Transcriptional Response to cAMP in the Liver

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CHAPTER 23

Transcriptional Response to cAMP in the Liver

Maria Agnese Della Fazia, Giuseppe Servillo, Paolo Sassone-Corsi

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23.1

Introduction

The complex mechanisms that govern gene expression involve a number of nuclear proteins mostly implicated in various steps of RNA transcription [9, 48, 62, 96]. Many of the proteins controlling transcription are under the direct control of intracellular signaling pathways [53]. A number of essential liver functions are directed by the cyclic AMP-dependent signaling pathway [26]. Various factors acting on liver (e.g., hormones) bind to specific receptors and via G proteins regulate the intracellular concentration of this second messenger [36]. Thus, the understanding of the signaling pathways and of the molecular events leading to regulated gene expression by cyclic AMP is central to the interpretation of liver physiological responses.

23.2

Cyclic AMP Transduction Pathway

Cyclic AMP(cAMP) derives from a molecule of ATP by action of the enzyme adenylate cyclase (AC). A multigene family of at least nine components en- codes various forms of AC. AC is a plasma-membrane-bound enzyme constituted by two clusters of six transmembrane segments [52, 90, 100]. At least nine closely related isoforms of AC, AC1–AC9, have been cloned and characterized in mammals. These share a wide homology in their structure. Two cytoplasmic domains of ACs, C1 and C2, constitute the catalytic site; this is specific for each subtype and it is subjected to regulation. The different AC isoforms present a tissue-specific distribution. In particular, in the liver the isoforms AC4, AC5, AC6, AC7 and AC9 are present. The most abundant isoform in the liver is AC6 [23]. Different external stimuli, which act on specific receptors coupled to trimeric G proteins (G proteins are constituted by three chains: α, β and γ), change AC activity [37, 98] (Fig. 23.1). A

number of G protein subunit isoforms exist, some displaying differential tissue distribution, particu- larly in the liver [67]. Regulation of the intracellular concentration of cAMP relies on what type of receptor is activated; indeed, depending on the coupling of receptors to G proteins, these will elicit either activation or inhibition of AC function [10, 69].

AC activation results in increased cAMP levels, which then trigger the cAMP-dependent protein kinase A (PKA). In the absence of cAMP, PKA is a tetrameric holoenzyme constituted of two regulatory and two catalytic subunits (R and C subunits) [5, 35, 101]. Multiple isoforms of both R and C subunits have been identified [94].

Binding of cAMP on the two regulatory subunits (RI and RII) results in the dissociation of the protein complex (Fig. 23.1). The activated catalytic subunits are released from cytoplasmic anchoring sites and become able to phosphorylate a variety of cytoplasmic and nuclear substrates on serine sites within the canonical peptidic setting X-Arg-Arg-X-Ser-X (where X is any amino acid) [59, 69, 83]. RI subunits contain binding sites for MgATP. Binding of MgATP stabilizes the holoenzyme by raising the threshold of cAMP concentration required to cause activation and enhancing holoenzyme reassociation. RII subunits do not bind MgATP, but are targets of au- tophosphorylation, which destabilizes and activates the holoenzyme.

23.3 Cyclic AMP

and Gene Transcription in the Liver

23.3.1

cAMP-Responsive Promoter Element

The analysis of promoter sequences of several cAMP-responsive genes allowed the identification of a promoter element that could mediate transcriptional activation in response to increased levels of

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intracellular cAMP. The cAMP-responsive element (CRE) [59, 108] is constituted by a consensus palin- dromic sequence of eight base pairs (TGACGTCA, see Table 23.1) [59]. Importantly, several genes essential for liver physiology are under the control of the cAMP signaling pathway (Table 23.1). Relevant examples include the genes involved in gluconeogenesis, such as tyrosine aminotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK), serine dehydratase (SDH) and glucose-6-phosphatase (G-6-P), which all contain CREs in their promoter and present cAMP-inducible expression [7, 65, 66, 87, 97]. The CRE is frequently placed proximal to the transcriptional start site. Recently, by using a genome-wide analysis of CRE-binding protein (CREB) target genes, it was shown that the positional con- servation of CREB-binding sites is crucial to iden- tify true target genes. Strikingly, the presence of a proximal TATA box seems essential for CREB-mediated activation [14].

A number of proteins have been found to bind a CRE sequence [74, 89]. The identification of the first CREB [49] paved the way for the identification of a family of related factors.

23.3.2

CRE-Binding Protein Family

The discovery of nuclear factors binding to CRE sites revealed a class of proteins bearing common structural and functional characteristics [74, 89].

All CRE-binding factors belong to the bZip (basic domain-leucine zipper) class. These proteins share a highly conserved bZip domain, a α-helical coiled- coil structure with an adjacent basic domain, necessary for dimerization and DNA binding.

The CREB family members share little similarity at the level of primary amino acid sequence outside the bZip region. However, CREB, activating tran-

Fig. 23.1. Schematic representation of the cAMP signal trans- duction pathway and its coupling to transcription factors controlling gene expression. Once ligands bind membrane receptors, they activate coupled G-proteins (G) which in turn stimulate the activity of the membrane-associated adenylyl cyclase (AC).

AC converts ATP to cyclic AMP. Increased intracellular levels of cAMP cause the dissociation of the tetrameric protein kinase A (PKA) complex into the active catalytic subunits (C) and the regulatory subunits (R). Catalytic subunits migrate into the nucleus and phosphorylate (P) and induce transcriptional activatiors such as CREB and CREM. Phosphorylation allows the recruiting

of CBP (CREB binding protein). CREB and CREM binding to cAMP responsive elements (CRE) found in promoters of cAMP-responsive genes is a key step in transcriptional activation. Products of cAMP-inducible genes are involved in several physiophatologi- cal function in liver, such as differentiation, hormonal response and proliferation. An intronic, cAMP-inducible promoter (ps) of the CREM gene directs the synthesis of ICER (Inducible cAMP Early Repressor) which down-regulates the expression of CRE- containing promoters, including P2, generating a negative autoregulatory transcriptional loop. P1, CREM promoter; CAREs cAMP autoregulatory elements present in intron P2.

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scription factor 1 (ATF-1) and CREM (CRE modulator) have similar functional domains in their structural organization [34, 44, 49].

In CREM, alternative splicing is extensive as more than ten isoforms are generated. In addition, CREM displays alternative polyadenylation, alternative use of two promoters and alternative transla- tion, all determining different functional isoforms [33, 89].

Alternative isoforms of CRE-binding factors are obtained, mostly by alternative splicing. The transcription factors CREMτ, CREB and ATF-1 act as activators [42, 44, 82]; CREMα, β, γ and CREB-2 as repressors [17, 33, 55]. The repressor ICER (inducible cAMP early repressor), the only inducible repressor of the class, deserves special mention. This is generated by an alternative, cAMP-inducible promoter located within an intron of the CREM gene [72, 89, 95].

23.3.3

Transcriptional Activation

The CREB family of activator proteins mediates transcriptional activation by two independent,

well-conserved regions [89]. The first, termed kinase inducible domain (KID) or phosphorylation box (P-box), contains a serine residue within a consensus phosphorylation site for PKA at position 133 in CREB and 117 in CREM [21, 22, 42, 59, 64]. The second region is constituted by two glutamine-rich domains, termed Q1 and Q2, which flank the P-box [59, 89]. The phosphorylation event is a prerequisite for turning these transcription factors into powerful activators.

The serine phosphorylation within the P-box represents the direct link between cAMP signaling and activation of gene expression. It has been demonstrated that other kinases are able to phosphorylate the same serine, which thereby represents a conver- gence site of various signaling pathways [18, 19, 22, 38, 93]. For example, in Hep-G2 and 3T3-L1 cells, insulin induces CREB phosphorylation and stimulates transcriptional activity [56]. CREB has also been involved in the signaling pathway activated by the nerve growth factor (NGF). The activated NGF receptor stimulates the activity of the small GTP- binding protein Ras [41]. Activation of Ras triggers the MAPK pathway, which includes the MAP kinase kinase (MEK) and the ribosomal S6 kinase pp90^rsk [12]. Although MAPK and MEK do not phosphorylate CREB directly, the use of cells expressing a dominant-interfering Ras mutant has revealed the involvement of this pathway for CREB phosphorylation upon NGF induction [38]. Indeed, the involvement of a CREB kinase with characteristics similar to pp90^rsk has been proposed. pp90^rsk is likely to be responsible for CREB phosphorylation in human melanocytes [6], although a distinct member of the RSK family, p70^s6k, also possesses CREB phosphorylation activity [20] (Fig. 23.2).

CREB has been shown to be phosphorylated upon activation of the stress pathway involving the p38/MAPKAP-2 kinases [99]. Thus, various signaling pathways converge to modulate gene expression via the same transcriptional regulator, CREB.

The role of the two glutamine-rich domains, Q1 and Q2, also appears central. Other transcription factors, such as AP-2 and Sp-1 [16, 107], contain Q-rich regions. These domains are thought to mediate the interaction with other components of the transcriptional machinery. The contribution of Q2 to activation function seems more significant with respect to Q1 [89]. The demonstration of the role of the Q2-rich domain is offered by splicing isoforms of CREM and by ATF-1 [8, 63], both lacking Q1, which are still able to activate transcription [82]. Thus, the P-box region and Q2 are sufficient for induction.

The identification of the CREB-binding protein (CBP) increased the understanding of the mechanism by which the cAMP pathway controls tran-

Table 23.1. CRE location in promoter of liver-inducible genes

Gene CRE location in the promoter

G-6-Pase -136/-129

-161/-152

PEPCK -90/-83

SDH -1129/-1122

TAT -3651/-3644

CREM (ICER) -148/-140 -136/-129 -116/-109 -105/-98

Cyclin A -80/-73

c-fos -66/-59

G-6-Pase glucose-6-phosphatase, PEPCK phosphoenolpyru- vate carboxykinase, SDH serine dehydratase, TAT tyrosine ami- notransferase, CREM cyclic AMP-responsive element modulator, ICER inducible cyclic AMP early repressor.

CHAPTER 23: Transcriptional Response to cAMP in the Liver

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scription [11]. CBP is a protein of 265 KDa that in- teracts selectively with the phosphorylated form of CREB; it presents two zinc finger domains, a glutamine-rich domain and PKA consensus sites.

Upon phosphorylation CREB binds to CBP, an interaction that facilitates the binding of CBP to TFIIB, a factor directly linked to RNA polymerase II [58].

Protein p300 shares similar regulatory functions to CBP [2, 28] in differentiation [30], cell growth [29], apoptosis and DNA repair [39]. Both CBP and p300 interact with the basal transcription factors including TFIIB, TBP and a RNA helicase. Several other transcription factors (e.g., Jun, Fos, MyoD, p53, NF-κB) and some nuclear receptors interact with CBP/p300 [54]. CBP has an intrinsic or associated histone acetyltransferase activity (HAT), establishing a direct link with chromatin remodeling [4, 40, 57, 58, 76]. The HAT function of CBP establishes a strong link between signaling and chromatin modifications. The role of chromatin remodeling in liver physiology is still poorly understood, although it surely represents a fascinating area of research.

Another protein, ACT (activator of CREM in tes- tis), is able to interact with and modulate the CREM transcriptional activity. This protein is expressed exclusively in male germ cells and is constituted by four complete LIM domains and one amino-termi- nal half LIM motif. ACT shares a high degree of homology with a family of proteins expressed in heart and skeletal muscle. ACT is able to perform its func-

tion independently of CREM phosphorylation at Ser-117 – and thus of the interaction with CBP. This suggests an alternative way to elicit activation by the transcription factors of the CREB class, possibly bypassing classical signaling pathways [32]. As ACT defines a novel class of tissue-specific co-activators, it would be of great interest to define whether proteins of this type may operate in the liver. Recently, the mechanism by which the co-activator transducers of regulated CREB activity (TORCs) stimulate CREB activity in a phosphorylation-independent manner has been studied. TORCs appears to interact with the bZIP domain and enhance the recruit- ment of TAF_II130 to the promoter. The interaction between CREB and TORCs suggests a role played by TORCs in controlling the active and the inactive cellular pools of CREB [13].

23.3.4

Mechanisms of Repression

Dephosphorylation represents one of the most important molecular mechanisms involved in the negative regulation of CREB. Genes under CREB control are likely to be downregulated by the induced dephosphorylation of CREB itself. Protein phosphatases, presumably PP-1 and PP-2a, have been involved in the dephosphorylation process.

Some in vitro results appear to support this hypoth-

Fig. 23.2. Signal transduction routes leading to phosphoryla- tion of ser-133 and ser-117 of CREB and CREM. Various signaling cascades are activated by external stimuli. Crosstalk between pathways is indicated by arrows. Dashed lines indicate the pres- ence of intermediate kinases (not shown). CaMKIV Ca²⁺-calmod-

ulin-dependent kinase IV, ERK extracellular regulated kinase, MAPKAP-K2 MAP-kinase-activated protein kinase 2, MSK mi- togen- and stress-activated kinase, p70S6K p70 S6 kinase, PI-3K phosphoinositide 3-kinase, PKA cyclic AMP-dependent protein kinase, RSK-2 ribosomal S6 kinase 2

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esis. However, the signals required to trigger phosphatase activity and its regulation remain obscure.

Thus, the role played by phosphatases in the in vivo regulation of CREB function still needs further in- vestigation.

In contrast to the remaining members of the CRE-binding factor family, whose expression seem to be constant and ubiquitous [44, 88], several studies have demonstrated that differential transcript processing plays a central role in the regulation of CREM expression [89]. Three isoforms (CREMα, -β, -γ) generated from a GC-rich housekeeping promoter (P1) lack the activation domain and thereby act as antagonists of cAMP-responsive transcription by either competing for binding to CREs or by blocking CREB by heterodimerizing with it [89].

The requirement of a de novo produced repressor was suggested to justify the rapid transcriptional downregulation of early response genes following activation. The CREM isoform ICER fulfils all the physiological requirements for such function. ICER is a small protein of 120 amino acids consisting only of the CREM bZip domain. ICER synthesis is directed by an alternative promoter (P2) lying within an intron near the 3' end of the CREM gene (Fig. 23.1).

The P2 promoter is strongly inducible by cAMP as it contains two pairs of closely spaced CREs [72]. cAMP treatment induces rapid and transient increase of ICER expression characteristic of the early response gene class [72]. ICER turns off its own expression by repressing the activity of the P2 promoter, establishing a negative autoregulatory loop. ICER, of course, once synthesized, also represses other cAMP-induc- ible genes whose promoter contains a CRE (c-fos [73], cyclin A [61], TAT, PEPCK) [92]. The dynamic and versatile ICER inducibility, combined with its tissue- and developmental-specific expression pattern, provides a remarkable frame to the molecular interpretation of various physiological regulations with oscillatory behavior [60].

23.4

Role of cAMP in Liver

Genes essential for liver physiology and involved in gluconeogenesis are under control of the cAMP signaling pathway. The genes encoding TAT, PEPCK, SDH and G-6-P contain CREs in their promoter and are cAMP-inducible [7, 65, 66, 80, 97]. TAT and PEPCK share common features and constitute use- ful models to study cAMP-responsive transcription in the liver. Glucagon stimulates the expression of both genes by acting through the cAMP pathway [43, 45, 75].

Increased levels of cAMP in hepatocytes lead to CREB phosphorylation and activation of TAT synthesis [106]. CREB also binds to the CRE-1 in the promoter of the PEPCK gene, playing a role in both basal and cAMP-stimulated expression [78]. Other transcription factors, such as C/EBPαC/EBPβ and DBP bind to the PEPCK promoter and are thought to synergize with CREB [78, 85]. Thus, one important issue concerns the interplay that cAMP-responsive transcription factors have with other proteins regulating liver-specific promoters. This issue has not been explored satisfactorily yet, but it is likely that future experiments will reveal functional interactions with liver-specific transcription factors.

Indeed, cooperation between CREB and the co-activator PGC-1 has been described in liver of fasted animals. During the activation of the gluconeogenic pathway, CREB modulates the expression of PEPCK mediated by catecholamines and glucagon. Follow- ing prolonged stimulation of neoglucogenesis, CREB further activates neoglucogenic genes by overex- pressing PGC-1 [46]. Similarly to gluconeogenesis, CREB controls lipid mobilization in the liver of fasted animals. Indeed, CREB appears to regulate hepatic lipid metabolism by repressing the expression of PPARγ. Thus, CREB acts as a kind of molecular balance in the liver controlling gluconeogenesis and fatty acid oxidation during fasting [47].

Many functions of hepatocytes are governed by cAMP. In hepatoma cells induced to proliferate or treated with forskolin, there is a remarkable induction of CREB phosphorylation and a subsequent induction of ICER expression [91]. Importantly, the same pattern is observed in vivo by treating rats with intraperitoneal injection of cAMP. In these animals, ICER expression is powerfully induced at 2, 4 and 8 h following treatment with cAMP. These ob- servations suggest that CREM may play a role in the control of the hepatocytes response to cAMP [91].

Another interesting role that CREB appears to play in the liver does not involve hepatocytes, but stellate cells and cholangiocytes. Hepatic stellate cells play a pivotal role in the pathogenesis of hepatic fibrosis. Activation of stellate cells leads to a proliferation with an excessive production of col- lagen type I. Studies performed on proliferation of stellate cells have demonstrated that phosphorylation of CREB regulates the cell cycle by acting as an inhibitor of proliferation [51].

Finally, a characteristic CRE has been found in the gene promoter of cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is only expressed on bile duct epithelium and not in hepatocytes. CFTR is a Cl^– channel mediating ion transport in bile duct epithelium. Genetic defects in CFTR are responsible for cystic fibrosis, which develops in

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hepatobiliary disease, one of the most important causes of mortality of these patients. These notions highlight the importance of the role played by CREB in controlling the expression of CFTR at the level of cholangiocytes [68].

23.5

Cyclic AMP and Proliferation, Role in Liver Regeneration

The role played by cAMP in proliferation is some- what unclear as in some cases it acts as a promoting factor, whereas in others it appears to function as a suppressor. In fact, in primary rat hepatocytes glucagon increases cAMP levels and stimulates DNA synthesis in cooperation with EGF and insulin [102], whereas in long-term experiments cAMP synergizes with glucocorticoids in inhibiting hepatocyte DNA replication [104, 105]. It has been demonstrated that cAMP concentration appears to be critical for its effect. Glucagon at low concentrations induces DNA synthesis, while an opposite effect is seen at high concentrations. Cyclic AMP acts on hepatocyte proliferation mainly at two cell cycle checkpoints:

it facilitates the transition from G₀/early G₁ to the pre-replicative period and exerts an inhibitory effect before the G₁/S border [102]. The action of different hormones and growth factors [70, 71, 81, 102]

on hepatocyte proliferation suggests convergent interactions between the cAMP-dependent signaling pathway and other transduction routes. The co-activation of cAMP and other signaling systems in hepatocytes increases or delays DNA synthesis.

cAMP acts as an inhibitor of proliferation by interfering in vitro with the MAPK pathway in rat fibrob- lasts [15] and it is crucial during the proliferation of residual hepatocytes that occurs following hepatec- tomy. Other factors, such as β-adrenergic agonists, increase cAMP levels in response to partial hepate- ctomy (PH) [26] and seem to stimulate DNA synthesis. It is possible that the balance between stimulatory and inhibitory effects of cAMP is closely coupled to the co-action of other factors (e.g., glucocorticoid, prostaglandins, vasopressin, insulin, etc.).

A remarkable feature of liver regeneration is the increase in intracellular cAMP levels during the proliferation of residual hepatocytes following PH [27].

In fact, cAMP peaks during the first hours following PH and elevated levels of cAMP also correlate with the proliferation of liver cells at birth [26]. These notions emphasize the critical role that cAMP-responsive transcription factors are likely to play in liver regeneration.

Following PH there are interesting changes in the expression of PKA subunits that occur in residual hepatocytes (during liver regeneration). The protein levels of the RIα and RIIα subunits increase in the first hours following PH, while the expression of catalytic subunit remains constant [31]. Similar results have been obtained in hepatocytes stimulated in vitro with cAMP [50].

Two distinct peaks of intracellular concentration of cAMP take place during liver regeneration.

The first peak occurs 2–6 h following PH. The second precedes the first round of mitosis and has been associated with hepatocyte proliferation [26]. In the first hours following PH, ICER expression was shown to be rapidly and transiently induced, suggesting a role for this transcriptional repressor in the regulation of the early proliferation phase. These results provide clear evidence that CREM exerts a pivotal role in the proliferation of hepatocytes [92, 103].

In the liver the role of CREM has been defined by the use of mutant mice in which the CREM gene was targeted by homologous recombination. In CREM- deficient mice there is a significant delay in the first round of mitosis during liver regeneration. Expres- sion of the genes encoding cyclins A, B, D1, E and cdc2 undergoes a switch confirming a delay in mitosis. The expression of liver-specific markers (TAT and PEPCK) is affected, as well expression of the im- mediate-early gene c-fos [92]. The protein product of the c-jun gene combines with the Fos protein to constitute the transcription factor AP-1, which has been involved in various proliferative processes. No differences in jun expression with respect to normal mice have been observed (our unpublished data), indicating that alteration of one of the AP-1 components is sufficient to cause a delay in hepatocyte proliferation.

The role of CREM as a cAMP-responsive factor in liver physiology is thus quite attractive. It is note- worthy that the promoter regulatory regions of several genes whose expression is altered in the CREM- deficient mice – cyclin A, cyclin D1, TAT, PEPCK and c-fos – contain CRE sequences [24, 61, 77, 84], establishing a direct link with the regulatory function of the CREM products. CREB seems to play an important role in mediating the response to prostaglandins in liver regeneration. Indeed, it seems that CREB activation following PH is crucial for the correct timing of liver regeneration [86].

In addition, CREB might be involved in the control of tumor progression in hepatocellular carcino- ma, as it seems to regulate angiogenesis and induce survival of the cells [1]. Moreover, CREB appears to be an important target of pX protein of hepatitis B virus. pX binds unphosphorylated CREB and activates the transcription by bypassing the require-

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ment of Ser-133 phosphorylation. The increased activation of CREB leads to an upregulation of cAMP- responsive genes, an event that could be responsible for the development of liver cancer following viral infection [25, 79]. Involvement of CREB in proliferation is suggested by association of CREB phosphorylation with mitochondrial dysfunction. CREB has been implicated in “retrograde signaling” events between functionally impaired mitochondria and nuclear transcription [3].

23.6 Conclusion

The cAMP transduction pathway plays a pivotal role in directing the function of the liver. A number of physiological functions are controlled by expressed genes that are regulated by cAMP. The interplay between CREB/CREM and different transcription factors finely regulates in the liver the expression of genes that present a CRE in their promoter. The liver regeneration is governed by a highly specialized program of gene expression. The understanding of the intracellular signaling routes leading to the modulation in the activity of specific transcription factors is essential. Signals to transcription factors are utilized to integrate the information required to direct synchronized waves of hepatocyte proliferation.

A central role for the cAMP signaling transduction cascade in this process has been long proposed, and evidence involving cAMP-responsive transcription factors is now present.

The implication of CREM as a key regulator of the liver regeneration process stresses the role of cAMP as a critical second messenger. Yet, clear links with cell proliferation are not well established. The direct control that CREM appears to exert on cyclin gene expression is of interest.

Many open questions remain: what are the target genes of cAMP-responsive transcription factors?

What is the role played by crosstalks among signaling pathways? In what way does cAMP influence the cell cycle of proliferating hepatocytes? Various experimental approaches will be needed to elucidate further the molecular mechanisms governing normal and pathological liver physiology. The available technology of targeted disruption of specific genes in the mouse by homologous recombination, espe- cially if coupled to tissue-specific and conditional mutagenesis, will greatly help our understanding and is likely to provide crucial information for future biomedical applications.

Acknowledgments

We thank all the members of the Sassone-Corsi laboratory for help and discussions. This study was supported by grants from the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Université Louis Pasteur, La Ligue contre le Cancer, Association pour la Recherche sur le Can- cer and the Human Frontiers Scientific Programme (RG-240). Work in G. Servillo’s laboratory is supported by a grant from the Associazione Italiana Ricerca sul Cancro.

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