder for which no cure exists. One therapeutic approach is transplantation of myogenic progenitors to restore dystrophin to damaged muscle, but this approach is limited by poor engraftment of cultured myoblasts. Transient upregulation of CCAAT/enhancer-binding protein ß in primary myoblasts using the phosphodiesterase isobutylmethylxanthine (IBMX) increases satellite cell marker expression in cultured myoblasts, improves their migration, and increases their survival after transplantation. When transplanted into C57BL/10ScSn-mdx/J mice , IBMX-treated myoblasts restored dystrophin expression and were able to occupy the satellite cell niche more efficiently than controls. A myoblast culture approach that reprograms myoblasts to a more primitive state, resulting in improved transplantation outcomes and reinvigorating research into myoblast transplantation as a viable therapeutic approach, is described.

Mdm2 promotes myogenesis through the ubiquitination and degradation of CCAAT/enhancer-binding protein ß.

Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein ß (C/EBPß) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPß protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPß protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPß for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPß levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPß, suggesting that the regulation of C/EBPß turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPß for degradation by the 26 S proteasome.

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.

Interplay between positive and negative activities that influence the role of Bicoid in transcription.

The Drosophila mophogenetic protein Bicoid (Bcd) can activate transcription in a concentration-dependent manner in embryos. It contains a self-inhibitory domain that can interact with the co-repressor Sin3A. In this report, we study a Bcd mutant, Bcd(A57-61), which has a strengthened self-inhibitory function and is unable to activate the hb-CAT reporter in Drosophila cells, to analyze the role of co-factors in regulating Bcd function. We show that increased concentrations of the co-activator dCBP in cells can switch this protein from its inactive state to an active state on the hb-CAT reporter. The C-terminal portion of Bcd(A57-61) is required to mediate such activity-rescuing function of dCBP. Although capable of binding to DNA in vitro, Bcd(A57-61) is unable to access the hb enhancer element in cells, suggesting that its DNA binding defect is only manifested in a cellular context. Increased concentrations of dCBP restore not only the ability of Bcd(A57-61) to access the hb enhancer element in cells but also the occupancy of the general transcription factors TBP and TFIIB at the reporter promoter. These and other results suggest that an activator can undergo switches between its active and inactive states through sensing the opposing actions of positive and negative co-factors.

The co-activator CREB-binding protein participates in enhancer-dependent activities of bicoid.

Bicoid (Bcd) is a transcriptional activator required for early embryonic patterning in Drosophila. Despite extensive studies, it currently remains unclear how Bcd activates transcription and what proteins participate in its activation process. In this report, we describe experiments to analyze the role of the Drosophila co-activator dCBP in Bcd-mediated activation. In Drosophila S2 cells, the Bcd activity is increased by the co-transfection of plasmids expressing dCBP and reduced by double-stranded RNA-mediated interference against dCBP. We further show that Bcd and dCBP can interact with each other and that Bcd-interacting domains of dCBP can cause dominant negative effects on Bcd activity in S2 cells. Our comparison of two Bcd-responsive enhancers, hunchback (hb) and knirps (kni), reveals a differential role of dCBP in facilitating Bcd activation. A dCBP mutant defective in its histone acetyltransferase activity exhibits a reduced, but not abolished, co-activator function for Bcd. Our chromatin immunoprecipitation experiments show that dCBP can increase not only the occupancy of Bcd itself at the enhancers but also the recruitment of general transcription factors to the promoter. Together, these experiments suggest that dCBP is an enhancer-dependent co-activator of Bcd, facilitating its activation through multiple mechanisms.

A composite motif of the Drosophila morphogenetic protein bicoid critical to transcription control.

Bicoid is a molecular morphogen-controlling embryonic patterning in Drosophila. It is a homeodomain-containing protein that activates specific target genes during early embryogenesis. Our recent studies have identified a domain of Bcd located outside its homeodomain and referred to as a self-inhibitory domain that can dramatically repress its own ability to activate transcription. Here we present evidence that the self-inhibitory function is evolutionarily conserved. A systematic analysis of this domain reveals a composite 10-amino acid motif with interdigitating residues that regulate Bcd activity in opposite manners. Mutations within the Bcd motif can exert their respective effects when the self-inhibitory domain is grafted to an entirely heterologous activator, but they do not affect DNA binding in vitro or subcellular localization of Bcd in cells. We further show that the self-inhibitory domain of Bcd can interact with Sin3A, a component of the histone deacetylase co-repressor complex. Our study suggests that the activity of Bcd is intricately controlled by multiple mechanisms involving the actions of co-repressor proteins.

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription.

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.

The activity of the Drosophila morphogenetic protein Bicoid is inhibited by a domain located outside its homeodomain.

The Drosophila morphogenetic protein Bicoid (Bcd) is a homeodomain-containing activator that stimulates the expression of target genes during early embryonic development. We demonstrate that a small domain of Bcd located immediately N-terminally of the homeodomain represses its own activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. We further show that this domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. We also provide evidence to suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es).