John M O'

Neil D Perkins
email: n.d.perkins@bristol.ac.uk

Ron Hay

Thr-435 Phosphorylation Regulates RelA (p65) NF-κB subunit transactivation

Keywords: RelA (p65), Cxcl2 (MIP2), HDAC1, NF-κB, Histone Acetylation, TNFα ANOVA, Analysis of Variance, ChIP, Chromatin Immunoprecipitation, DBD, DNA Binding Domain, DMEM, Dulbecco's Modified Eagle's Medium, ELISA, Enzyme-Linked ImmunoSorbent Assay, EMSA, Electrophoretic Mobility Shift Assay, HAT, Histone Acetyltransferase, HDAC, Histone Deacetylase, IκB, Inhibitor of NF-κB, IKK, IκB Kinase

Phosphorylation of the RelA (p65) NF-κB subunit has been previously shown to modulate its ability to induce or repress transcription. Here we have investigated the consequences of Thr-435 phosphorylation within the Cterminal transactivation domain of RelA. We confirm that Thr-435 is phosphorylated in cells and is induced by TNFα treatment. Mutational analysis of this site revealed gene specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 mRNA levels in reconstituted Rela-/-mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation, associated with decreased recruitment of the histone deacetylase HDAC1. Moreover, mutation of this site disrupted RelA interaction with HDAC1 in vitro. Thr-435 phosphorylation of promoter bound RelA was also detected at NF-κB target genes following TNFα treatment in wild type MEFs. Phosphorylation at this site therefore provides an additional mechanism through which the specificity of NF-κB transcriptional activity can be modulated in cells.

Introduction

Members of the NF-κB transcription factor family are known to regulate a variety of cellular processes including inflammatory and immune responses, cell survival, cell differentiation and cell proliferation [START_REF] Hayden | Shared principles in NF-κB signaling[END_REF]. Furthermore, dysregulation of NF-κB signalling has been implicated in the development and progression of a multitude of diseases, particularly conditions involving chronic inflammation or compromised immunity and cancer [START_REF] Courtois | Mutations in the NF-κB signaling pathway: implications for human disease[END_REF][START_REF] Kumar | Nuclear factor-κB: its role in health and disease[END_REF]. Mammalian NF-κB is a multigene family composed of five members, capable of forming a variety of homo-and heterodimeric complexes through their highly conserved N-terminal Rel homology domain (RHD) [START_REF] Gilmore | Introduction to NF-κB: players, pathways, perspectives[END_REF]. In unstimulated cells NF-κB dimers are primarily found as inactive, cytoplasmic complexes. Classical activation of NF-κB occurs in a well defined, IκB Kinase (IKK) dependent manner, typically culminating in the release of RelA/p50 heterodimers from inhibitory IκB proteins, enabling dimer translocation to the nucleus and transcriptional modulation of NF-κB target genes [START_REF] Perkins | Integrating cell-signalling pathways with NF-κB and IKK function[END_REF].

The RelA (p65) subunit contains a potent transactivation domain (TAD), which allows recruitment of co-transcriptional regulators and components of the basal transcriptional machinery to gene targets [START_REF] O'shea | Regulation of the RelA (p65) transactivation domain[END_REF]. Numerous post-translational modifications to RelA have been reported and varying effects on transcriptional activity, protein interactions, stability and degradation have been demonstrated [START_REF] Perkins | Post-translational modifications regulating the activity and function of the nuclear factor κB pathway[END_REF]. Phosphorylation of sites within RelA's TAD lead to both increased and decreased levels of transcriptional activity, with the precise effect dependent on the context and gene target [START_REF] Adli | IKK-i/IKKε Controls Constitutive, Cancer Cell-associated NF-κB Activity via Regulation of Ser-536 p65/RelA Phosphorylation[END_REF][START_REF] Buss | Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-κB at Serine 536 Is Mediated by Multiple Protein Kinases Including IκB Kinase (IKK)-α, IKKβ, IKKε, TRAF Family Member-associated (TANK)binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATAbinding Protein-associated Factor II31-mediated Interleukin-8 Transcription[END_REF][START_REF] Buss | Phosphorylation of Serine 468 by GSK-3β Negatively Regulates Basal p65 NF-κB Activity[END_REF][START_REF] Campbell | Active repression of antiapoptotic gene expression by RelA(p65) NF-κB[END_REF][START_REF] Chovolou | Downregulation of NF-κB activation in a H4IIE transfectant insensitive to doxorubicin-induced apoptosis[END_REF][13][START_REF] Jiang | The NF-κB Activation in Lymphotoxin β Receptor Signaling Depends on the Phosphorylation of p65 at Serine 536[END_REF][START_REF] Mattioli | Inducible Phosphorylation of NF-κB p65 at Serine 468 by T Cell Costimulation Is Mediated by IKKε[END_REF][START_REF] Mao | GCN5 is a required cofactor for a ubiquitin ligase that targets NF-κB/RelA[END_REF][START_REF] O'mahony | Human T-cell Lymphotropic Virus Type 1 Tax Induction of Biologically Active NF-κB Requires IκB Kinase-1-mediated Phosphorylation of RelA/p65[END_REF][START_REF] Rocha | p53-and Mdm2independent repression of NF-κB transactivation by the ARF tumor suppressor[END_REF][19][START_REF] Wang | Tumor Necrosis Factor α-induced Phosphorylation of RelA/p65 on Ser529 Is Controlled by Casein Kinase II[END_REF]. The effects these modifications exert over protein-protein interactions has not been extensively studied. However, site-specific phospho-dependent increased binding to the co-transcriptional regulators TAFII31 (TATA-boxbinding-protein-associated factor 31) and HDAC1 (Histone deacetylase 1) have been shown [START_REF] Buss | Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-κB at Serine 536 Is Mediated by Multiple Protein Kinases Including IκB Kinase (IKK)-α, IKKβ, IKKε, TRAF Family Member-associated (TANK)binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATAbinding Protein-associated Factor II31-mediated Interleukin-8 Transcription[END_REF][START_REF] Campbell | Active repression of antiapoptotic gene expression by RelA(p65) NF-κB[END_REF]. Additionally, components of the ubiquitin ligase complex COMMD1 and cullin 2 were found to bind to RelA via GCN5, in a phospho site-specific manner, thereby directing RelA ubiquitination and degradation at certain promoters following TNFα stimulation [13,[START_REF] Mao | GCN5 is a required cofactor for a ubiquitin ligase that targets NF-κB/RelA[END_REF].

The mechanisms behind RelA's ability to specifically regulate endogenous target gene expression are continually being uncovered. Activation of certain endogenous target genes was recently shown to occur by two distinct modes; one involving the direct interaction of RelA with the Trap-80 mediator complex subunit and subsequent recruitment RNA Pol II; and the other via RelA's ability to regulate promoter occupancy of secondary transcription factors [START_REF] Van Essen | Two modes of transcriptional activation at native promoters by NF-κB p65[END_REF]. The murine chemokine Cxcl2 (MIP2) was found to be regulated in a Trap-80 independent manner and RelA was shown to regulate the recruitment of the secondary transcription factor SP1 to this promoter [START_REF] Van Essen | Two modes of transcriptional activation at native promoters by NF-κB p65[END_REF]. Here we present evidence for RelA's ability to induce changes in the acetylation state of histones at the Cxcl2 promoter. Furthermore, we demonstrate that this effect is influenced by phosphorylation at Thr-435 within RelA's TAD, which modulates RelA's interaction with HDAC1. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Experimental

Cell Culture Human U-2 OS and HEK 293 cells were obtained from the American Type Culture Collection (ATCC). Immortalised mouse embryonic fibroblast (MEF) cells and immortalised Rela-/-MEFs were provided by Professor Ron Hay (University of Dundee). RelA null MEFs were stably reconstituted with vector alone (Control), wild type RelA (RelA), RelA with Thr-435 mutated to an alanine (T435A) or an aspartic acid (T435D). Stably reconstituted Rela-/-MEFs were selected with 2 µg/mL puromycin and subsequently maintained in 0.5 µg/mL puromycin.

Preparation of viral stocks

Viral stocks for lentiviral infections were generated by transfecting 5 µg of viral envelope (pCMV-VSV-G), packaging (pCMVΔR8.91) and vector (PIRESpuro-deNotI and derivatives) plasmids into HEK 293 cells. Media containing the viral stocks was removed after 48 hours. The media was centrifuged at 1200 rpm for 3 minutes and filtered through a 0.45 µm filter to remove cells. Viral stocks were then aliquoted, snap frozen and stored at -80 ºC.

Generation of Stable Cells

Infections were performed by mixing 4 µg/mL polybrene (Sigma) with 30 µL virus stock in a total volume of 3 mL media. This was used to infect a 50 % confluent 10 cm plate of Rela-/-MEFs by incubating overnight at 37 ºC. On the second day the media containing the virus was removed and the plate was washed twice with PBS before the addition of fresh media. On the third day the cells were split 1:5. On the fourth day cells were selected by replacing media with puromycin containing media (2 µg/mL) and when all cells in the mock infection plate had died the puromycin concentration was lowered to 0.5 µg/mL. This concentration was used to maintain stably expressing cell lines. In all experiments pools of reconstituted MEF cells were used.

Quantitative PCR (qPCR) QPCR reactions were performed on Rotor-Gene 3000 and 6000 machines (Corbett Research). Ct values were calculated using Rotor-Gene 6 software and all values were calculated relative to GAPDH or Input levels using the Pfaffl method [22].

Primers

RT-PCR Bcl2l1 (Bcl-xl)

FWD: 5'-GATGCAGGTATTGGTGAGTCG-3', REV: 5'-GCTCTCGGCTGCTGCATT-3'; Cxcl1 (KC) FWD: 5'-CTGGGATTCACCTCAAGAAC-3', REV: 5'-GAAGCCAGCGTTCACCAGAC-3'; Cxcl2 (MIP2) FWD: 5'-TCAAGGGCGGTCAAAAAGTT-3', REV: 5'-TCCTCCTTTCCAGGTCAGTTA-3'; Cxcl3 FWD: 5'-CCTACCAAGGGTTGATTT-3', Tagged RelA was generated by inserting a N-Terminal PK-TAG, in a pcDNA3.1 vector. All point mutations were generated by PCR overlap extension, except for the Gal4 fusion mutants, which were generated in a single PCR step, and were sequenced prior to use.

Other Assays

Chromatin-immunoprecipitation, co-immunoprecipitation, reporter gene assays, western blot and EMSA analysis were performed essentially as described previously [START_REF] Rocha | p53-and Mdm2independent repression of NF-κB transactivation by the ARF tumor suppressor[END_REF][START_REF] Barré | A cell cycle regulatory network controlling NF-κB subunit activity and function[END_REF][START_REF] Chapman | A novel form of the RelA nuclear factor κB subunit is induced by and forms a complex with the proto-oncogene c-Myc[END_REF]. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Results

RelA Thr-435 is phosphorylated following calyculin A and TNFα treatment.

Protein Phosphatase 4 (PP4) mediated Thr-435 dephosphorylation was previously proposed to enhance RelA-mediated activation following cisplatin treatment, implying that phosphorylation at this site has a negative effect on RelA's activity [START_REF] Yeh | Suppression of MEK/ERK signaling pathway enhances cisplatininduced NF-κB activation by protein phosphatase 4-mediated NF-κB p65 Thr dephosphorylation[END_REF]. In order to determine the phosphorylation status of Thr-435 in cells, a phospho-specific antibody against this site was raised and ELISA analysis confirmed that the purified antibody was specific for this phosphorylated epitope (Data not shown). Treating U-2 OS osteosarcoma cells with the serine/threonine phosphatase inhibitor calyculin A, resulted in increasing levels of phosphorylation at this site, masked by background bands in direct western but clearly visible after immunoprecipitation with a RelA antibody (Fig. 1A). It was subsequently found that there was weak induction of phosphorylation at this site in U-2 OS cells following stimulation with the wellcharacterised NF-κB activator, TNFα (Figure 1B). However, rapid, Thr-435 phosphorylation was detected in mouse embryonic fibroblast (MEF) cells following TNFα stimulation (Fig. 1C).

Role of Thr-435 on RelA transcriptional activity.

To investigate potential effects on RelA's transcriptional activity, mediated through the Thr-435 residue, both phospho-null (T435A) and phospho-mimicking (T435D) mutations of this site were created. These mutants were initially generated as TAD fusion proteins with the DNA binding domain (DBD) of Gal4, thereby allowing only TAD-dependent effects to be investigated. Significantly, when transfected into U-2 OS cells, the T435A mutation resulted in a dramatic increase in transcriptional activity, while p-142 (t 9 (u 1 (t) -2 0 stably expressing wild type and mutant forms of RelA at equivalent levels were generated (Fig. 2A).

It is known that deletion of Rela results in embryonic lethality from extensive foetal hepatocyte apoptosis, due to increased sensitivity to TNFα [START_REF] Beg | Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB[END_REF]. Loss of RelA also sensitises MEFs to TNFα-induced cell death and this can be rescued by stably expressing RelA [START_REF] Beg | An Essential Role for NF-κB in Preventing TNF-α-Induced Cell Death[END_REF]. Both the wild type and Thr-435 mutant RelA reconstituted cells were rescued from TNFα-induced apoptotic cell death, as revealed by the absence of Poly (ADP-ribose) polymerase (PARP) cleavage, an indicator of caspase mediated cell death (Fig. 2B), indicating that phosphorylation at this site is unlikely to affect this important function of RelA [START_REF] Kaufmann | Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis[END_REF].

Stimulation with TNFα induces the activation of the canonical NF-κB pathway, resulting in the rapid translocation of RelA complexes to the nucleus of the cell and enhanced DNA binding to κB sites in responsive genes, such as IL8 [START_REF] Hayden | Shared principles in NF-κB signaling[END_REF][START_REF] Kunsch | NF-κB subunit-specific regulation of the interleukin-8 promoter[END_REF]. DNA binding in response to canonical pathway activation in the reconstituted cells were investigated by EMSA analyses. Although a low level of basal NF-κB DNA binding to an IL8 κB oligo could be seen in unstimulated RelA reconstituted cells, this was significantly induced following TNFα stimulation (Fig. 2C). Furthermore, mutating Thr-435 did not affect TNFαinduced DNA binding. Therefore, these reconstituted cell lines contain functional, TNFα inducible RelA, capable of regulating the expression of the anti-apoptotic genes necessary for rescue from TNFα induced cell death.

Regulation of NF-κB gene targets

To analyze the potential effects, mediated through Thr-435 on known NF-κB gene targets, qPCR analysis was performed on cDNA generated from these reconstituted cell lines. Initially, known NF-κB target genes; Bcl2l1 (Bclxl); Cxcl1 (KC); Cxcl2 (MIP2) & Tnfaip3 (A20) [START_REF] Chen | The Rel/NF-κB Family Directly Activates Expression of the Apoptosis Inhibitor Bcl-xL[END_REF][START_REF] Hoffmann | Genetic analysis of NF-κB/Rel transcription factors defines functional specificities[END_REF][START_REF] Krikos | Transcriptional activation of the tumor necrosis factor α-inducible zinc finger protein, A20, is mediated by κB elements[END_REF][START_REF] Ouaaz | A critical role for the RelA subunit of nuclear factor κB in regulation of multiple immune-response genes and in Fas-induced cell death[END_REF] were investigated in unstimulated cells, to allow analysis of Thr-435 mutation on basal level expression. In agreement with previous studies, expression of the chemokines Cxcl1 and Cxcl2 was severely diminished in the absence of RelA and was found to be enhanced following reconstitution with all forms of RelA (Fig. 3A&B) [START_REF] Van Essen | Two modes of transcriptional activation at native promoters by NF-κB p65[END_REF][START_REF] Hoffmann | Genetic analysis of NF-κB/Rel transcription factors defines functional specificities[END_REF][START_REF] Ouaaz | A critical role for the RelA subunit of nuclear factor κB in regulation of multiple immune-response genes and in Fas-induced cell death[END_REF]. However, some differences between the Thr-435 mutant forms of RelA were observed. Cxcl2 expression was dramatically increased in the phospho-mimicking T435D cell line, indicating that phosphorylation at this site may play a role in the induction of this gene (Fig. 3A). In agreement with this, a Cxcl2 promoter luciferase reporter plasmid was also found to have higher expression levels with the T435D mutant (Fig. 3E). Similar enhancement of mRNA expression by the T435D mutation was not observed for other members of the CXC chemokine superfamily whose expression was detectable in these cell lines (Supp. Fig. 1). Expression levels of the CXC chemokine Cxcl1 was significantly reduced in the unphosphorylatable T435A cell line, suggesting that phosphorylation at this site is important for basal expression of this gene (Fig 3B). Levels of the ubiquitin editing enzyme Tnfaip3 also became elevated following the introduction of RelA, but no significant differences were detected between the wild type and Thr-435 mutant cell lines (Fig. 3C). Finally, expression of the anti-apoptotic gene Bcl2l1 was reduced following reconstitution with RelA (Fig. 3D).

Taken together, these results indicate that the Thr-435 residue regulates the transcriptional activity of full length RelA in a gene specific manner. To begin a more mechanistic evaluation of these effects, the role played by the T435D mutant in the upregulation of Cxcl2 expression levels was investigated using chromatin immunoprecipitation (ChIP) analysis.

Protein occupancy at the Cxcl2 promoter in reconstituted cell lines.

Increased expression of the murine Cxcl2 gene is known to occur in a RelA dependent manner, following exposure to a variety of NF-κB inducing stimuli, including TNFα, LPS and hypoxia [START_REF] Van Essen | Two modes of transcriptional activation at native promoters by NF-κB p65[END_REF][START_REF] Hoffmann | Genetic analysis of NF-κB/Rel transcription factors defines functional specificities[END_REF][START_REF] Lee | Sp1-associated activation of macrophage inflammatory protein-2 promoter by CpGoligodeoxynucleotide and lipopolysaccharide[END_REF][START_REF] Zampetaki | Hypoxia induces macrophage inflammatory protein-2 (MIP-2) gene expression in murine macrophages via NF-κB: the prominent role of p42/ p44 and PI3 kinase pathways[END_REF]. The Cxcl2 promoter contains not only an NF-κB binding motif, but also SP1 and AP1 binding motifs, all of which are required for optimal promoter activation [START_REF] Lee | Sp1-associated activation of macrophage inflammatory protein-2 promoter by CpGoligodeoxynucleotide and lipopolysaccharide[END_REF][START_REF] Widmer | Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 α, and MIP-1 β, members of the chemokine superfamily of proinflammatory cytokines[END_REF]. ChIP analysis revealed that, in unstimulated cells, consistent with enhanced levels of gene expression, elevated levels of RelA, RNA Pol II and SP1 were found at the Cxcl2 promoter in the T435D cell line (Fig. 4A-C). In contrast, enhanced binding of the T435D mutant form of RelA was not observed at either the Cxcl1 or Tnfaip3 promoters (Sup. Fig. 2A&B). This indicated that Cxcl2 promoter specific recruitment is regulated by the T435D mutation and that this is not an intrinsic effect on DNA-binding, consistent with the earlier EMSA analysis (Fig. 2C).

We next examined effects on histone modification at the Cxcl2 promoter and enhanced acetylation of Histones H3 (Lysine 9 & 14) and H4 (Lysine 12), both markers for active regions of transcription, were observed following the introduction of wild type RelA or the T435D mutant (Fig. 4D&E). In the T435D cells, acetylated Histone H3 levels were higher than those found in the phospho-null T435A cells, but not significantly higher than in the wild type RelA cells (Fig. 4D). However, acetylated Histone H4 levels were significantly higher in the T435D cells, compared to both wild type RelA and T435A mutant cells, consistent with increased expression of this gene in the T435D cell line (Fig. 4E). To determine if the reduced acetylation detected in the T435A cell line results from enhanced binding of a deacetylase enzyme, levels of HDAC1 binding to the Cxcl2 promoter were determined and enhanced HDAC1 binding was detected at this promoter (Fig. 4F). These results illustrate that mutating Thr-435, in RelA's TAD, influences the ability of RelA and other transcription factors, such as SP1 and Pol II, to bind specifically to the Cxcl2 promoter. Additionally, differences in the acetylation status of histone tails at this promoter were also detected and can be accounted for, at least in part, by elevated levels of HDAC1 binding to the promoter in the T435A cell line.

HDAC1 interaction with RelA.

To investigate potential differences in HDAC1 interaction with RelA, coimmunopreciptiation analyses were performed using PK-Tagged wild type and Thr-435 mutant forms of RelA, along with HA-Tagged HDAC1. Significantly, the phosphomimicking T435D mutation decreased HDAC1 interaction with RelA, corroborating data obtained through ChIP analysis of the Cxcl2 promoter (Fig. 4G). As TNFα stimulation is known to result in decreased HDAC1 interaction with RelA [39], this data is consistent with Thr-435 phosphorylation contributing to this process.

Regulation of NF-κB gene targets following TNFα stimulation

Taken together, this data suggested that the effect of the T435D mutation was to mimic the effect of TNFα stimulation on the RelA dependent regulation of Cxcl2 expression. Therefore, we next investigated the effects of TNFα stimulation on the RelA reconstituted MEFs. Significantly, a RelAdependent, TNFα induced upregulation of both Cxcl1 and Cxcl2 was observed (Fig. 5A &B). In the case of Cxcl1 this induction resulted in similar levels of mRNA in the reconstituted cells, bypassing the reduced basal expression seen in the T435A cell line. However, in the case of Cxcl2 there were differences in the induction of this gene between the Thr-435 mutants. Cells containing wild type RelA showed a robust induction in Cxcl2 mRNA levels, reaching levels similar to that found basally in the T435D cell line, suggesting that this mutation did indeed mimic the effects of TNFα stimulation for this NF-κB gene target. This increase was much reduced in cells containing the T435A mutant. The already enhanced levels of expression seen with the T435D mutant were somewhat further stimulated by TNFα treatment, as might be expected from the enhanced translocation of RelA to the nucleus under these conditions. These results are consistent with RelA Thr-435 phosphorylation playing an important role in the TNF dependent induction of Cxcl2 expression.

Effects on other NF-κB target genes were also examined. RelA also enhanced Tnfaip3 expression following TNFα treatment, but significant differences were not detected between wild type and Thr-435 mutants (Fig. 5C). However, little effect of TNFα was seen on Bcl2l1 mRNA levels (Fig. 5D). This result is consistent with ChIP analysis, where we could not observe RelA binding to the Bcl2l1 promoter in the reconstituted MEF cell lines (Data not shown), suggesting that in this context this gene is not a RelA target and the effects we see on its expression are indirect. We also examined the expression levels of the other members of the CXC chemokine superfamily expressed in these cells, before and after TNFα stimulation (Supp. Fig. 1). Interestingly, although some effects with Thr-435 mutation are seen, these are associated with reductions in the levels of gene expression (see Cxcl3 and Cxcl7 for example). However, in this analysis, no other genes were found with the enhanced levels of expression, seen with Cxcl2, associated with Thr-435 phosphorylation. This confirmed that in this context, the effect of Thr-435 phosphorylation on RelA dependent gene expression is highly specific.

Protein occupancy at the Cxcl2 promoter in wild type MEFs.

We next investigated endogenous RelA binding to the Cxcl2 promoter in wild type MEFs. As expected, ChIP analysis revealed enhanced RelA binding following TNFα stimulation (Fig. 6A). Importantly, and consistent with its predicted role as a regulator of Cxcl2 expression, we also detected enhanced phospho-Thr-435 RelA bound to the Cxcl2 promoter following TNFα stimulation (Fig. 6B). Levels of RelA binding to the Cxcl2 promoter, both before and after TNFα stimulation, were comparable between the reconstituted MEFs and wild type MEFs (Supp. Fig. 3). Furthermore, consistent with the earlier data from the reconstituted MEFs, specific Thr-435 dependent effects at the Cxcl2 promoter were also observed in the wild type MEFs after TNFα stimulation, with acetylated Histone H4 levels being significantly enhanced while levels of HDAC1 present were reduced. However, some differences were observed between these experimental systems with acetylated Histone H3 levels not being seen to increase in the wild type MEFs (Fig. 6C-E).

Since this ChIP analysis indicated that Thr-435 modified RelA is bound to the Cxcl2 promoter, we investigated what proportion of total promoterassociated RelA this represented. To determine this, the ChIP samples were immuno depleted with the phospho-Thr-435 antibody prior to RelA immunoprecipitation and PCR analysis. Significantly, preclearing Thr-435 modified RelA negated the TNFα induced increase in RelA binding to the Cxcl2 promoter but had little effect on RelA binding to the Cxcl1 promoter, indicating that a very specific pool of activated RelA binds to and regulates the expression of Cxcl2 (Fig. 6F).

Taken together, these results support the data obtained from the reconstituted MEF cell lines and demonstrate that phosphoryation of Thr-435 plays an important role in the regulation of Cxcl2 mRNA expression in wild type MEF cells following TNFα stimulation.

Discussion

Although Thr-435 had been identified as a putative phosphorylation site in RelA's transactivation domain, subject to dephosphorylation by PP4, this had relied upon in vitro phosphorylation assays and no analysis of modification in cells was performed [START_REF] Yeh | Suppression of MEK/ERK signaling pathway enhances cisplatininduced NF-κB activation by protein phosphatase 4-mediated NF-κB p65 Thr dephosphorylation[END_REF]. Therefore, we generated a phospho specific antibody against Thr-435 which allowed the detection of endogenous RelA phosphorylation at this site following calyculin A treatment of U-2 OS cells (Fig. 1A). Phosphorylation was also detected following TNFα treatment in MEF cells and at the Cxcl1, Cxcl2 and Tnfaip3 promoters following ChIP analysis (Fig. 1C, 6B and Supp. Fig. 2). Although this analysis establishes Thr-435 phosphorylation as a regulator of RelA transactivation, the identitity of the kinase responsible is currently unknown. Phospho-site prediction algorithms identify Thr-435 as a potential CK2 or polo like kinase 1 (PLK1) target. Interestingly, CK2 was previously shown to phosphorylate RelA at Ser-529 in response to TNFα treatment [START_REF] Wang | Tumor Necrosis Factor α-induced Phosphorylation of RelA/p65 on Ser529 Is Controlled by Casein Kinase II[END_REF]. However, phosphorylation of Thr-435 by CK2 could not be verified empirically during the course of this study (data not shown). Identification of the Thr-435 kinase is an important question that should be investigated in future studies. Furthermore, Thr-435 phosphorylation in response to other NF-κB inducers is also currently unknown and will require further analysis. That this site is only weakly phosphorylated after TNFα stimulation in U-2 OS cells implies a possible cell type specific role in vivo.

In contrast with the dramatic transcriptional effects seen with Gal4 fusion proteins (Fig. 1D), full-length RelA versions of Thr-435 phosphomutants did not reproduce significant effects on transcription, when used with a selection of standard NF-κB reporter plasmids (Fig. 1E and data not shown). Consistent with this, analysis of endogenous gene expression revealed that, at least in this context, the effects of Thr-435 phosphorylation are highly promoter specific, suggesting that this modification can help provide the specificity and selectivity required to produce a context dependent NF-κB transcriptional response to a stimulus. Interestingly, the effects seen with Thr-435 mutation on the endogenous Cxcl2 gene were also mirrored with a Cxcl2 promoter luciferase reporter (Fig. 3E). By contrast, other members of the CXC superfamily of genes did not display Cxcl2 like sensitivity to RelA Thr-435 phosphorylation (Supp. Fig. 1) and gene expression array analysis will be required to identify other target genes similarly regulated by modification at this site.

These observations suggest that it is the specific organisation of the Cxcl2 promoter that confers Thr-435 regulation. Importantly, immuno depletion, prior to ChIP analysis revealed that the majority of RelA bound to the Cxcl2 promoter was Thr-435 phosphorylated, in contrast to the Cxcl1 promoter where most RelA was not modified at this site (Fig. 6F). Moreover, the T435D mutant shows elevated levels of Cxcl2 promoter binding, not seen with Cxcl1 or Tnfaip3 (Fig. 3A effect is due to an alteration in site specific RelA DNA binding. Rather, it is more likely to arise through effects on transcriptional coregulators required to appropriately modify Cxcl2 promoter chromatin structure. Such effects may be mediated by the corepressor HDAC1, whose binding to RelA is inhibited by the T435D mutation (Fig. 4G). However, we cannot rule out effects on other interactions and it is entirely possible that Thr-435 phosphorylation not only decreases HDAC1 binding but also enhances binding to an, as yet unidentified, histone acetyltransferase (HAT). We also investigated binding of the CBP and TIP60 HATs to the Thr-435 mutants, as a potential explanation for the variations in histone acetylation observed, but no differences in their occupation of the Cxcl2 promoter were detected in the RelA T435D reconstituted cells and no differences in binding to Thr-435 mutant forms of RelA were observed (data not shown). However other TNFα induced interactions, with additional co-transcriptional proteins such as SIRT6, Pin1, which is required for optimal Cxcl2 expression, and RPS3 [40][41][START_REF] Wan | Ribosomal protein S3: a KH domain subunit in NF-κB complexes that mediates selective gene regulation[END_REF] were not investigated during the course of this study and may also emerge as being regulated by Thr-435 phosphorylation.

Our investigation of the consequences of RelA Thr-435 phosphorylation shows similarities and differences to previous analyses of point mutations to known RelA post-translational modification sites. Phosphorylation at S276 is known to play a role in activating RelA and regulating its interaction with either the co-activators p300/CBP or HDAC corepressors [START_REF] Zhong | The phosphorylation status of nuclear NF-κB determines its association with CBP/p300 or HDAC-1[END_REF]. Reconstituting Rela-/-MEFs, with a non-phosphorylatable S276A mutant form of human RelA, similarly resulted in distinctive expression profiles of NF-κB target genes, potentially due to decreased p300 recruitment [START_REF] Anrather | cis-Acting Elementspecific Transcriptional Activity of Differentially Phosphorylated Nuclear Factor-κB[END_REF][START_REF] Chen | NF-κB RelA Phosphorylation Regulates RelA Acetylation[END_REF][START_REF] Dong | Repression of gene expression by unphosphorylated NF-κB p65 through epigenetic mechanisms[END_REF][START_REF] Nowak | RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes[END_REF]. Furthermore, the S276A mutation was found to severely impair RelA's ability to rescue Rela-/-MEFs from TNFα-mediated cell death [START_REF] Okazaki | Phosphorylation of serine 276 is essential for p65 NF-κB subunit-dependent cellular responses[END_REF], an effect not found with Thr-435 mutation. Phosphorylation at S536 is known to affect RelA's activity and reconstituting Rela-/-MEFs with a nonphosphorylatable S536A mutant form of human RelA was shown to decrease LPS induced NF-κB activation and also to reduce p300 recruitment, subsequently decreasing TNFα activation [START_REF] Chen | NF-κB RelA Phosphorylation Regulates RelA Acetylation[END_REF][START_REF] Yang | IKK β plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide[END_REF]. Reconstituting Rela-/-MEFs with a phospho mimicking S536D mutant produced enhanced binding to TAFII31 and enhanced transcriptional activity, reciprocating effects of IL1 induced NF-κB activation [START_REF] Buss | Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-κB at Serine 536 Is Mediated by Multiple Protein Kinases Including IκB Kinase (IKK)-α, IKKβ, IKKε, TRAF Family Member-associated (TANK)binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATAbinding Protein-associated Factor II31-mediated Interleukin-8 Transcription[END_REF]. Finally, phosphorylation at S468 has been reported to have both inhibitory and activatory effects on transactivation and reconstituting Rela-/-MEFs with a non-phosphorylatable S468A mutant form of human RelA has been shown to have a negative effect on both IL1-and TNFα-induced NF-κB transactivation [19]. TNFα-induced Ser-468 phosphorylation was also found to regulate ubiquitin mediated, proteasomedependent removal of chromatin-bound RelA on certain NF-κB gene targets [13]. However, this effect was not observed for Cxcl2, suggesting other modifications, such as the Thr-435 phosphorylation, identified in this study, will have a primary regulatory role.

Taking these results together with the conclusions of previous studies, it is becoming clearer that phosphorylation of RelA helps to programme its transcriptional functions by determining its activity at select target genes through modulating interactions with co-activators and co-repressors. Here we have identified a role for Thr-435 phosphorylation, which, following TNFα stimulation, modulates the interactions with HDAC1 and selectively regulates NF-κB dependent gene expression. This adds to the number of such modulatory phosphorylation events controlling NF-κB activity. Given the possible side effects associated with total NF-κB inhibition as a clinical therapy, drugs that selectively regulate RelA activity through affecting its posttranslational modifications, have the potential to act as effective therapies for the wide range of diseases associated with aberrant NF-κB activity.

Biochemical

  Journal Immediate Publication. Published on 15 Dec 2009 as manuscript BJ20091630 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091630

  Biochemical Journal Immediate Publication. Published on 15 Dec 2009 as manuscript BJ20091630 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091630A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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  and Supp Fig 2A & B), suggesting that this modification facilitates RelA recruitment to the Cxcl2 promoter. Given the location of this motif in the transactivation domain, we consider it unlikely that Biochemical Journal Immediate Publication. Published on 15 Dec 2009 as manuscript BJ20091630 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091630 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  this

Biochemical

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Biochemical

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Figure Legends Figure 1 :

 1 Figure LegendsFigure 1: RelA Thr-435 is a phospho site that affects Transactivation Domain activity. (A) U-2 OS cells were stimulated for the indicated times with calyculin A (50 nM). 40 µg of whole cell extract was analysed by western blot using the P-Thr-435 or RelA antibodies (upper panel). 350 µg whole cell extract was immunoprecipitated with RelA antibody and samples were western blotted with P-Thr-435 antibody (lower panel). Membranes were then stripped and reprobed with total RelA antibody. (B) U-2 OS cells were stimulated with calyculin A (50 nM) for 15 minutes or TNFα (20 ng/mL) as indicated. 40 µg whole cell extract was western blotted with P-Thr-435 antibody. Membrane was then stripped and reprobed with total RelA antibody. Densitonmetry analysis indicates increase in intensity of the phospho band in relation to untreated sample, normalized to RelA input levels. (C) MEF cells were stimulated with TNFα (20 ng/mL) as indicated. 100 µg whole cell extract was immunoprecipitated with P-Thr-435 antibody and samples were western blotted with total RelA antibody. (D) U-2 OS cells were transfected with 1.5 µg Gal4 E1B luciferase reporter plasmid and 750 pg of expression plasmids encoding the Gal4 DBD, the Gal4 DBD fused to amino acids 428-551 of RelA [RelA TAD], amino acids 428-551 of RelA with Thr-435 mutated to alanine [T435A] and amino acids 428-551 of RelA with Thr-435 mutated to aspartic acid [T435D]. A western blot demonstrating expression levels of Gal4 fusion proteins is shown. Luciferase assay results are expressed as fold activation over Gal4 DBD control, relative to wild type RelA TAD. Presented here are mean ± SEM, n=8, ANOVA was performed followed by a Tukey-Kramer multiple comparisons test using Prism 4 software (GraphPad). * P<0.05 and *** P<0.001 compared to wild type TAD. (E) U-2 OS cells were transfected with 1 µg 3x κB ConA luciferase reporter plasmid and 0.6 µg of expression plasmids encoding empty RSV vector, full length wild type RelA [RelA], RelA with Thr-435 mutated to alanine [T435A] and RelA with Thr-435 mutated to aspartic acid [T435D]. A western blot demonstrating expression levels of RelA proteins is shown. Luciferase assay results are expressed as fold activation over empty RSV plasmid. Data presented here are the mean ± SEM, n=3. ANOVA was performed followed by a Tukey-Kramer multiple comparisons test using Prism 4 software (GraphPad).

Figure 2 :

 2 Figure 2: Creation of stable cell lines constitutively expressing wild type and mutant forms of RelA. (A) Western blot analysis of RelA protein levels in stably reconstituted Rela-/mouse embryonic fibroblasts (MEF). (B) Introduction of RelA protects cells from TNFα-induced cell death. Whole cell extracts were prepared from reconstituted MEF cells stably expressing wild type and mutant forms of RelA, either unstimulated or stimulated with

Figure 5 :

 5 Figure 5: Expression levels of NF-κB target genes following TNFα treatment. (A-C) TNFα stimulation differentially increases mRNA levels of Cxcl2, Cxcl1 and Tnfaip3 in the reconstituted cells. (D) TNFα stimulation does not significantly alter the expression of Bcl2l1 in the reconstituted cells. RNA was extracted from reconstituted MEFs cells, either unstimulated or stimulated with TNFα (40 ng/mL) for 30 minutes. Total cDNA was prepared and qPCR analysis was performed using primers to mouse (A) Cxcl2, (B) Cxcl1, (C) Tnfaip3, (D) Bcl2l1 and Gapdh control.All results were normalized to Gapdh levels and expressed as fold induction relative to the level of gene expression in control cell line. Data presented here are the mean ± SEM, n=3. Two-way ANOVA was performed followed by a Bonferroni post-test to compare replicate untreated and TNFα means, using Prism 4 software (GraphPad). * P<0.05 and ** P<0.01.

Figure 6 : 2 Biochemical

 62 Figure 6: ChIP analysis of the murine Cxcl2 promoter in wild type MEFs. (A&B) TNFα stimulation increases binding of RelA and RelA phosphorylated at Thr-435 to the Cxcl2 promoter region. (C) TNFα stimulation does not significantly increase levels of acetylated Histone H3 (K9/14) at the Cxcl2 promoter. (D) Elevated levels of acetylated Histone H4 (K12) were found at the Cxcl2 promoter following TNFα treatment. (E) HDAC1 binding to the Cxcl2 promoter was reduced after TNFα treatment. qPCR analysis was performed using primers to the mouse Cxcl2 initiation site. (F) Depleting samples of RelA phosphorylated at Thr-435 with phosphospecific antibody prior to performing ChIP analysis negates RelA binging to the Cxcl2 promoter, but not the Cxcl1 promoter following TNFα treatment. All results were normalized to Input levels and control antibodies. Data presented here are the mean enrichment levels relative to Untreated cells ± SEM, (A-C) n=7, (D&E) n=4, (F) n=3. Students t test was performed using Prism 4 software (GraphPad). * P<0.05, ** P<0.01 and ***P<0.001.

  Biochemical Journal Immediate Publication. Published on 15 Dec 2009 as manuscript BJ20091630 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091630 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We thank Mary Collins, Ron Hay, Michael Hottiger and Tony Kouzarides for supplying reagents and all members of the NDP Laboratory for their advice and support. We thank Sonia Rocha and Stephan Roberts for their critical reading of this manuscript.

funded by a Wellcome Trust Four-Year studentship at the University of Dundee and by Cancer Research UK [Programme Grant C1443/A4201] at the University of Bristol.

Funding

TNFα (40 ng/mL) for 4 hours. Western blot analysis was performed for PARP cleavage. (C) TNFα-induced DNA binding was not affected by mutating Thr-435. Nuclear protein extracts were prepared from reconstituted MEF cells stimulated with TNFα (40 ng/mL) for the indicated times and 5 µg nuclear extract was subjected to EMSA analysis using 32 P labeled IL8 and OCT1 oligonucleotides.


Balazs Visegrady
email: balazs.visegrady@aok.pte.hu

Laura M Machesky
email: l.machesky@beatson.gla.ac.uk

Tomczak, K. K V L Lehtokari

Myopathy-causing Actin Mutations Promote Defects in Serum Response Factor Signaling

AM-Actin myopathy NEM-Nemaline Myopathy IRM-Intranuclear Rod Myopathy ACTA1-alpha skeletal muscle actin MADS box -a transcription factor named after the genes in which the sequence element for recognition was first identified (MCM1, Agamous, Deficiens, SRF) SRF-serum response factor MAP kinase-mitogen activated protein kinase Crm1-chromosome region maintenance 1 protein Cyt D-cytochalasin D Jasp-Jasplakinolide LMB

Introduction

Mutations in skeletal muscle actin and actin myopathy

Nemaline myopathy is a congenital neuromuscular disorder, which results in muscle weakness and wasting similar to the more common and more well-studied disease muscular dystrophy. Nemaline myopathy is diagnosed partly by the presence in muscle biopsies of nemaline bodies, inclusions of sarcomere proteins deposited in the muscle tissue around the z-line. The appearance of the muscle biopsy specimens has been used to classify these myopathies further into three subclasses: AM actin myopathy, IRM intranuclear rod myopathy and NEM nemaline myopathy. NEM, AM and IRM are caused primarily by mutations in the muscle thin filament proteins, namely nebulin, alpha skeletal muscle actin (ACTA1), -and -tropomyosin, troponin-2 and perhaps surprisingly, cofilin-2 [1], which is not a typical thin filament protein, reviewed in [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF]. Mutations in ACTA1 account for around 20% of NEM patients and the mutations span more than 140 different residues of the ACTA1 protein [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF][START_REF] Laing | Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1)[END_REF]. The disease manifests itself in various forms, ranging from severe, where infants are dependent on a respirator from birth, to mild, where the symptoms present during childhood and patients have relatively normal motor milestones, but even the mild disease may be progressive [4].

The mutations in ACTA1 that cause NEM, AM or IRM do not obviously localise to any specific region(s) of the actin molecule, but rather they are found throughout the 3D structure [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF][START_REF] Laing | Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1)[END_REF][START_REF] Sparrow | Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1)[END_REF]. This may help to explain why thus far characterization of more than 30 different actin mutants that cause NEM, AM or IRM have not shown very clear correlations between the disease and any specific biochemical or cell biological defect of the mutant actin. It may also be that 30 mutants is still such a small proportion of the total number described that we need to analyse more mutations before a clear pattern emerges. With these caveats in mind, some interesting patterns have emerged, such as the observation that ACTA1-L94P and ACTA1-G259V, which cause severe recessive myopathy, show protein folding defects leading to lack of functional muscle actin [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]. Around half of the mutants tested thus far show a Biochemical Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited reduced ability to co-polymerize with wild-type actin [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF]. Many of the mutants also form aggregates and rods in the nucleus and cytoplasm of cells in culture that are reminiscent of those seen in patient biopsy samples [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF]. However, whether these structures are related to nemaline bodies in more than just appearance remains to be explored. Expression of several different mutant actins leads to cell death with cell rounding and bleb formation, suggesting that apoptotic cell death may be a major feature of muscle wasting seen in NEM patients [START_REF] Vandamme | alpha-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells[END_REF].

Muscle differentiation and development is regulated by multiple transcription factor families. SRF, serum response factor is a MADS box transcription factor, sharing a common DNA-binding motif with other MADS box proteins, that is highly expressed in skeletal muscles and controls growth and differentiation in multiple tissues. One of the main mechanisms of SRF-mediated activation is through recognition and binding with a DNA motif known as a CArG box (Figure 1E). The CArG box is a 10 base pair sequence with the consensus CCW 6 GG [START_REF] Sun | Defining the mammalian CArGome[END_REF]. Several genes whose expression is regulated by SRF binding to CArG boxes in their promoter region have been identified. Many of these targets affect muscle growth and development, including ACTA1 itself, muscle creatine kinase, dystrophin, tropomyosin, myogenin, MyoD and myosin light chain 1/3 [START_REF] Arsenian | Serum response factor is essential for mesoderm formation during mouse embryogenesis[END_REF]. SRF also forms complexes with other transcription factors involved in muscle development, such as myogenin and MyoD [10]. SRF activates the expression of c-fos, another transcription factor that regulates proliferation and differentiation [START_REF] Treisman | Ternary complex factors: growth factor regulated transcriptional activators[END_REF]. At the promoter of c-fos, SRF interacts with other transcription co-factors such as members of the ternary complex factor (TCF) proteins and controls transcription via MAP-kinase signaling [START_REF] Treisman | Ternary complex factors: growth factor regulated transcriptional activators[END_REF]. Myocardin related transcription factors also interact with SRF in muscle-specific Rho signaling and actin assembly pathways that are independent of its control by TCF [12][START_REF] Miralles | Actin dynamics control SRF activity by regulation of its coactivator MAL[END_REF][START_REF] Selvaraj | Megakaryoblastic leukemia-1/2, a transcriptional co-activator of serum response factor, is required for skeletal myogenic differentiation[END_REF]. Overall, SRF is involved in many transcriptional signaling cascades and cooperates with other transcription factors to regulate fundamental elements of embryogenesis, including muscle differentiation and growth.

The co-factor for SRF activation, MAL, has three actin binding RPEL sequences and binds directly to unpolymerized (globular, or G-) actin (Figure 1E). Activation of actin assembly via serum stimulation leading to Rho GTPase activation causes MAL release from the G-actin as it polymerises and allows the released MAL to activate SRF-mediated transcription. Recent studies show that high rates of nuclear export via the Crm1 exporter maintain MAL in the cytoplasm of resting cells [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF] and Figure 1E. G-actin sequesters MAL in the nucleus and in the cytoplasm and prevents SRF activation (Figure 1E). Stimulation by serum blocks export of MAL and induces its accumulation in the nucleus. Simultaneous depletion of free nuclear and cytoplasmic actin via activation of actin assembly and also regulation of the MAL-actin interaction (by an unknown mechanism) leaves more free nuclear MAL that can form active complexes with SRF for transcription [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF] (Figure 1E). It is not understood how actin regulates nuclear export of MAL or whether actin-MAL complexes travel together when going into and out of the nucleus [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF].

To date, many myopathy-causing actin mutations have been found to affect actin assembly and cytoskeletal dynamics in cultured cells [START_REF] Feng | Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies[END_REF]. Since the actin cytoskeleton is intimately linked with the SRF (serum response factor) pathway, we hypothesized that patients with actin mutations could have defects in SRF signaling as well as in actin assembly and cytoskeletal function in their muscle cells. We therefore A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited investigated the effects of 12 mutants of ACTA1 that have previously exhibited a range of biochemical and cell biological defects, to look for defects in SRF signaling. Based on our in vitro studies, we postulate that patients with actin mutations may have defects in SRF signaling that could lead to defects in muscle growth and development and we propose that this is a potential previously overlooked side-effect of actin mutation that should be explored further for possible therapeutic benefit in myopathy patients.

Results

Actin myopathy mutations affect the serum response factor signaling pathway in C2C12 myoblasts

When SRF signaling is activated by serum stimulation, cells show increased localization of MAL in the nucleus and enhanced transcription of SRF reporter genes [START_REF] Sotiropoulos | Signalregulated activation of serum response factor is mediated by changes in actin dynamics[END_REF] (Figure 1E). We hypothesized that mutations in actin that cause myopathy might affect the SRF transcriptional programme either directly via altered interactions of the mutant G-actin with MAL or indirectly via disruption of normal cellular actin dynamics. We monitored the nuclear accumulation of MAL in response to serum stimulation using an immunoflourescence microscopy assay and we measured SRF activity in cells expressing mutant actins with a biochemical luciferase reporter assay [12]. Cells expressing only wild type ACTA1 showed a dramatic relocalisation of MAL to the nucleus following serum stimulation, with 5% of resting cells and around 80% of stimulated cells showing nuclear MAL accumulation (Figure 1A andB). The luciferase activity in this assay increased around 2.5-fold for cells expressing wildtype ACTA1 upon serum stimulation (Figure 1 C and D). Cells expressing mutant actins displayed cytoplasmic or nuclear rods or aggregates as previously reported [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF] (e.g. Fig. 1.A, ACTA1-H40Y green nuclear rods) as well as some localization of mutant actin to normal actin structures (e.g. Figure 1A and [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]). We did not generally find any colocalisation between actin stress fibres, rods or aggregates and MAL, which localised evenly in the cytoplasm and the nucleus (Figure 1A). Cells expressing any of the mutant actins showed less nuclear accumulation of MAL in the presence of serum than cells expressing wild-type actin (Figure 1B). The luficerase reporter assay, which measures the level of SRF activation, also reflected this trend (Figure 1C andD) with only one mutant, ACTA1-I357L showing slightly higher than normal levels of SRF activation. In the absence of serum, cells expressing most of the mutants had a similar level of SRF activation to the wild-type except one construct, the ACTA1-H40Y which had an approximately 2-fold higher number of cells showing nuclear accumulation of MAL (Figure 1B) but still a slightly reduced or similar level of luciferase reporter activity (Figure 1C). The mutant ACTA1-R183G showed nearly no serum-stimulation of nuclear accumulation of MAL and only modest stimulation of luciferase SRF reporter activity (Figure 1B,C).

Since mutant ACTA1-H40Y showed an increased basal level of nuclear MAL accumulation and a somewhat reduced response to serum stimulation and mutant ACTA1-R183G was severely blunted in serum responsiveness, we characterized these mutants further. We compared ACTA1-H40Y with ACTA1-G15S (Fig. 2 A), a mutant not associated with disease, but previously shown to promote the nuclear accumulation of MAL in cultured cells and elevate basal activity of SRF but not serum responsive activity [START_REF] Posern | Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL[END_REF]. We found 50% nuclear MAL accumulation caused by ACTA1-G15S that does not change after treating cells with serum. In contrast, cells expressing ACTA1-H40Y show 60% and 25% nuclear MAL localised with and without serum, respectively, so ACTA1-H40Y appears to be somewhat more sensitive to serum stimulation than ACTA1-G15S but less than the wild type actin. It is interesting to note that ACTA1-G15R, a naturally occurring nemaline myopathy mutant, retains some serum sensitivity, unlike ACTA1-G15S (Figure 1). We treated the cells in presence of serum with the actin monomer sequestering drug LatB to find out whether the nuclear MAL translocates back to the cytoplasm to interact with the higher concentration of G-actin induced by LatB. LatB treatment results in rapid cytoplasmic relocalization of MAL in serum treated wild type cells ( [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF] and Figure 1E). We did not find any change in the distribution of MAL after 40 min LatB treatment of actin ACTA1-H40Y cells (Figure 2A). Thus we conclude that ACTA1-H40Y mutant actin promotes a higher than normal accumulation of MAL in the nucleus, but does not increase SRF reporter activity and is relatively insensitive to LatB treatment. Perhaps the LatB fails to induce depolymerisation of ACTA1-H40Y and thus cells expressing ACTA1-H40Y fail to show cytoplasmic sequestration of MAL in response to LatB.

Since actin cycles through the nucleus and the MAL-actin complex is exported via the nuclear exporter Crm1 ( [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF] and Figure 1E), we tested the effect of inhibiting Crm1 on the nuclear localization of ACTA1-R183G to determine whether this mutant is preventing MAL accumulation in the nucleus or is causing more rapid export of MAL. We found that without serum approximately 30% of cells show accumulation of MAL in the nucleus and this doesn't significantly change with addition of LMB for 5 or 15 minutes (Figure 2B). Thus we conclude that actin R183G is inhibiting the import of MAL into the nucleus.

Response of cells expressing myopathy mutant ACTA1 to cytochalasin D or jasplakinolide treatment

We used Cytochalasin D (CytD) and jasplakinolide (Jasp) to further probe the defects in the SRF response that we observed with the myopathy mutant actins. CytD and Jasp both induce rapid nuclear accumulation of MAL in wild type cells (Figure 1A) [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF]. Both drugs are actin-specific and commonly used to alter cytoskeletal dynamics. However they act in completely different ways; Jasp stabilizes actin filaments while CytD binds to barbed ends of filaments and prevents polymerization, as well as having some interactions with actin monomers and oligomers at higher concentrations [START_REF] Sampath | Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments[END_REF]. Jasp induces nuclear MAL via stabilization of filaments and dissociation of actin monomer from MAL to allow faster nuclear import [START_REF] Miralles | Actin dynamics control SRF activity by regulation of its coactivator MAL[END_REF][START_REF] Sotiropoulos | Signalregulated activation of serum response factor is mediated by changes in actin dynamics[END_REF]. The mechanism by which CtyD induces nuclear MAL accumulation is less clear, but it appears to disrupt the MAL-actin complex and promote MAL accumulation in the nucleus [START_REF] Sotiropoulos | Signalregulated activation of serum response factor is mediated by changes in actin dynamics[END_REF]. The actin myopathy-related mutations investigated in this study would not be predicted to alter the binding site of Jasp or CytD, based on their position in the 3D structure of ACTA1 [START_REF] Lorenz | Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm[END_REF]. We tested the mutant actin constructs using 15 min drug treatment, where significant effects of each drug on the actin cytosketon were observed (data not shown) (Fig. 3). All the mutants tested show a similar response to Jasp or CytD treatment with respect to the appearance of F-actin in phalloidin stained cells (data not shown). Cells expressing G15R, H40Y, I64N, and N115S mutant ACTA1 show lower activation of SRF in the presence of either drug, while ACTA1-R183G and ACTA1-D286G show higher SRF reporter gene activation than the wild type ( A c c e p t e d M a n u s c r i p t expressing ACTA1-I136M, G182D and I357L show SRF overactivation while M132V and G268R have wild type like activation. Thus we conclude that a subset of myopathy mutations induce significant alterations in SRF activity and that different mutants have different sensitivity to the actin-binding drugs Jasp and CytD. In general, 9/12 mutants showed a reasonable drug response, indicating that whatever the cause of low SRF activity, it could be partially overcome with drug treatment to disrupt the MAL-actin interaction. This could represent an interesting opportunity for further studies on the effect of drug treatment on altered transcriptional programmes of myopathy muscles.

Interactions of mutant actin with SRF RPEL 2 domain

One explanation for the altered SRF response in cells expressing mutant actins might be that mutations in actin cause altered binding to MAL. Mutants showing increased affinity for MAL would be expected to have an inhibitory effect on the nuclear translocation and/or on SRF activation once translocated (Figure 1E). Mutants showing decreased affinity for MAL might be expected to promote increased basal levels of MAL activation and decreased overall serum responsiveness. We thus tested whether some of the myopathy mutants that showed altered SRF reporter responses showed altered binding capacity to the actin-binding RPEL domain of MAL. GSTtagged RPEL 2 domain containing RPEL 2 and 3 motifs bound to agarose beads were incubated with in vitro translated wt or mutant actin. Seven actin mutants which had impaired activation of the SRF response, MAL accumulation or drug response were tested (Figure 4). We found that all the mutants tested could bind to the RPEL 2 domain. Two mutants, G268R and D286G bound with relatively higher affinity while H40Y showed lower affinity for RPEL 2 than the wild type. Thus these three mutants may show altered SRF activity due to a direct change in their affinity for MAL.

Changes in levels of G-actin/F-actin ratio in cells expressing mutant actins

Another possible explanation for how mutant actins might affect SRF activity is that their expression in the context of wild-type actin co-expression (as occurs in most myopathy patients) could alter the balance of polymerized F-actin and unpolymerized G-actin. Excessive G-actin might sequester MAL in the cytoplasm or nucleus and prevent serum response factor activation of transcription [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF] and Figure 1E. Since many myopathy mutants showed a lessened ability to copolymerize with wild-type actin in biochemical assays [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF], we tested the effect of mutant actin expression on the G/F actin ratio in cells using a previously established flow cytometry sorting assay based on fluorescently labeled DNAse-I binding to the G-actin and fluorescently labeled phalloidin binding to the F-actin as a readout of levels [START_REF] Posern | Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor[END_REF]. Of five mutants tested (G182D, I136M, G268R, I357L and D286G), we found that the G/F actin ratio was only significantly altered in G268R, with a 1.4-fold increase in the G-actin (Fig. 5). Thus, expression of mutant actins does not seem to generally cause a significant alteration in the balance between G/F actin, but may in some cases increase the Gactin pool, which could potentially contribute to low serum responsiveness.

Discussion

Our study suggests that the ability of mutant actins to activate SRF responses in developing muscle might be a major, yet thus far relatively unexplored, area for consideration in understanding actin-based myopathies. The idea that altered SRF signaling could cause myopathy symptoms has precedent in the literature. Charvet and colleagues used a skeletal muscle specific deletion of SRF to show that mutant mice displayed growth retardation and loss of muscle mass when SRF was deleted in post-mitotic myofibers, but not satellite cells (muscle stem cells) [START_REF] Charvet | New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways[END_REF]. Mice showed severe myofiber hypotrophy, which resembled myopathy. SRF depletion altered the overall gene expression patterns of the mutant muscle, with upregulation of myogenin and downregulation of many of the genes controlled by SRF in vitro [START_REF] Charvet | New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways[END_REF]. This suggests to us that altered SRF responses in patients with actin-based myopathy could lead to altered gene expression programmes and a similar muscle degeneration phenotype. Strikingly, the authors described that SRF-depleted myofibers had extensive sarcomere disorder and they pointed out that they frequently observed "numerous fragments of Z-disks resembling rods that are observed in nemaline myopathies (NEM) and are thought to be accumulations of sarcomeric proteins." They pointed out that defects closely resembled the phenotype of patients with NEM caused by mutations in thin filament protein encoding genes and that several of the thin filament encoding genes have CArG (SRF response) boxes in their promoters [START_REF] Charvet | New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways[END_REF]. Thus mutant actin expression could alter major gene expression programmes in the muscle and possibly enhance the myopathy phenotype.

The control of SRF by actin is a complex cycle and is still an area of active investigation, but a molecular understanding of how actin dynamics modulates SRF transcription is emerging [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF][START_REF] Posern | Actin' together: serum response factor, its cofactors and the link to signal transduction[END_REF]. We explored the molecular mechanisms of effects of mutant actins on SRF stimulation for selected mutants that showed alterations in SRF responses. In particular, we showed that mutant ACTA1-H40Y, which conferred on cells a higher than normal basal level of SRF activity (Figure 1), showed low serum responsiveness and low serum-stimulated nuclear MAL accumulation. The inability of LatB to remove ACTA1-H40Y from the nucleus may be due to reduced binding of the drug to mutant actin or to the tendency of ACTA1-H40Y to form nuclear rods (See Figure 1A and [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]) that could be more resistant to LatB than single filaments. However, since ACTA1-H40Y still retains around half of the wild-type level of serum responsiveness, it is only partially interfering with the ability of wildtype actin to respond in the SRF pathway and in the absence of serum, it actually promotes SRF activity. Cells expressing this mutant retained relatively normal enhancement of SRF activity in response to Jasp and CytD (Figure 3), indicating that it did not have a strong dominant negative effect on overall ability of cellular actin to polymerize (Jasp) or of CytD to promote nuclear MAL translocation and SRF activation. Interestingly, H40Y actin showed some reduction in MAL binding in an in vitro translation assay, indicating that lower affinity for MAL could be the cause for the observed higher than normal basal SRF activity of the mutant (Figure 1C). This is the opposite of G15S actin, which is reported to have a higher than normal affinity for MAL [START_REF] Posern | Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL[END_REF]. This is the first in vitro defect identified for H40Y actin to our knowledge, as it showed relatively normal responses in the actin dynamics assays we carried out previously [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF] despite the appearance of nuclear rods. We conclude that patients with H40Y mutations are good candidates to test for altered SRF activity in their affected muscle tissue, since both basal and stimulated activity is affected by this mutant actin.

Another mutant that we chose for further study was R183G, which showed significantly reduced nuclear MAL accumulation and SRF serum responsiveness in expressing cells (Figure 1). Wild-type actin is normally shuttled out of the nucleus a nuclear export signal and via the CRM1 exporter (Figure 2C and [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF]). Nuclear export of actin promotes the low basal SRF activity of cells with wild type actin, as inhibiting CRM1 with leptomycin B causes an increase of nuclear actin-MAL complex (Figure 1E and [START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF][START_REF] Posern | Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor[END_REF]). However, MAL did not accumulate in the nucleus in response to LMB in the R183G mutant expressing cells, indicating that the defect is likely to be due to reduced entry of MAL into the nucleus in R183G expressing cells. R183G actin expressing cells showed a relatively normal response to Jasp treatment, indicating that this actin is capable of polymerization induced by Jasp and thus nuclear accumulation of MAL. This mutant also had a relatively normal response to CytD, indicating perhaps that the normal serum-induced or signal-induced actin assembly pathway of this mutant is aberrant, but that drug stimulation can overcome this deficiency. ACTA1-R183G showed normal binding to the MAL RPEL domain in vitro, as well. Taken together, the data suggest that it could be worth investigating a possible aberrant interaction with the actin binding protein STARS, which mediates the dissociation of MAL-actin complexes in response to serum stimulation to promote nuclear MAL accumulation and SRF activation [12]. This also suggests that patients with ACTA1-R183G could likely have defective SRF responses.

Cells expressing ACTA1-G268R had increased levels of G-actin, perhaps leading to sequestration of MAL in the cytoplasm. ACTA1-G268R showed reduced ability to copolymerize with wild type actin in a previous study [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF] and here we found that expression of ACTA1-G268R led to an increase in the G/F actin ratio in cells and a lower than normal serum response in an SRF reporter assay. ACTA1-G268R was not defective in binding to the MAL RPEL motifs, nor to other actin binding proteins, nor apparently was it unable to polymerize in response to addition of jasplakinolide to cells (Table 1). This mutant likely binds to MAL and sequesters it in the cytoplasm, thus dampening the SRF response, due to its relatively weaker ability to polymerize in response to signals that activate Rho.

Overall, most of the mutants that we tested showed a dampened SRF response pathway, with the majority having lower nuclear MAL accumulation and lower SRF reporter activity in response to serum stimulation. These mutants also mostly had previously shown actin co-polymerization defects in biochemical assays [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]. All of the mutants that we tested were capable of interaction with the MAL RPEL motif and indeed previous studies showed interaction with vitamin D binding protein, which interacts with a similar site to MAL in the hydrophobic cleft and close to the subdomain 3 ledge [START_REF] Mouilleron | Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL[END_REF]. Significantly, 5 mutants (V163L, R183G, G268R, H40Y and D286G) showed low serum response in the SRF reporter assay but a significant increase in SRF activity upon treatment with Jasp or CytD, indicating that even though the actins conferred a defect in SRF responsiveness to serum, this could be overcome with drug treatment. Thus, we conclude that mutations which lead to reduced polymerization capacity in general might cause a dampened SRF response in myopathy patients and that perhaps the SRF response ought to be further investigated to see if enhancement SRF pathways via drug treatment could be of any benefit therapeutically. Some of the mutants, V163L, I136M, G268R, H40Y and I64N have been reported to cause blebbing and eventual cell death when expressed in cultured myoblasts over several days and after the cells fuse to form myotubes [START_REF] Vandamme | alpha-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells[END_REF]. It is also possible that the lowered SRF response in some of these mutants (all but I136M, which had a normal SRF response) could be contributing to the cell death phenotype observed and that cell survival may be a major factor which is affected by SRF signaling.

The idea that patients with NEM have altered gene expression programmes is not new. A study of 10 patients with actin mutations leading to NEM showed that they had altered levels of several sarcomeric proteins by western blot analysis [START_REF] Ilkovski | Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms[END_REF]. Some upregulated proteins (e.g. -filamin, -actinin, myotilin and desmin) may accumulate in the nemaline bodies deposited near the z-disks, while other proteins, such as nebulin were found to be reduced. Perhaps interestingly, desmin [START_REF] Mericskay | An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells[END_REF], -filamin and -actinin [START_REF] Sun | Defining the mammalian CArGome[END_REF] are CArG box proteins and are thus likely to be regulated by SRF. The group of Beggs has done expression profiling of NEM muscle samples from patients and found that they have altered satellite (muscle stem cell) cell numbers and altered glycolytic enzyme transcription [26]. However, they did not specify the nature of the mutation (e.g. actin versus other thin filament proteins) and they did not also highlight whether SRF responsive genes were specifically affected. Expression of ACTA1 in myoblasts changed the gene expression patterns for a number of muscle genes [START_REF] Gunning | Alpha-skeletal actin induces a subset of muscle genes independently of muscle differentiation and withdrawal from the cell cycle[END_REF] and the authors pointed out that this may be through regulation of G/F actin ratios which will affect SRF signaling. Now that a comprehensive CArG-ome of CArG box containing SRF responsive genes has been defined at least by one group [START_REF] Sun | Defining the mammalian CArGome[END_REF] it will be interesting to check whether there is a trend in any particular actin mutations of myopathy patients to correlate with altered expression of CArG-ome genes.

Materials and methods

Plasmids

Expression vectors encoding C-terminal EGFP-tagged or N-terminal Myc-tagged wt or ACTA1 mutations were made with the Quick Change site directed mutagenesis kit (Stratagene, UK) as described previously [START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]. The expression vectors for pEF-FLAG-MAL and the luciferase reporter SM22 promoter were reported previously [START_REF] Arai | STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription[END_REF][START_REF] Li | Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells[END_REF]. C-terminal EGFP-tagged ACTA1 G15S mutant derivative was made with pEGFP vector as template, a 5' primer containing XhoI and a 3' primer containing EcoRI site and appropriate primers.

Cell culture and transfection C2C12 cells were cultured at 37 ºC in humidified 5% CO 2 incubator in a proliferation medium composed of Dulbecco's modified Eagle's medium (DMEM 41966, Gibco BRL, UK) supplemented with 10% fetal bovine serum (Sigma, UK). Transfections were made using Lipofectamine 2000 transfection reagent (Gibco BRL, UK) according to the manufacturer's protocol. Cells were generally used for experiments 48 hours after transfection.

Immunofluorescence and microscopy C2C12 cells were plated on glass coverslips than washed three times in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Free aldehyde groups were blocked with 50 mM NH 4 Cl for 10 min and cells were permeabilized with PBS containing 0.1% Triton X-100 for 4 min. Cells were incubated with 9E10 anti-Myc antibody for 20 min at room temperature followed by Texas-red anti-mouse secondary antibody. For FLAG detection cells were blocked with 10% bovine serum albumin (BSA) and were incubated with M2 monoclonal anti-FLAG antibody for 2 h containing 3% BSA. Cells were washed with PBS for 5 min between each step. Immunostained cells were examined with Zeiss microscope (Zeiss, Jena, Germany) using 63x objective. At least 100 cells were counted in each independent experiment (or 20 cells in the case of control experiments) and statistics were calculated using three independent transfections. Images were recorded with a Hamamatsu C4880 camera (Bridgewater, NJ) and processed using Photoshop software (Adobe, USA).

Luciferase assay C2C12 cells (10 x 10 5 cells/well) were plated on 6-well plates and were cotransfected with encoding vectors of 3 g wt or mutants ACTA1, 3 g FLAG-MAL and 1 g luciferase reporter SM22. After 12h incubation the cells were serum starved for 24 hours and 20% serum was added for 4 hours. In case of drug treatment after 24 hours serum starving 2.5 M cytochalasin D (CytD), 0.5 M jasplakinolide (Jasp), 20 nM leptomycin B was added to the cells for the time specified separately. 1 M latrunculin B (LatB) was applied after 4 hours 20% serum treatment. Finally the cells were harvested and luciferase activities were measured according to the manufacturer's instructions. All the assays have been performed three times and the values have been normalized to the values obtained for wild type actin.

GST-pulldown assay

In vitro transcription translation reaction was used to express 35 S labeled wt or mutant actin in reticulocyte lysate (Promega, UK) according to manufacturer's instructions.

In the reactions 0.02 Ci 35 S -methionine and 1 g DNA per 25 l reaction was used. After 1.5 hours incubation at 30°C the products were analyzed on sodium dodecyl sulfate (SDS) gel followed by autogradiography. The Pfam-defined RPEL2 and RPEL3 motifs (RPEL 2 domain) containing 85 amino acids from position 21 in MAL isoform 1 has been cloned into pGEX-4T-2 vector as template, a 5'primer containing the EcoRI and the 3' primer containing the XhoI site. The RPEL 2 domain protein has been expressed in BL21 cells than bound to GST-beads. 25 l of the in vitro transcription translation reaction of wt or mutant actin was centrifuged at 100,000 rpm for 20 min in a Beckman ultracentrifuge to remove aggregates. 5 l of the supernatant was taken for control. The supernatant then was incubated with 10 g of RPEL 2 domain bound to agarose beads for one hour at room temperature. The agarose beads were sedimented and the supernatant was removed. The agarose beads were washed three times then resuspended in 20 l PBS. The final pellets, supernatants and control were analyzed on 4-12% sodium dodecyl sulfate (SDS) gel followed by autoradiography. The band intensities were measured with ImageJ software and the values were normalized to the control. All mutants were analyzed three times. Statistical analysis was made at P < 0.05 using the Student's t-test in Microsoft Excel.

Flow cytometry 5x10 5 C2C12 cells were transected with Myc-tagged ACTA1 constructs. Cells were pelleted at 300g for 5 minutes than resuspended in 100 µl 4% paraformaldehyde for 10 min. Cells were washed with 1 ml FACS buffer (5% fetal calf serum, 0.02% NaN 3 , 0.5% saponin (Invitrogen) in PBS) and sedimented. 9E10 anti-Myc primary antibody was applied for 30 min, followed by Marina blue conjugated anti-mouse secondary

Data Analysis and Statistics

At least three independent experiments were performed for each assay. Error bars are standard deviations in all cases. The statistical analysis was made at P < 0.05 with the unpaired Student's t-test using Microsoft Excel. [START_REF] Posern | Actin' together: serum response factor, its cofactors and the link to signal transduction[END_REF][START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF]). Arrow 1 shows the G-actin-MAL complex dissociating as actin polymerises and MAL is released. This free MAL then diffuses into the nucleus to interact with SRF and co-activate transcription. This is the reaction induced by serum and also by drug treatment with Jasplakinolide (Jasp) or cytochalasin D (CytD

Biochemical

  Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641

Biochemical

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  Fig 3 A). Cells Biochemical Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641

Biochemical

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Biochemical

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  via

Biochemical

  Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited Finally, coverslips were washed three times in water and mounted onto a slide with 5 l Mowiol (Calbiochem) plus Antifade.

Biochemical

  Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited antibody, Alexa fluor 488 deoxyribonuclease I and Texas Red-X phalloidin (Invitrogen) was added for 30 min. Cells were washed and sedimented between each step. The samples were analyzed with BD FACS Aria cell sorter system. 10x10 3 blue positive cells were used for analysis and the green (530nm) and red (610nm) intensities were measured and the average values were calculated.

Figure legends Figure 1

 1 Figure legends Figure 1 Cells expressing mutant actin show impaired subcellular MAL distribution and SRF activation. (A) Representative immunofluorescence pictures of C2C12 cells cotransfected with EGFP-ACTA1 and mutants (wt-ACTA1, ACTA1-H40Y, ACTA1-R183G) or Myc-actin (green) and MAL (red) in response to serum stimulation. (B) Nuclear MAL accumulation in presence (black bars) or absence (white bars) of serum expressing wt or mutant EGFP-ACTA1 and MAL revealed by counting the cells observed in immunofluorescence (At least 100 cells were counted per experiment and three independent experiments were performed). Serum induced SM22 luciferase activation of C2C12 cell line expressing GFP-(C) or Myc-ACTA1 (D), MAL and SM22 luciferase reporter. Error bars are standard deviations from three independent experiments. Asterisks indicate significant difference from the control value at P< 0.05 in the unpaired Student's t-test. (E) Schematic showing the relationship between actin assembly/disassembly and nuclear shuttling of MAL (summarized from[START_REF] Posern | Actin' together: serum response factor, its cofactors and the link to signal transduction[END_REF][START_REF] Vartiainen | Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL[END_REF]). Arrow 1 shows the G-actin-MAL complex dissociating as actin polymerises and MAL is released. This free MAL then diffuses into the nucleus to interact with SRF and co-activate transcription. This is the reaction induced by serum and also by drug treatment with Jasplakinolide (Jasp) or cytochalasin D (CytD). Arrow 2 shows that when actin depolymerises, due to serum starvation or treatment with Latrunculin B (LatB), actin monomer can sequester MAL in a complex that cannot participate in SRF co-activation. Arrow 3 shows that free MAL can enter the nucleus, where free MAL interacts with SRF and actin bound MAL does not. It isn't known if actin-MAL complexes can enter the nucleus together, but there is actin in the nucleus and it can form complexes there with MAL. Arrow 4 shows that free MAL can be exported from the nucleus by the CRM1 transporter and that this reaction is inhibited by leptomycin B (LMB). It is unclear whether actin-MAL complexes can be co-transported out of the nucleus. Arrow 5 shows that free nuclear MAL can interact with SRF and mediate transcription of CArG box containing target genes.

Figure 2

 2 Figure 2 Effect of mutants H40Y and R183G on nuclear MAL accumulation. C2C12 cells were tested for nuclear MAL after expressing MAL and wt, G15S or H40Y ACTA1 (A) or ACTA1-R183G (B) and treated with serum and latrunculin B (LatB, 1 M) or leptomycin B (LMB, 20 nM) as indicated. Wild type (wt) or G15S ACTA1 were used as comparisons for ACTA1-H40Y. At least 20 cells were counted per experiment and three independent experiments were performed per condition. Error bars are standard deviations.

Figure 3

 3 Figure 3 Different responses of myopathy mutants to disruption of the actin cytoskeleton SRF activation was monitored after jasplakinolide (white bars) or cytochalasin D (black bars) treatment. C2C12 cells were cotransfected with MAL, luciferase-reporter SM22 and GFP-actin (A) or Myc-actin (B). After serum starvation for 15 minutes, jasplakinolide (0.5 M) or cytochalasin D (2.5 M) treatment was applied. The luciferase activation is shown relative to cells expressing wt-ACTA1 in serum-starved conditions. Error bars are standard deviations from at least three independent experiments and asterisks label the values statistically different from the control value with significance at P < 0.05 (unpaired Student's t-test).

Figure 4

 4 Figure 4 Actin binding to MAL-RPEL 2 domain is not greatly altered for selected mutants (A) The 35 S-labelled wt or mutant actin from in vitro translation assays was incubated with GST-RPEL 2 domain bound to agarose beads and then sedimented. The pellet has been analyzed on SDS page and exposed to film and the band intensities were measured. (B) Error bars are standard deviations from at least three independent experiments and asterisks label the values significantly different from the control value with statistical significance at P < 0.05 (unpaired Student's ttest).

Figure 5

 5 Figure 5Ratios of filamentous/monomeric actin in cells expressing selected mutants. C2C12 cells were transfected with myc-tagged ACTA1 constructs and fixed in formaldehyde and permeablized. Myc was detected with 9E10 primary and Marina Blue secondary antibody, while Alexa 488 DNAseI and Texas Red phalloidin were used to detect G-actin and F-actin respectively. Blue myc-positive cells were used for analysis and the relative binding to Alexa 488 (G-actin) and Texas Red (Factin) were compared. The relative ratio of G-actin/F-actin is shown in the bar graphs with the wild type ACTA1 expressing cells normalized to 1 and the error bars indicate the standard deviation from at least three independent experiments, while the asterisk denotes statistical significance at P < 0.05 (unpaired Student's t-test).

  ). Arrow 2 shows that when actin depolymerises, due to serum starvation or treatment with Latrunculin B (LatB), actin monomer can sequester MAL in a complex that cannot participate in SRF co-activation. Arrow 3 shows that free MAL can enter the nucleus, where free MAL interacts with SRF and actin bound MAL does not. It isn't known if actin-MAL complexes can enter the nucleus together, but there is actin in the nucleus and it can form complexes there with MAL. Arrow 4 shows that free MAL can be exported from the nucleus by the CRM1 transporter and that this reaction is inhibited by leptomycin B (LMB). It is unclear whether actin-MAL complexes can be co-transported out of the nucleus. Arrow 5 shows that free nuclear MAL can interact with SRF and mediate transcription of CArG box containing target genes. Biochemical Journal Immediate Publication. Published on 20 Jan 2010 as manuscript BJ20091641 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091641 A c c e p t e d M a n u s c r i p t

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Table 1

 1 Summary of resultsCategories of mutants are shown as from the study by Costa et al.[START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF] and we have added the new data from this study. Asterisks indicate data previously shown in[START_REF] Costa | Myopathy mutations in alphaskeletal-muscle actin cause a range of molecular defects[END_REF]. nd= not done.

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Acknowledgements

We thank Heidi Welch and Christophe Ampe at U. Ghent, Belgium for several of the mutant constructs. We thank Eric Olson and the University of Texas Southwestern Medical Center, Department of Molecular Biology for the gift of the SM22 luciferase reporter construct. We thank Tom Gilbey at the Beatson Institute for his excellent assistance with running the FACS samples. This work was founded by the Muscular Dystrophy Campaign grant RA3/583/3 and by an MRC senior research fellowship and a core CRUK grant to L.M.M.