María Tomé

Pedro López-Romero

Carmen Albo

Juan Carlos Sepúlveda

Benjamín Fernández-Gutiérrez

Ana Dopazo

Ph.D Antonio Bernad
email: abernad@cnic.es.

Ph.D Manuel A González
email: magonzalez@cnic.es.

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells Running title: miR-335 controls hMSC activation

Keywords: microRNA, mesenchymal stem cell, cell proliferation, cell migration, cell differentiation, osteogenesis hMSC: human mesenchymal stem cells. fdr: false discovery rate

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses between undifferentiated and differentiated bone marrow-derived hMSCs revealed that miR-335 is significantly downregulated upon hMSC differentiation, being coexpressed in with its host coding-gene MEST. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, which has been described as a positive regulator of MSC self-renewal, and downregulated by interferon gamma (IFN-γ), a pro-inflammatory cytokine that plays an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 is important for the maintenance of the immature state and its downregulation is critical for the acquisition of reparative MSC phenotypes.

INTRODUCTION

Mesenchymal stem cells (MSCs) are multipotent mesoderm-derived somatic stem cell precursors of non-hematopoietic connective tissues that are present in the stroma of virtually all mammalian organs, especially bone marrow and subcutaneous fat (reviewed in [START_REF] Bernardo | Mesenchymal stromal cells[END_REF]). Upon activation by tissue damage, MSCs contribute to tissue repair processes through a multitude of activities, including cell proliferation, differentiation and migration, and the regulation of angiogenesis and immune responses. There is growing evidence, both in animal and clinical models, that administration of ex vivo-expanded human MSCs (hMSCs) has potential to ameliorate degenerative disorders, including cardiovascular, neurodegenerative, and autoimmune and inflammatory diseases; however, the specific molecular mechanisms underlying this therapeutic potential remain mostly unknown.

MicroRNAs (miRNAs) are a family of small (18-24 nucleotide), single-stranded noncoding RNAs which regulate gene expression in eukaryotic cells by controlling the translation (usually by repression), stability and localization of specific mRNA targets. Hundreds of conserved miRNA genes are known in many species, making miRNAs, after transcription factors, the second most diverse family of gene expression regulators. Computational predictions of miRNA targets indicate that each miRNA regulates hundreds of mRNAs, and that approximately one third of all mammalian protein-coding genes are regulated by miRNAs [START_REF] Berezikov | Phylogenetic shadowing and computational identification of human microRNA genes[END_REF]. Functional studies show that miRNAs participate in virtually each cellular process investigated, and that alterations in their expression levels might underlie human diseases, including cardiovascular disease and cancer. There are also data indicating that mammalian miRNAs can be imported into the nucleus [START_REF] Hwang | A hexanucleotide element directs microRNA nuclear import[END_REF] or even secreted from cells within small exocytic particles (4), suggesting the existence of currently unknown functions for this class of molecules.

Experimental evidence shows that miRNAs are critical regulators of stem cell biology. In mouse embryonic stem (ES) cells, knockout of Dicer or DGCR8, two key protein factors involved in miRNA biogenesis, impairs cell growth, with cells accumulating in G1, and severely reduces differentiation capacity [START_REF] Kanellopoulou | Dicerdeficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[END_REF][START_REF] Wang | DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal[END_REF]. Additionally, expression profiling demonstrates that ES cells express a unique repertoire of miRNAs compared with differentiated cell types [START_REF] Suh | Human embryonic stem cells express a unique set of microRNAs[END_REF]. Molecular functions of ES cell-specific miRNAs have begun to be elucidated, and the results confirm their critical involvement in the regulation of ES cell pluripotency and self-renewal. MiRNAs also play an important role in controlling ES cell differentiation and commitment. As example, miR-134, miR-296 and miR-470 are upregulated by retinoic acid, and modulate ES cell differentiation through specific targeting of Nanog, Oct4 and Sox2 mRNAs [START_REF] Tay | MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation[END_REF].

To date, relatively few studies have examined miRNA function in MSCs. Gene expression microarray profiling has identified several miRNAs that are regulated during differentiation of MSCs into different cell lineages. Some of these miRNAs have been identified as regulators of osteogenic differentiation, including miR-125b (9), miR-26a [START_REF] Luzi | Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor[END_REF], miR-196a [START_REF] Kim | miR-196a Regulates Proliferation and Osteogenic Differentiation in Mesenchymal Stem Cells Derived From Human Adipose Tissue[END_REF], miR-204/211 [START_REF] Huang | MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation[END_REF], and miRs -148b, -27a, and -489 [START_REF] Schoolmeesters | Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells[END_REF]. Also, miR-335 has been recently identified as the most upregulated miRNA in bone marrow-derived human MSCs (hMSCs) in comparison with skin fibroblasts [START_REF] Bae | Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts[END_REF], which are otherwise phenotypically similar to hMSCs. However, the specific targets of most of these miRNAs remain to be determined, and the possible role of miRNAs in other therapeutically relevant MSC activities, such as in cell migration and proliferation, is unknown.

Here, we sought to identify miRNAs that may regulate the activation of the reparative phenotype of hMSCs, as well as some of their target genes.

Results

miR-335 is downregulated upon hMSC differentiation. We hypothesized that key miRNA regulators of MSC activation/differentiation in tissue repair should be expressed in the undifferentiated state, and downregulated early upon exposure to a differentiation signal. To identify these putative miRNAs, we used Agilent microarrays to obtain differential miRNA expression profiles of undifferentiated bone marrow-derived hMSCs versus the same cells cultured in the presence of adipogenic or osteogenic media. We also profiled human skin fibroblasts, since the target miRNAs should be expressed at comparatively low levels in more developmentally-restricted mesenchymal cell types. As we aimed to identify miRNAs potentially involved in the initial steps of hMSC activation/differentiation, cells were exposed to differentiation media for a relatively short period (nine days), instead of the 21-days commonly used for in vitro MSC differentiation assays.

Signal processing is a critical step in the analysis of the results of miRNA microarray experiments. We used a normalization algorithm that incorporates quantile normalization between arrays [START_REF] Lopez-Romero | Processing of Agilent microRNA array data[END_REF] to estimate a processed microRNA signal for the Agilent arrays. The quantile normalization, when applied to the background-corrected signal, showed significantly lower variability between replicates than the total gene signal normalized by the 75% percentile (Supplementary Figure S1).

The results showed no significant regulation (false discovery rate, fdr<15%) of miRNAs previously described as regulators of osteogenic (miR-26a, miR-27a, miR-125b, miR-148b, miR-196a, miR-489) or adipogenic differentiation (miR-103, miR-107, miR-143) under any of the conditions tested (Supplementary Figure S2A and Supplementary Table S1). Gene enrichment analysis of the predicted targets of miRNAs up-or downregulated in at least two conditions (see Methods) showed a significant (p<1E-06) enrichment for genes involved in angiogenesis or signaling by Wnt, integrins, PDGF, cadherin, endothelin and TGF-β (Supplementary Figure S2B and Supplementary Table S1). MiR-335 was the only miRNA significantly downregulated in all three "differentiated" cell populations (Figure 1a). Fold change (log2) values were as follows: fibroblast vs. undifferentiated hMSCs, M=-5.684 (fdr=0.008); osteogenic differentiation vs. undifferentiated, M=-2.434 (fdr=0.029); adipogenic differentiation vs. undifferentiated, M=-1.141 (fdr=0.103). After validating the microarray results by real-time RT-PCR (Figure 1b), we selected miR-335 for further characterization.

MiR-335 levels were also significatively reduced in human osteoblasts (Saos cell line) after differentiation (Supplementary Figure S4). hMSCs from different tissues express higher levels than fibroblasts of miR-335 and its host gene MEST. To determine whether relatively high expression of miR-335 is specific to bone marrow-derived hMSCs or is a common characteristic of hMSCs, we analyzed miR-335 expression levels in hMSC populations obtained from bone marrow, subcutaneous adipose tissue and articular cartilage (Figure 2b). All hMSC isolates were tested at passage 5-7 at the same cell density (8000 cells/cm 2 ), and were assayed for their differentiation potential to osteogenic, adipogenic, and chondrogenic lineages in the presence of specific differentiation factors (data not shown). All three populations express significantly higher levels of miR-335 than dermal fibroblasts, and miR-335 expression levels were significantly higher in hMSCs isolated from bone marrow or articular cartilage than in hMSCs derived from adipose tissue.

MiR-335 is encoded by the second intron of the MEST (mesoderm specific transcript homolog) gene (Figure 2a) [START_REF] Kobayashi | Human PEG1/MEST, an imprinted gene on chromosome 7[END_REF]. MEST expression, determined by real-time RT-PCR, correlated with the levels of mature miR-335 (Figure 2b; Spearman's r=0.5769, p=0.0008), suggesting that the different hMSC isolates co-express mature miR-335 with its host gene and not through a MEST-independent mechanism (such as a specific promoter or posttranscriptional regulation).

MEST expression levels also correlated with the levels of miR-335 under all other conditions tested in this study (Supplementary Figure S5). miR-335 impairs hMSC proliferation, migration and differentiation. We next analyzed the effect of miR-335 overexpression in bone marrow-derived hMSCs. hMSCs were transduced with the lentiviral vector pLV-EmGFP-MIR335, which encodes the genomic sequence spanning miR-335, or with a control vector (pLV-EmGFP-Mock). Transduced cells were purified to >95% homogeneity (gfp-positive cells) by FACS. To avoid non-specific effects due to lentiviral gene silencing or to a high proviral copy number per cell, a multiplicity of infection of 5 was used, and only cells with medium-level gfp expression were selected (Supplementary Figure S3A). Real-time RT-PCR demonstrated a ~3-fold increase in miR-335 expression in pLV-EmGFP-MIR335-transduced cells compared with controls (Supplementary Figure S3B). Overexpression of miR-335 was not associated with any apparent changes in cell morphology or size (not shown).

When cultured over several passages, miR-335-overexpressing hMSCs showed a significant reduction in their proliferative activity compared with control cells (Figure 3a). However, miR-335 overexpression did not cause significant alterations to cell cycle kinetics (not shown) or the rate of apoptosis (Figure 3b). hMSCs overexpressing miR-335 also showed an impaired migratory response to stimulation with fetal bovine serum (Figure 3c). Consistently, wild-type hMSCs transfected with a miR-335 inhibitor (Anti-miR TM -335, Ambion) showed increased migratory activity compared with cells transfected with a negative control Anti-miR TM oligonucleotide (Figure 3c, Supplementary Figure S3C). MiR-335-overexpressing hMSCs also showed impaired migratory activity in an in vitro wound-healing assay compared with control cells (see Supplementary information videos). These results demonstrate that miR-335 is a negative regulator of hMSC migration.

The effect of miR-335 expression on hMSC differentiation capacity was monitored by comparing the in vitro differentiation responses of control and miR-335-transduced hMSCs to exposure to adipogenic or osteogenic stimuli for three weeks. Both adipogenic and osteogenic differentiation were significantly reduced by miR-335 overexpression. In particular, in vitro osteogenesis was almost completely abolished (Figure 3d), indicating a role for miR-335 in the regulation of hMSC differentiation programs. Analysis of expression of the differentiation markers osteopontin (osteogenic differentiation) and PPARγ (adipogenic differentiation) confirmed the inhibitory effect of miR-335 in both processes (Figure 3e).

MiR-335 expression levels are regulated by signals that control MSC migration and differentiation.

The apparent regulatory role of miR-335 in hMSC suggested that it might be regulated by signals known to control MSC biology. We therefore tested if canonical Wnt signaling, a negative regulator of MSC differentiation [START_REF] Boland | Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells[END_REF], affected miR-335 expression levels. Bone marrow-derived hMSCs treated with 10 mM LiCl or Wnt3a-containing conditioned medium upregulated miR-335 expression (Figure 4a, left panel). Moreover, addition of Dkk1 (100 ng/ml), a specific inhibitor of canonical Wnt signaling, significantly decreased miR-335 expression in the presence of exogenous Wnt3a (Figure 4a, right panel).

Upregulation of miR-335 expression by Wnt3a was also observed in adipose-derived hMSCs and in skin fibroblasts (Figure 4b), although in a different extension, suggesting that this effect is common to all mesenchymal cell types.

We next evaluated the effect on miR-335 expression of IFN-γ signaling, another key regulator of MSC activity. IFN-γ has numerous actions on MSC, including the induction of immunorregulatory activity, cell migration and osteogenic differentiation [START_REF] Krampera | Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells[END_REF][START_REF] Duque | Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis[END_REF]. Treatment of bone marrow-derived hMSCs with human IFN-γ for 48 h significantly decreased expression of miR-335 (Figure 4c) in a dose-dependent manner, consistent with the lowered miR-335 expression during cell migration and osteogenic differentiation.

To identify possible binding sites in the MEST locus for transcription factors involved in β-catenin or IFNγ signaling pathways, we performed a multispecies (human, mouse and dog) sequence homology analysis for conserved binding sites for LEF/TCF (transcription factors involved in β-catenin signaling) and STAT1 (involved in IFNγ signaling). MULAN analysis with a matrix similarity of 0.95 showed one possible LEF1 and one possible TCF4 site in the 5 kb region upstream of the MEST transcriptional start site (corresponding to the longest human splicing variant), both mapping at -2.675 kb (Supplementary Figure S6). A similar analysis, with a matrix similarity of 0.90, identified three conserved potential STAT1 binding sites at -4.440, -4.384, and -4.303 kb (Supplementary Figure S6).

Potential miR-335 targets in hMSCs are enriched in transcription regulators of cell movement and differentiation. In order to identify potential miR-335 targets, we used a two-step approach (Figure 5a). First, we used three different miRNA target prediction programs, miRanda [START_REF] Enright | MicroRNA targets in Drosophila[END_REF], TargetScan [START_REF] Lewis | Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[END_REF] and PicTar [START_REF] Krek | Combinatorial microRNA target predictions[END_REF], to obtain a list of putative miR-335 targets. As these programs use different target scoring systems, we combined the three lists in a single non-redundant gene list (a total of 1838 genes, of which 1607 were mapped). Gene enrichment analysis using the PANTHER database revealed significant enrichment (p<0.001) for molecular functions related to transcriptional and post-transcriptional gene regulation, and for biological processes (p<1E-06) also related to regulatory activities, including mRNA transcription, protein modification, and signal transduction (Supplementary Figure S7).

Second, we performed a microarray gene expression analysis (Agilent Whole Human

Genome Microarray Kit) on hMSCs transduced with pLV-EmGFP-MIR335 or pLV-EmGFPmock. We focused on downregulated genes since mRNA expression levels tend to correlate negatively with the expression levels of their specific miRNAs [START_REF] Sood | Cell-type-specific signatures of microRNAs on target mRNA expression[END_REF]. Using this approach, we identified 489 genes with decreased expression in miR-335-transduced cells that are thus candidate direct or indirect targets of this miRNA in hMSCs. Of these genes, 62 were also contained in the previously obtained list of predicted miR-335 targets (Supplementary Table S2), and were therefore classed as probable direct targets. This set of 62 genes was significantly enriched (p<0.05) for transcription factors (21%) and membrane-bound signaling molecules (6.5%). A detailed analysis with the Ingenuity® Pathways Analysis software (IPA, Ingenuity® Systems, http://www.ingenuity.com) showed a predominant enrichment for functions involved in cell development, cell movement, gene expression, and the cell cycle (Benjamini-Hochberg multiple test-corrected p-value=1.37E-02-8.82E-02).

Other significantly enriched biological functions included cell signaling, cell growth and proliferation, cell morphology, cell commitment, and cell death (Table 1). Using IPA, the two more representative gene regulatory networks were constructed, respectively containing 11 of the 20 potential miR-335 target genes involved in cellular movement (score 27) and 15 of the 17 genes involved in gene expression (score 42) (Figure 5b).

One of the predicted targets of miR-335 is RUNX2, a key transcription factor involved in osteogenesis. Although miRNAs can bind to coding regions and even to 5´regulatory regions, a single miR-335 binding site is predicted by the miRanda algorythm at nucleotides 3545-3567 in all the known RUNX2 transcript variants (GenGank Accession Numbers NM_001024630, NM_001015051, NM_004348), corresponding to the 3'UTR region.

A western blot analysis of RUNX2 demonstrated that its protein levels are reduced in hMSCs by miR-335 exogenous overexpression (Figure 6a). To determine if miR-335 directly modulates RUNX2 expression levels in hMSCs, we cloned the full-length RUNX2 3'UTR downstream of the Renilla luciferase gene as a reporter, and assayed its expression in hMSCs transfected with a synthetic precursor or a specific inhibitor of miR-335. UTR reporters corresponding to SOX4 and UBE2F genes were used as positive and negative controls, respectively, for the miR-335 inhibitory activity [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. Transfection of the miR-335 synthetic precursor significantly reduced the expression of UTR reporters for SOX4 and RUNX2, but not the expression of the negative control gene UBE2F (Figure 6b, upper panel). Transfection with the specific inhibitor of miR-335 significantly increased the expression levels of UTR reporters for SOX4 and RUNX2, but did not affect the levels of UBE2F (Figure 6b, bottom panel). These results indicate that miR-335 controls RUNX2 expression in hMSCs by direct binding to its 3'UTR.

Finally, we have quantified the relative expression levels of RUNX2 after osteogenic differentiation in hMSCs overexpressing miR-335. The results of these experiments (Figure 6c) showed a significant reduction of RUNX2 expression levels in the cells transduced with the lentiviral vector encoding miR-335, compared to those transduced with a control lentiviral vector, which is in good agreement with the rest of the data indicating that RUNX2 is a target of miR-335 in hMSCs.

Discussion

Our results support a central role for miR-335 in the gene regulatory network that controls the tissue-repair activities of hMSCs. Expression of this miRNA is high in undifferentiated multipotent hMSCs compared with their differentiated cell progeny, and is regulated by major signaling pathways that control MSC biology, such as those controlled by Wnt3a and IFNγ.

The set of predicted miR-335 targets is enriched in genes whose products are regulators of cellular movement and gene expression, including RUNX2, involved in the control of osteogenic differentiation.

There is growing evidence that miRNAs play a critical role in the biology of all stem cell types, including ES cells, germline stem cells, and somatic stem cells (SSCs). SSCs, which are present in adult somatic tissues, are usually less well-defined than embryonic or germline stem cells because a single tissue often contains several distinct SSC populations (or, as in the case with MSCs, similar SSCs can be isolated from different tissues) and they often cannot be purified to homogeneity. For these reasons, it has been difficult to identify the specific roles of a miRNA in a particular SSC population. Most miRNAs described as regulators of SSC biology are inducers of cell differentiation/commitment or inhibitors of self-renewal and proliferation. These include miR-203 in epidermal stem cells [START_REF] Yi | A skin microRNA promotes differentiation by repressing 'stemness[END_REF], and miR520h and others in hematopoietic stem cells [START_REF] Georgantas | CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control[END_REF]. It also appears that miRNAs are involved in the self-renewal or maintenance of at least some types of SSCs, since targeted deletion of Dicer in mouse epidermis causes altered morphogenesis of hair follicles, with loss of stem cell markers [START_REF] Andl | The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles[END_REF].

Our results demonstrate that miR-335 is downregulated in hMSC cultures undergoing adipogenic or osteogenic differentiation, and is also expressed at comparatively lower levels in primary skin fibroblasts, which are mesenchymal cells with a limited differentiation potential. Our data are in complete accordance with previous work identifying miR-335 as the most upregulated miRNA in bone marrow-derived hMSCs in comparison with fibroblasts [START_REF] Bae | Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts[END_REF].

Until recently, the only known specific biological role for miR-335 is in human breast cancer, where it has been proposed as a metastasis suppressor [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. Expression of miR-335 is lost in the majority of primary breast tumors from patients who relapse, and this is associated with poor distal metastasis-free survival [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. These results are in good agreement with our findings demonstrating that miR-335 is a negative regulator of hMSC migration. MiR-335 downregulation is also observed in drug-resistant ovarian cancer cells [START_REF] Sorrentino | Role of microRNAs in drug-resistant ovarian cancer cells[END_REF], cultured chondrocytes (versus cartilage tissue) (29), ethanol-treated neural progenitors [START_REF] Sathyan | Competing interactions between micro-RNAs determine neural progenitor survival and proliferation after ethanol exposure: evidence from an ex vivo model of the fetal cerebral cortical neuroepithelium[END_REF], multiple myeloma cells and tumors [START_REF] Ronchetti | An integrative genomic approach reveals coordinated expression of intronic miR-335, miR-342, and miR-561 with deregulated host genes in multiple myeloma[END_REF], and muscle regeneration [START_REF] Greco | MicroRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells[END_REF]. In contrast, miR-335 is highly expressed in obesity models [START_REF] Nakanishi | The upregulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice[END_REF] and in fetal lung compared with adult tissue [START_REF] Williams | Maternally imprinted microRNAs are differentially expressed during mouse and human lung development[END_REF]. During the preparation of this manuscript, a communication describing the direct targeting of RB1 by miR-335 has been published [START_REF] Scarola | miR-335 directly targets Rb1 (pRb/p105) in a proximal connection to p53-dependent stress response[END_REF]. In this study, the authors establish the important role of this miRNA in the induction of p53-dependent cell cycle arrest after DNA damage. These results are in accordance with our findings demonstrating that miR-335 is a negative regulator of hMSC proliferation, and, taken together with our results, suggest an important role of RB1 activity in regulation/activation of hMSC tissue repair activities. miR-335 is encoded by the second intron of the maternally imprinted gene MEST (human chromosome 7q32) [START_REF] Kobayashi | Human PEG1/MEST, an imprinted gene on chromosome 7[END_REF]. Our results demonstrate that both miR-335 and MEST are coordinately upregulated in hMSCs from three different tissues (bone marrow, subcutaneous fat and articular cartilage) in comparison with skin fibroblasts. Moreover, adipose-derived hMSCs expressed significantly lower levels of miR-335 than the two other hMSC populations analyzed, indicating that the miRNA expression level in hMSC is dependant on the source tissue. The biological relevance of this finding remains to be determined, but our results suggest that miR-335 expression levels could be used to discriminate between hMSCs and fibroblasts, or even between different MSC populations with diverse differentiation potentials. Future studies are needed to confirm this finding.

We found no significant regulation of miRNAs previously described as regulators of osteogenic differentiation (miR-26a, miR-27a, miR-125b, miR-148b, miR-196a, miR-489, miR-204/211) or adipogenic differentiation (miR-103, miR-107, miR-143) under any of the conditions tested. This apparent discrepancy with published data might be related to the fact that our microarray analyses were performed on hMSCs at early differentiation stages, unlike other studies that used terminally differentiated MSCs. Also, the method we used for signal processing (quantile normalization) [START_REF] Lopez-Romero | Processing of Agilent microRNA array data[END_REF] probably contributed to an overall reduction in the variation of gene expression between samples.

The finding that miR-335 expression impairs the proliferative and migratory capacities of primary bone marrow-derived hMSCs and inhibits both adipogenic and osteogenic differentiation suggests that miR-335 is part of a common regulatory pathway for cell proliferation, migration and differentiation in MSCs. The existence of a partially overlapping regulatory circuitry for these biological processes has already been demonstrated in MSCs [START_REF] Boland | Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells[END_REF], as well as in other SSC lineages [START_REF] Ge | Coupling of cell migration with neurogenesis by proneural bHLH factors[END_REF]. The role of canonical Wnt signaling pathway (Wnt3a) as a positive regulator of miR-335 expression in hMSCs agrees with evidence showing that Wnt3a inhibits osteogenic differentiation in MSCs [START_REF] Boland | Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells[END_REF]. However, the observed reduction in proliferation capacity in hMSCs overexpressing miR-335 suggests that the reported proliferative effect of Wnt3a acts via miR-335-independent mechanisms. Also, it has been reported that Wnt3a induces ECM invasion in hMSCs [START_REF] Neth | Wnt signaling regulates the invasion capacity of human mesenchymal stem cells[END_REF], although it inhibits it in murine MSCs [START_REF] Karow | Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression[END_REF]. Further studies are needed to determine the effects of Wnt3a in the migration capacity of hMSCs in different regulatory conditions, and if they are related to miR-335 up-regulation.

In contrast with the effect of Wnt3a, IFNγ decreased miR-335 expression in hMSCs.

Considered together with the anti-migratory and differentiation effects of miR-335, this action might account, at least partially, for the previously described pro-migratory and proosteogenic activities of IFNγ [START_REF] Duque | Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis[END_REF]. This cytokine plays a key role in the therapeutic activity of MSCs, as it is involved in the activation of their immunomodulatory phenotype [START_REF] Krampera | Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells[END_REF]. IFNγ signaling, both intercellular and autocrine, might thus regulate an unsuspected large number of genes in MSCs through the inhibition of miR-335 expression. At this time, we do not know the precise molecular mechanism by which IFNγ downregulates miR-335 expression in MSC, but it might involve an indirect effect through its previously described inhibitory action on the canonical Wnt signaling pathway [START_REF] Fillmore | Nmi (N-Myc interactor) inhibits Wnt/beta-catenin signaling and retards tumor growth[END_REF].

Comparative genomics is a powerful new tool for identifying and characterizing functional sequences. The presence in the 5' upstream region of the MEST locus of highly conserved potential LEF1 and TCF4-binding sites (95% similarity between human, mouse and dog) and potential STAT1-binding sites is compatible with the regulation of the MEST/miR-335 locus by both Wnt3a and IFNγ; however, further studies are needed to confirm these putative transcription factor binding sites.

Consistent with our experimental results, the set of 62 potential direct gene targets of miR-335 is significantly enriched in genes related to cell movement and gene expression, pointing to a key regulatory role of miR-335 in MSC biology. In particular, we have experimentally validated RUNX2, a transcription factor essential for osteogenic differentiation, as a direct miR-335 target. Recently, miR-204 and miR-211 have been described as negative regulators of RUNX2 [START_REF] Huang | MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation[END_REF]. Taken together, these results demonstrate that osteogenic differentiation of mesenchymal precursors is regulated by multiple miRNAs through the control of RUNX2 expression levels. Since miR-335 is differentially expressed in MSCs derived from different tissues and donors, it is tempting to speculate that such a difference in miR-355 expression might correlate with their action on bone homeostasis in physiological or in pathological conditions. Further studies are needed to evaluate this possibility.

To our knowledge, this is the first demonstration of the involvement of a specific miRNA in the coordinated regulation of MSC proliferation, migration and differentiation. Our findings are summarized in the model presented in Figure 7. miR-335 is down-regulated in MSCs in response to tissue damage signals, such as IFNγ, which leads to de-repression of miR-335 target genes involved in MSC proliferation, migration, and osteogenic differentiation. These results have implications for the understanding of the major molecular mechanisms controlling the therapeutic activity of hMSCs versus their maintenance in an undifferentiated state, and strongly suggest an important role of miR-335 in tissue homeostasis.

Materials and Methods

Cell Culture. This study was carried out according to guidelines of the Instituto de Salud Carlos III (Spain). hMSCs from bone marrow, adipose tissue, and articular cartilage were isolated and characterized as previously described [START_REF] De La Fuente | Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells[END_REF]. Human skin fibroblasts were obtained from Inbiobank (San Sebastián, Spain). The 293T and the Saos-2 cell lines were purchased from The American Type Culture Collection (CRL-11268, Manasas, VA). All cell lines were maintained in expansion medium, consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 g/l D-glucose, supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and 50 μg/ml gentamicin; all culture reagents were from Invitrogen (Carlsbad, CA). Cells were cultured in a humidified 37ºC incubator at 5% CO 2 .

For Wnt stimulation, cells were exposed to different concentrations of conditioned medium from Wnt3a-expressing QT6 cells. Control and Wnt3a-expressing QT6 cells were kindly provided by Dr. D. Büscher (Cellerix SL; Madrid, Spain).

When indicated, several factors (DKK1, R&D Systems, Minneapolis, MN; IFNγ, PeproTech, Rocky Hill, NJ) or chemicals (KCl/LiCl, Sigma-Aldrich, St. Louis, MO) were added to the hMSCs cells to evaluate its effect both on the modulation of miR-335 expression or on the several properties of the cells. In each case, the range of concentrations used is indicated.

Lentiviral Vectors. The lentiviral vectors pLV-EmGFP-MIR335 (encoding the human miR-335 gene) and pLV-EmGFP-Mock (encoding a non-specific shRNA sequence) were based on pRRLsin18.PPT.CMV.GFP.Wpre, kindly provided by Dr. Luigi Naldini [START_REF] Zufferey | Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery[END_REF]. Detailed construction procedures for both plasmids are provided in Supplementary Information. For apoptosis analysis, cells were harvested and centrifuged at 2000 x g for 5 min at room temperature, washed twice with PBS and resuspended in 1X binding buffer (10 mM HEPES/NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl 2 ) at a concentration of 1x10 6 cells/ml. 5 μl of Annexin V-DY634 (Immunostep, Salamanca, Spain) were added to 4x10 5 cells suspended in 400 μl of binding buffer. As a negative control, 2x10 5 cells were used without addition of Annexin V antibody. Cells were vortexed gently and incubated in the dark for 15 min at room temperature. Cells were centrifuged at 1000 x g for 5 min, and 1X binding buffer (500 μl) and PI (5 μl) were added to each tube. The samples were analyzed by FACScan (BD Biosciences) using the Cell Quest software (BD Biosciences).

Cell Differentiation Assays. hMSCs were seeded at 2x10 4 cells/cm 2 in expansion medium, and after 24 h medium was replaced by the corresponding induction medium. Osteogenic medium contained expansion medium supplemented with 10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 0.2 mM ascorbic acid. Adipogenic medium contained expansion medium supplemented with 0.01 μM dexamethasone, 0.5 mM IBMX (3-isobutyl-1-methyl xanthine), and 60 μM indomethacin. In all cases, induction medium was replaced every 3-4 days and, on day 9 or 21 (depending on the experiment) cells were processed for histochemical analysis. Cells were fixed with 70% ethanol (1 h, 4ºC) and stained (5 min). For osteogenic assays, cells were stained with 40 mM Alizarin Red, pH 4.1. Cells cultured in adipogenic medium were stained with 2% Oil Red O. Analysis of the expression of marker genes for osteogenic (osteopontin) and adipogenic (PPARγ) differentiation was performed by real-time RT-PCR as described above.

Cell Migration Assays. For transwell migration assays, hMSCs (1x10 4 ) were cultured in medium containing 0.2% FCS in a 24-well tissue culture insert with an 8 μm pore size membrane (BD Biosciences). Fresh culture medium containing 10% FCS was added to the bottom well. After 6 h, the filter membranes were fixed with 70% ethanol and mounted in mounting medium containing DAPI. Migration of hMSCs was determined by counting the number of DAPI-stained nuclei on the underside of the membrane under x 100 magnification, using CellProfiler TM image analysis software (http://www.cellprofiler.org).

For wound-healing assays, a "wound" was created in a cell monolayer by scraping with a sterile plastic tip, and time-lapse video recordings were made by sequentially capturing microscopic images at 30 min intervals over a total period of 46 h.

Western blot analysis.

Whole cell lysates for western blotting were extracted with lysis buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 0.25% sodium deoxycholate and protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). Protein samples were resolved by 10% SDS-PAGE and gels transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight at 4ºC with primary antibodies at the following dilutions: RUNX2, 1:500 (Sigma-Aldrich) and β-actin, 1:5000 (AbCam, Cambridge, MA). Signals were detected using the appropriate horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark). The blots were visualized by an enhanced chemiluminesence system (ECL; GE Healthcare) according to the manufacturer's instructions, and relative intensity of the specific bands were quantified by densitometry using the ImageJ software (http://rsb.info.nih.gov/ij/). UTR reporter assays. The full-length 3'UTRs of the putative miR-335 target gene RUNX2, as well as the negative control UBE2F and the positive control SOX4 genes were amplified from human genomic DNA (Novagen, Madison, WI, USA) and individually cloned into the Psicheck TM -2 dual luciferase reporter vector (Promega, Madison, WI, USA). hMSCs were then co-transfected with each reporter construct and with a miRNA synthetic precursor (Pre-miR TM -335 or Pre-miR TM Negative Control #2, Ambion) or antagonist (Anti-miR TM -335 or Anti-miR TM Negative Control #1, Ambion) at a final concentration of 50 nM as described above. Cells were lysed 30 h after transfection and the ratio of Renilla to firefly luciferase was measured with the dual luciferase assay (Promega). Normalized Renilla to firefly ratios were determined in the presence or absence of miR-335 inhibition.

Microarray Analyses. The Agilent Human microRNA Microarray v2.0 (G4470B, Agilent Technologies) was used to identify miRNAs highly expressed in undifferentiated hMSCs. miRNA expression profiles of undifferentiated hMSCs were compared with profiles of human skin fibroblasts and of the same hMSC lines after 9 days of adipogenic or osteogenic induction.

The Agilent Whole Human Genome Microarray Kit (G4112F, Agilent Technologies) was used to identify genes downregulated in hMSCs exogenously overexpressing miR-335. The mRNA expression profile of hMSCs transduced with lentiviral vector pLV-EmGFP-MIR335 was compared with that of hMSCs transduced with the control vector pLV-EmGFP-Mock.

A full description of the experimental procedures, data processing and statistical analysis used for both types of microarrays is included in the Supplementary information. All microarrays have been submitted to the Gene Expression Omnibus database at http://www.ncbi.nlm.nih.gov/geo; accession number GSE19232. Bioinformatic analysis. Computational miRNA target prediction analyses were carried out using the algorithms miRanda (http://www.microrna.org/microrna/home.do), TargetScan (http://www.targetscan.org) and PicTar (http://pictar.mdc-berlin.de).

Human, mouse and dog MEST loci were aligned, and the extent of DNA sequence homology was computed with the web-based program MULAN (http://mulan.dcode.org) [START_REF] Ovcharenko | Mulan: multiple-sequence local alignment and visualization for studying function and evolution[END_REF]. Using MULAN and multiTF (http://www.multitf.dcode.org) with the TRANSFAC professional V10.2 library database (http://www.biobase.de), binding sites for LEF1, TCF4 (0.95 matrix similarity) and STAT1 (0.9 matrix similarity) were predicted in the 5 kb upstream region of the human MEST locus.

  Lentiviral particles were produced by transient transfection of 293T cells[START_REF] Zufferey | Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery[END_REF].Lentiviral transduction was carried out by overnight incubation of 3x105 hMSCs in 10 cm plates with 3 ml culture medium containing lentiviral particles at a multiplicity of infection (MOI) = 5. Transduced (gfp+) cells were purified 48 h after transduction by fluorescenceactivated cell sorting (FACS) using a FACSAriaII SORP cell sorter (BD Biosciences). Transfection of miRNA precursors and inhibitors. Anti-miR TM miRNA inhibitors (Ambion, Austin, TX) are chemically modified, single stranded nucleic acids designed to specifically bind and inhibit endogenous miRNAs. Pre-miR TM miRNA precursors (Ambion) are synthetic double-stranded oligoribonucleotides designed to match the sequence of specific miRNA:miRNA* duplexes. Depending on the experiment, Anti-miR TM or Pre-miR TM oligos at a final concentration of 50 nM were transfected (Lipofectamine 2000, Invitrogen) into 2x10 4 hMSCs cultured in 24-well plates (BD Biosciences, Franklin Lakes, NJ) according to the oligonucleotide manufacturer's protocol. Real-Time Reverse Transcriptase Polymerase Chain Reaction. Total RNA was isolated from cultured cells with a miRNeasy Mini Kit (Qiagen, Valencia, CA). Human miR-335, MEST, SPP1 (osteopontin), PPARG, and RUNX2 transcripts were quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the corresponding TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA). RNU48 and GAPDH were used as endogenous normalization controls for miRNAs and protein-coding genes, respectively. Analysis of cell proliferation, cell cycle, and apoptosis. Cell proliferation was assessed by seeding 6x10 4 cells/2 ml/well in 6-well plates (Corning, Lowell, MA, USA) and culturing for 30 days. The medium was replaced every 3-4 days, and the cells were harvested and counted once a week. Cumulative population doubling (CPD) was calculated as log 10 (number of cells harvested) -log 10 (number of cells seeded)/log 10 (2). For cell cycle profiling, approximately 1x10 6 methanol:acetic acid-fixed cells were incubated with 10 μM propidium iodide and 10 μM RNase A for 1 h at 37°C and analyzed by flow cytometry using a Becton-Dickinson LSR cytometer. Data were analyzed with the Summit v4.3 software (Dako Inc, Carpinteria, CA, USA).

Table 1 .

 1 Statistical analyses of experimental data were conducted with Prism 3.0 (Graphpad Software Inc., San Diego, CA). Unless otherwise stated, data were compared by Most significantly enriched molecular and cellular functions found using the IPA software in the list of 62 genes identified as potential miR-335 targets in hMSCs (B-H pvalue: Benjamini-Hochberg multiple test-corrected p-value).

	Target	gene	functions	were	profiled	with	PANTHER	software
	(http://www.pantherdb.org/tools)	and	Ingenuity®	Pathways	Analysis	software
	(http://www.ingenuity.com).
	Statistical Analysis.

Acknowledgements

. We thank S. Calleja and R. Alvarez (CNIC) for technical help with microarrays and real-time RT-PCR; P. Fernández and J.C. Ramírez (CNIC) for lentivirus production; F. Cabo (CNIC) for bioinformatics and statistical support; C. Carreiro and A. Esteban (CNIC) for preparation and maintenance of plasmid stocks; M. García-Arranz (Hospital Univesitario La Paz, Madrid, Spain), C. Trigueros, A. Izeta and A. García (F. Inbiomed, San Sebastián, Spain) for biological samples; S. Bartlett (CNIC) for English revision; and M. Ramón for secretarial support. This work was supported by grants to AB from the Spanish Plan Nacional de Salud y Farmacia/CICYT (SAF 2008-02099), Comunidad Autónoma de Madrid (P-BIO-0306-2006) and Red de Terapia Celular del Instituto de Salud Carlos III (TerCel); MAG is supported by the "Miguel Servet" Program (CP07/00306) of the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, Spain); JCS is supported by the ACI-PLAN-E Program (Ministerio de Ciencia e Innovación, Spain). The CNIC is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation. Supplementary information accompanies the paper on the Cell Death and Differentiation website (http://nature.com/cdd)

the Mann-Whitney U-test for statistical significance. Results were considered statistically significant at P ≤ 0.05.

Conflict of interest

The authors declare no conflict of interest.


Jonathan Ham
email: j.ham@ich.ucl.ac.uk

Rosie Hughes

Mark Kristiansen

Irina Lassot

Solange Desagher

Roberto Mantovani

NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons

Keywords: apoptosis, sympathetic neuron, Bim, NF-Y, CBP/p300 NGF, nerve growth factor, ICB, inverted CCAAT box, MOMP, mitochondrial outer membrane permeabilisation, EMSA, electrophoretic mobility shift assay, ChIP, chromatin immunoprecipitation, GTP C1,

Introduction

The intrinsic (mitochondrial) pathway of apoptosis hinges on the balance of activity between proapoptotic and antiapoptotic members of the Bcl-2 superfamily. Bim is a BH3-only Bcl-2 family protein whose proapoptotic function is critical for apoptosis via the intrinsic pathway and Bim is one of just three BH3-only members that can bind to all five of their prosurvival relatives. [START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF] In addition, there is mounting evidence that Bim can induce mitochondrial outer membrane permeabilisation (MOMP) by binding to and directly activating the multidomain proapoptotic protein Bax. [START_REF] Chipuk | The BCL-2 family reunion[END_REF] During development, sympathetic neurons depend on target-derived NGF for their survival, and in its absence the cells die by apoptosis, usually within 24-48 hr. [START_REF] Deshmukh | Programmed cell death in neurons: focus on the pathway of nerve growth factor deprivation-induced death of sympathetic neurons[END_REF] Following NGF withdrawal, bim RNA and Bim protein levels increase rapidly and peak at around 16 hours later. [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF] The overexpression of Bim EL in sympathetic neurons is sufficient to induce the release of cytochrome c and apoptosis in the presence of NGF and sympathetic and sensory neurons isolated from bim -/-knockout mice are significantly protected from trophic factor withdrawal-induced death. [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Coultas | Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis[END_REF] Because Bim is a potent inducer of apoptosis in many cell types, its expression is tightly regulated. In models of neuronal cell death, in which the cells depend on NGF for survival, three transcriptional pathways have been described. Activation of the MLK-JNK-c-Jun pathway is a key event for bim upregulation in sympathetic neurons: the overexpression of a dominant negative c-Jun protein reduces the increase in bim mRNA and protein level that occurs after NGF withdrawal [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF] and the jun AA knock-in mutation, which eliminates the two major JNK phosphorylation sites in c-Jun, reduces the increase in Bim protein level after NGF withdrawal. [START_REF] Besirli | The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: identification of the nuclear pore complex as a potential target of the JNK pathway[END_REF] Furthermore, bim transcription is negatively regulated by PI3-K-Akt signalling: active Akt phosphorylates FOXO3a, which is sequestered in the cytoplasm by 14-3-3 protein and following NGF withdrawal FOXO3a translocates into the nucleus of sympathetic neurons and activates bim expression via two conserved FOXO sites. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] Moreover, FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] Additionally, bim is activated by a Cdk4-E2F-Myb pathway following NGF withdrawal in neuronally differentiated PC12 cells and this requires Myb binding sites in the bim promoter. [START_REF] Biswas | Bim is a direct target of a neuronal E2Fdependent apoptotic pathway[END_REF] Previous studies have therefore identified important transcriptional mechanisms by which bim is regulated following growth factor withdrawal, however, nothing is currently known about the bim core promoter.

The DNA region that contains the core promoter is critical for the recruitment and assembly of the RNA polymerase II transcription initiation complex. [START_REF] Juven-Gershon | Regulation of gene expression via the core promoter and the basal transcriptional machinery[END_REF] It is important to fully understand the bim core promoter architecture, since the regulatory pathways that activate or repress bim transcription must ultimately act upon this region. Here, we use NGF-dependent sympathetic neurons to investigate how the DNA binding transcription factor NF-Y and the coactivators CBP and p300 contribute to bim promoter function and activation following NGF withdrawal. Our results suggest a model in which NF-Y and CBP/p300, together with FOXO3a, are part of a multi-protein complex that is regulated by NGF.

Results

NF-Y binds to an inverted CCAAT box in the bim proximal promoter

Since bim has a TATA-less promoter we were interested to establish what factor(s) control its basal promoter function and how these regulate bim expression following NGF deprivation in sympathetic neurons. Using ConSite DNA analysis software [START_REF] Lenhard | Identification of conserved regulatory elements by comparative genome analysis[END_REF] we identified a conserved, inverted CCAAT box (ICB) [START_REF] Mantovani | A survey of 178 NF-Y binding CCAAT boxes[END_REF] that is located 29 bp or 54 bp upstream of the major transcriptional start site in rat and human bim, respectively (Figure 1). The major CCAAT box-binding protein is NF-Y, a heterotrimer formed by the interaction of three subunits, NF-YA, B and C. [START_REF] Mantovani | A survey of 178 NF-Y binding CCAAT boxes[END_REF] To investigate whether NF-Y can bind to the ICB in the bim promoter, we performed some electrophoretic mobility shift assays (EMSAs). An oligo containing a previously identified CCAAT box in the rat GTP cyclohydrolase I (GTP C1) gene [START_REF] Kapatos | Identification and Characterization of Basal and Cyclic AMP Response Elements in the Promoter of the Rat GTP Cyclohydrolase I Gene[END_REF] was used as a positive control (Figure 2a lanes 1-4). Initially EMSA experiments were performed with nuclear extracts from neuronally differentiated PC6-3 cells, a PC12 subline (Figure 2a). The nuclear extracts contained proteins that bound the GTP C1 CCAAT box (lanes 1-4) and this binding (marked NF-Y complex) was abolished by point mutations in the CCAAT box consensus (lanes 5,6). NF-YA is the regulatory subunit and is responsible for sequence specific DNA binding. [START_REF] Manni | Posttranslational regulation of NF-YA modulates NF-Y transcriptional activity[END_REF] Therefore, antibodies specific to the NF-YA subunit or to c-Jun (as a negative control) were used for supershift assays. The c-Jun antibody did not alter the binding pattern (lane 4), whereas the NF-Y antibody supershifted the entire protein complex (marked ss) (lane 3). The binding of NF-Y to the bim ICB (lane 8) was similar to the binding of NF-Y to the GTP C1 probe (lane 2), and the NF-Y complex behaved in an identical fashion when antibodies against NF-Y were added (lanes 3,9). We also performed EMSA experiments with whole cell extracts prepared from sympathetic neurons (Figure 2b). Extracts from sympathetic neurons maintained in the presence of NGF (lane 2) reproduced the binding pattern that was observed with extracts from differentiated PC6-3 cells in Figure 2a, and binding did not change following NGF withdrawal (lane 3). The NF-Y complex was supershifted with the NF-YA antibody (lanes 4,5) but not a negative control antibody (Fra-2; lanes 6,7) and binding was abolished by mutations in the ICB (lanes 9,10).

To confirm that NF-Y can bind to the endogenous bim promoter, we carried out chromatin immunoprecipitation (ChIP) assays using the NF-YA antibody or with an antibody against MyoD as a negative control (Figure 2c andd). Neuronally differentiated PC6-3 cells were maintained in the presence or absence of NGF for 16 hours. The binding of NF-Y to the ICB in the bim promoter was detected by PCR, using primers that amplify the region that contains the ICB. The negative control antibody to MyoD immunoprecipitated background levels of bim promoter chromatin, however this was significantly enriched by the antibody against NF-YA in cells maintained in the presence of NGF and following NGF withdrawal (Figure 2c andd).

Therefore, NF-Y is bound to the ICB in the rat bim promoter in the presence and absence of NGF, with no significant change in the amount of NF-Y bound to the promoter following withdrawal of NGF.

The ICB is critical for bim promoter function and contributes to the activation of bim transcription following NGF withdrawal

A bim-LUC reporter construct is significantly activated after NGF withdrawal in microinjected sympathetic neurons. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF][START_REF] Gilley | Evidence for increased complexity in the regulation of Bim expression in sympathetic neurons: involvement of novel transcriptional and translational mechanisms[END_REF] To determine whether the ICB is important for bim promoter activity, the point mutations that abolished NF-Y binding (Figure 2a and b) were introduced into the ATTGG pentamer within the bim-LUC construct (Figure 3a).

The bim-LUC or bim-LUC CCAAT(-) constructs were then microinjected into sympathetic neurons and luciferase activity was determined following 16 hours in the presence or absence of NGF. Mutation of the ICB resulted in an 80% decrease in basal promoter activity (Figure 3b). Following NGF withdrawal the bim-LUC reporter was activated significantly as previously reported. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF][START_REF] Gilley | Evidence for increased complexity in the regulation of Bim expression in sympathetic neurons: involvement of novel transcriptional and translational mechanisms[END_REF] The basal levels of the two constructs, in the presence of NGF, were then set to 1

and the induction factors of the constructs compared (Figure 3c). Mutation of the ICB significantly decreased the fold induction of the bim promoter following NGF withdrawal, from 2.37±0.19 to 1.50±0.13.

NF-Y activity is required for Bim expression and contributes to NGF withdrawal-induced death

To establish whether NF-Y activity is required for bim promoter function, sympathetic neurons were microinjected with the YA13 m29 expression vector along with guinea pig IgG (gp IgG) as a marker (Figure 4). YA13 m29 is a dominant negative mutant of the NF-YA subunit. [START_REF] Mantovani | Dominant negative analogs of NF-YA[END_REF] It can bind to the other NF-Y subunits but not to DNA and thus acts by forming inactive NF-Y trimers. The YA13 m29 construct was microinjected into sympathetic neurons and after 24 hours the cells were stained with an anti-NF-YA antibody. NF-YA immunostaining was considerably brighter in the nuclei of injected cells (Figure 4a) and over 90% of the injected neurons expressed high levels of NF-YA. Sympathetic neurons were then co-microinjected with YA13 m29, or the empty vector pSG5, along with the bim-LUC construct (Figure 4b). Expression of YA13 m29 completely inhibited the induction of bim-LUC following NGF withdrawal and also caused a significant decrease in the basal level of bim-LUC.

We then studied the effect of YA13 m29 on endogenous Bim protein levels (Figure 4c andd). Sympathetic neurons were microinjected with the YA13 m29 construct, or pSG5, and then cultured ±NGF for 16 hours, after which time the cells were fixed and stained with an anti-Bim antibody. Bim is present in the cytoplasm at low levels in the presence of NGF, but strongly increases in level following NGF withdrawal (Figure 4c). Following NGF deprivation, almost 60% of the cells injected with pSG5 expressed high Bim levels (Figure 4d). However, when the cells were injected with the YA13 m29 construct the percentage of cells expressing high levels of Bim was significantly reduced, to 10% (Figure 4d).

To investigate the role of NF-Y in NGF withdrawal-induced death, sympathetic neurons were microinjected with the YA13 m29 construct, or pSG5, together with Texas red dextran (TRD) as a marker. The neurons were then maintained in the presence or absence of NGF and the number of viable injected cells was counted at 0, 24, 48 and 72 hours. Viable neurons were morphologically normal and retained TRD in the nucleus (Figure 5a). At 72 hours following NGF withdrawal, the number of viable cells injected with the pSG5 control was below 20% (Figure 5b), whereas cells injected with YA13 m29 were partially protected from cell death (39% were viable) (Figure 5b). The injected neurons not protected by YA13 m29 died by apoptosis following NGF withdrawal (Supplementary figure S1). This data indicates that NF-Y activity is necessary for cell death in NGF-deprived sympathetic neurons.

CBP and p300 are important for activation of the bim promoter following NGF withdrawal

The ICB and NF-Y activity are necessary for bim promoter activation following NGF withdrawal, but the mechanism by which NF-Y functions has yet to be elucidated, since NF-Y is bound to the ICB in both the presence and absence of NGF. CBP and p300 are two distinct but highly homologous transcriptional coactivators. They can activate the transcription of a multitude of target genes and can interact with over 350 viral and mammalian proteins [START_REF] Kasper | Conditional knockout mice reveal distinct functions for the global transcriptional coactivators CBP and p300 in T-cell development[END_REF] including c-Jun, FOXO3a, Myb and NF-Y which activate bim transcription in sympathetic neurons. [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF][START_REF] Biswas | Bim is a direct target of a neuronal E2Fdependent apoptotic pathway[END_REF] We therefore investigated whether CBP/p300 are important for bim promoter activation following NGF withdrawal. In immunocytochemistry experiments we confirmed that CBP and p300 localise to the nucleus of sympathetic neurons, with approximately 95% of sympathetic neurons expressing high levels of CBP and p300 in the presence and absence of NGF (Supplementary figure S2). To study whether CBP and p300 are required for bim promoter activation, antibodies specific for CBP or p300 were coinjected into sympathetic neurons together with the bim-LUC construct (Figure 6a).

The microinjection of specific antibodies can be used as a suitable alternative to conventional protein knockdown techniques in these cells. [START_REF] Arias | Activation of cAMP and mitogen responsive genes relies on a common nuclear factor[END_REF][START_REF] Estus | Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis[END_REF][START_REF] Towers | The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons[END_REF] The bim-LUC construct was not significantly activated following NGF withdrawal when antibodies against CBP or p300 were co-injected, compared to the control rabbit IgG (Figure 6a).

We also performed a positive control antibody co-injection experiment using a CREB reporter construct pCRE-LUC (Figure 6b), which requires CBP and p300 for its activation by protein kinase A and CREB. Sympathetic neurons were co-injected with pCRE-LUC together with the CBP or p300 antibodies or rabbit IgG. Following injection the cells were treated with CPTcAMP, a membrane permeable cAMP analogue. The pCRE-LUC construct was significantly activated by CPTcAMP treatment when co-injected with IgG compared to the control construct without CRE sites, p-LUC. Importantly, injection of the CBP or p300 antibodies significantly decreased this induction.

Studies with p300 and CBP knockout mice indicate that CBP and p300 are present in cells at limiting concentrations. [START_REF] Yao | Gene dosagedependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300[END_REF][START_REF] Tanaka | Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein[END_REF] Therefore, overexpression of p300 or CBP should activate the bim promoter in the presence of NGF. Sympathetic neurons were co-microinjected with pRc/RSV-mCBP or the control empty vector pBJ9Ω, or CMVp300 or the control empty vector pcDNA3.1, together with the bim-LUC reporter construct (Figure 6c). Bim-LUC was significantly activated by overexpression of either CBP or p300. Interestingly, overexpression of p300 did not activate bim-LUC CCAAT (-) (Figure 6d) suggesting that the ICB must be intact for activation of the bim promoter by p300.

The amount of CBP/p300 bound to the bim promoter is regulated by NGF withdrawal

To confirm that p300 and CBP can bind to the endogenous bim promoter, we performed ChIP assays using differentiated PC6-3 cells and the p300 and CBP antibodies, along with antibodies against MyoD and Bim as negative controls (Figure 7a andb). The binding of p300 or CBP to the bim core promoter was studied by PCR using the primers that were previously used to study the binding of NF-Y to the ICB.

In the presence of NGF, there was some binding of p300 and CBP to the bim promoter but this was significantly enriched following NGF withdrawal (Figure 7a and b). Therefore, p300 and CBP are bound to the same region of the bim promoter as NF-Y and the amount of p300 and CBP bound to the promoter increases ~2.1 fold following NGF withdrawal.

CBP/p300 interact with NF-Y in the presence of NGF and following NGF withdrawal

To investigate whether NF-Y and CBP/p300 interact in extracts from NGF-dependent cells, co-immunoprecipitation (Co-IP) experiments were carried out (Figure 7c and Supplementary figure S3a). Nuclear extracts were prepared from differentiated PC6-3 cells cultured ±NGF for 16 hours. The nuclear extracts were immunoprecipitated with antibodies against p300, CBP, NF-YA or rabbit IgG. Immunoblotting was then performed using an NF-YA antibody (Figure 7c). Importantly, NF-YA was precipitated with antibodies against CBP and p300 but not the control rabbit IgG. In addition, the NF-Y antibody immunoprecipitated p300 and CBP (Supplementary figure S3a). These results demonstrate that NF-Y interacts with CBP and p300 in the nucleus of PC6-3 cells cultured in the presence or absence of NGF.

Following NGF withdrawal FOXO3a binds to the bim promoter and interacts with CBP/p300

Since FOXO3a contributes to the induction of bim transcription after NGF withdrawal [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] we investigated whether, like NF-Y, FOXO3a bound to CBP and p300, as reported in other systems. [START_REF] Mahmud | Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells[END_REF] First, we performed ChIP assays on differentiated PC6-3 cells using a FOXO3a antibody and MyoD and Bim antibodies as negative controls (Figure 7e, f and Supplementary figure S3c,d). The binding of FOXO3a to FOXO site 1 (located upstream of the ICB) and FOXO site 2 (located at the exon 1/intron junction) was studied by PCR. Following NGF withdrawal there was a large increase (~4.5 fold) in the amount of FOXO3a bound to each site (7e, f and Supplementary figure S3c,d).

We then investigated in co-IP experiments whether FOXO3a can interact with CBP/p300 in nuclear extracts prepared from differentiated PC6-3 cells cultured ± NGF (Figure 7d and Supplementary figure S3b). The lysates were immunoprecipitated with antibodies against p300, CBP, FOXO3a or rabbit IgG.

Immunoblotting was then performed using a FOXO3a antibody (Figure 7d). In -NGF extracts, FOXO3a was precipitated with antibodies against CBP and p300, but this was not the case in the presence of NGF, or in either condition for the control rabbit IgG. In the reverse experiment, CBP and p300 were precipitated with the FOXO3a

antibody, but again only after NGF withdrawal (Supplementary figure S3b).

The bim promoter is not activated in response to NGF withdrawal following the combined loss of NF-Y and FOXO binding sites

To assess the relative contribution of the FOXO and NF-Y binding sites in the bim promoter, we mutated both FOXO sites and the ICB within bim-LUC (Figure 8a).

We compared bim-LUC CCAAT(-) FOXO(dm) to the wild type bim-LUC in microinjection experiments with sympathetic neurons (Figure 8b). Loss of both FOXO sites, together with the ICB, significantly reduced the basal promoter level, as shown previously for bim-LUC CCAAT(-). Interestingly, loss of all 3 sites completely abolished the induction of bim-LUC following NGF withdrawal. This indicates that the two FOXO sites are required for the residual induction that is seen with bim-LUC CCAAT(-) after NGF deprivation.

We also investigated the effect on cell survival after NGF withdrawal of inhibiting both NF-Y and FOXO activity. YA13 m29 was co-expressed with the FKH (DBD), and TRD was used as a marker. The FKH (DBD) construct expresses the DNA-binding domain (DBD) of FOXO3a, which is a specific inhibitor of FOXO transcriptional activity. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] Following injection, the neurons were withdrawn from NGF for 72 hours and the number of viable injected cells was determined (Figure 8c). In accordance with Figure 5b, at 72 hours following NGF withdrawal, the number of viable cells injected with the control (pSG5 + pcDNA.1) was approximately 15%, whereas cells that were injected with YA13 m29 were partially protected from cell death (~40% of cells were viable at 72 hours -NGF). As demonstrated previously by Gilley et al. (2003) neurons injected with the FKH (DBD) construct were better protected (58% of cells were viable at 72 hours -NGF). Co-expression of both YA13 m29 and FKH (DBD) did not significantly increase cell survival compared to injection of the FKH (DBD) construct alone (Figure 8c). This is probably because the FKH (DBD) construct alone already gives the maximum protection observed in this microinjection assay, similar to that conferred by Bcl-2, dominant negative c-Jun or the JNK binding domain of JIP-1. [START_REF] Ham | A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death[END_REF][START_REF] Kristiansen | Mkp1 is a c-Jun target gene that antagonises JNK-dependent apoptosis in sympathetic neurons[END_REF]

Discussion

We have studied the role of a conserved inverted CCAAT box in the bim core promoter that is bound by NF-Y. This is the first bim promoter element that has been shown to be important for basal promoter function and since the bim promoter does not contain a TATA box, it is likely that the NF-Y-binding-ICB plays an important role in pre-initiation complex assembly as reported for other TATA-less promoters. [START_REF] Kabe | NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes[END_REF] In addition, the ICB is critical for bim promoter activation following NGF withdrawal.

We used the YA13 m29 dominant negative mutant [START_REF] Mantovani | Dominant negative analogs of NF-YA[END_REF][START_REF] Agostino | Gain of function of mutant p53: The mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation[END_REF][START_REF] Benatti | A balance between NF-Y and p53 governs the pro-and anti-apoptotic transcriptional response[END_REF] to establish that NF-Y activity is required for bim promoter function and expression of the endogenous Bim protein. Importantly, we also demonstrated that expression of YA13 m29 partially protects sympathetic neurons from NGF withdrawal-induced death (~40% of the cells are viable at 72 hr -NGF). This level of protection is similar to the effect of a complete bim -/-knockout on neuronal survival. [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Coultas | Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis[END_REF] This partial protection is likely to be because the other BH3-only proteins that increase in level after NGF withdrawal, Puma and Dp5, can partially compensate for the loss of Bim. [START_REF] Putcha | Men are but worms: neuronal cell death in C elegans and vertebrates[END_REF][START_REF] Ham | BH3-only proteins: key regulators of neuronal apoptosis[END_REF][START_REF] Wyttenbach | The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons[END_REF] NF-Y can recruit the transcriptional cofactors CBP and p300 to DNA to potentiate transcription. [START_REF] Salsi | Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function[END_REF] Since the amount of NF-Y bound to the bim promoter does not increase after NGF withdrawal, we hypothesised that the recruitment of transcriptional cofactors to the bim promoter may be regulated by NGF withdrawal.

We found that co-injection of antibodies against CBP or p300 inhibits the induction of bim-LUC after NGF withdrawal and that CBP and p300 bind to the region of the bim promoter that is bound by NF-Y, and that the amount of CBP/p300 associated with bim significantly increases after NGF withdrawal.

Since CBP and p300 do not bind DNA directly [START_REF] Chan | p300/CBP proteins: HATs for transcriptional bridges and scaffolds[END_REF] , they must interact with proteins that are already bound to the bim promoter. We have shown that mutation of the ICB abolishes the activation of the bim-LUC reporter by p300 and that NF-Y interacts with CBP and p300 in extracts from neuronal cells. This suggests that CBP/p300 may activate the bim promoter through protein-protein interactions with NF-Y bound to the ICB. Furthermore, studies in other systems have demonstrated that CBP/p300 can interact with the other regulatory factors, c-Jun, FOXO and Myb, [START_REF] Mahmud | Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells[END_REF][START_REF] Lee | Adenovirus E1A downregulates cJun-and JunBmediated transcription by targeting their coactivator p300[END_REF][START_REF] Bessa | Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator[END_REF] that activate bim transcription in neurons. We have shown that FOXO3a binds to two conserved FOXO sites in the bim promoter following NGF withdrawal. In addition, we have demonstrated that FOXO3a interacts with CBP and p300 in extracts from neuronal cells, in the absence of NGF.

CBP and p300 can act as a 'bridge' to connect sequence-specific transcription factors to the basal transcription machinery [START_REF] Chan | p300/CBP proteins: HATs for transcriptional bridges and scaffolds[END_REF] and their large size allows multiple proteins to bind to them simultaneously. It is possible that a multi-protein enhanceosome-like structure [START_REF] Merika | Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription[END_REF] may form on the bim promoter following NGF withdrawal, since we have shown that CBP/p300 can bind to NF-Y and FOXO3a, and NF-Y is known to recruit the basal transcription machinery and RNA polymerase II to the promoters of CCAAT box-containing genes. [START_REF] Kabe | NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes[END_REF] It will be interesting to establish whether this mechanism is also critical in other cell types since the ICB is common to the rat, mouse and human bim genes and NF-Y, FOXO3a and CBP/p300 are widely expressed. Preliminary experiments using mouse cerebellar granule neurons suggest that the ICB is important for bim transcription in these cells (Supplementary figure S4).

In summary, our data supports a model (Figure 9) in which NF-Y is bound to the ICB in the presence of NGF and recruits some CBP/p300 to the bim promoter. In sympathetic neurons deprived of NGF, FOXO3a translocates from the cytoplasm into the nucleus and binds to the two conserved FOXO sites that flank the ICB. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] CBP/p300 binding to the bim promoter increases significantly after NGF withdrawal because CBP/p300 can be recruited by FOXO3a as well as NF-Y. Therefore, following NGF withdrawal, NF-Y and FOXO3a (and potentially other factors such as c-Jun and Myb) recruit CBP/p300 to form a stable multi-protein complex that increases the rate of bim transcription (Figure 9). Such protein-protein interactions would therefore integrate the different signalling pathways that cooperatively activate the bim promoter following survival factor withdrawal.

Materials and methods

Plasmid constructs

The bim-LUC reporter consists of a 5.2 kb fragment, containing the region immediately 5′ to the rat bim initiator codon, sub-cloned into pGL3-Basic. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] The bim-LUC CCAAT(-) construct was generated using the oligonucleotides 5′-GAGCCCGCGGTGAGCTCGCGCCGGAGCGGG-3′ and 5′-CCCGCTCCGGCGCGAGCTCACCGCGGGCTC-3′. The altered bases (underlined) create four point mutations in the inverted CCAAT box. The bim-LUC CCAAT(-) FOXO(dm) construct was generated by incorporating point mutations into FOXO site 1 and FOXO site 2 within bim-LUC CCAAT(-) using oligonucleotides described previously. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] Mutagenesis was carried out using the QuikChange II XL site-directed mutagenesis kit (Stratagene, Agilent, Santa Clara, CA, USA). YA13 m29 is a dominant negative mutant of the NF-YA subunit [START_REF] Mantovani | Dominant negative analogs of NF-YA[END_REF] cloned into the expression vector pSG5 (Stratagene). The expression vector for p300 was supplied by Upstate (Millipore, Billerica, MA, USA). It consists of the human p300 cDNA cloned into pCMVβ (CMVp300). The expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), which contains the CMV promoter, was used as an empty vector control. The expression vector for CBP was kindly provided by Professor Tony Kouzarides (The Gurdon Institute, Cambridge, UK). It contains the mouse CBP cDNA cloned into pRc/RSV (pRc/RSV-mCBP). The expression vector pBJ9Ω, which is also under the control of the RSV promoter, was used as an empty vector control (also supplied by T. Kouzarides). The CREB reporter construct pCRE-LUC and the control without CRE sites, pLUC, were supplied by Stratagene. FKH (DBD) [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] is the DNA-binding domain of FOXO3a cloned into the expression vector pcDNA.1 (Invitrogen).

Cell culture

Sympathetic neurons were isolated from the superior cervical ganglia (SCG) of 1-dayold Sprague Dawley rats and cultured as described previously. [START_REF] Whitfield | Methods for culturing primary sympathetic neurons and for determining neuronal viability[END_REF] Cells were maintained in SCG medium supplemented with 2.5S NGF (Cedarlane, Burlington, Canada) at 50 ng/ml, and fluorodeoxyuridine and uridine (both from Sigma-Aldrich, St Louis, MO, USA), each at 20 μM. PC6-3 cells, a subline of the PC12 cell line 37 , were cultured as described previously. [START_REF] Towers | The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons[END_REF] For differentiation, PC6-3 cells were maintained in medium containing 2% horse serum, 1% FCS and NGF (Promega, Fitchburg, WI, USA) at 100 ng/ml, for 5-7 days. In NGF withdrawal experiments, cells were rinsed twice with medium (without NGF) and then re-fed with medium containing an anti-NGF antibody at 100 ng/ml (Chemicon, Millipore). For experiments with pCRE-LUC and pLUC, sympathetic neurons were re-fed with +NGF medium containing 500 μM CPTcAMP (Sigma-Aldrich).

Cerebellar granule neurons (CGNs) were prepared from 7-day-old mice and maintained in medium containing 25 mM KCl, as described previously. [START_REF] Desagher | Genes regulated in neurons undergoing transcription-dependent apoptosis belong to signaling pathways rather than the apoptotic machinery[END_REF][START_REF] Lassot | Trim17, a novel E3 ubiquitinligase, initiates neuronal apoptosis[END_REF] In KCl deprivation experiments, neurons were washed once and then incubated in serum-free BME supplemented with L-Gln, HEPES, antibiotics and 1 μM of the NMDA antagonist (+)-MK-801, and 5 mM KCl (K5 medium) to induce apoptosis.

Microinjection

Sympathetic neurons were cultured in vitro for 5-7 days and then microinjected as described previously. [START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF] Approximately 150 cells were injected per coverslip and each injection experiment was repeated at least 3 times. Plasmids were injected into the nucleus of each cell and over 90% of cells survived injection.

Dual luciferase assays were carried out on injected cells using the Dual Luciferase reporter assay system (Promega). Cells were harvested for luciferase assays at 16 hours following NGF withdrawal.

For antibody coinjection experiments, the Santa Cruz (Santa Cruz, CA, USA) p300 (C-20:Sc-585 X) and CBP (C-20:Sc-583 X) antibodies were diluted in PBS (-)

and centrifuged in Microcon YM-3 centrifugal filters (Millipore) to remove sodium azide. Antibodies were injected at a final concentration of 1 μg/μl and purified rabbit immunoglobulin in PBS (Jackson ImmunoResearch, West Grove, PA, USA) was used as a control.

In cell survival assays, neurons were injected with the YA13 m29 expression vector or the control empty vector (pSG5) at 200 ng/μl, along with 5 μg/μl of Texas red dextran (70,000 MW; Molecular Probes, Invitrogen).

Transfection of CGNs

CGNs cultured in 24-well plates were transfected at 5 days in vitro with 0.5 μg of pRL-TK together with 1.5 μg of either the bim-LUC or bim-LUC CCAAT(-) constructs, using calcium phosphate precipitation as described previously. [START_REF] Lassot | Trim17, a novel E3 ubiquitinligase, initiates neuronal apoptosis[END_REF] After 24 hours the cells were either left untreated (K25) or deprived of KCl (K5). Four hours after deprivation, transfected neurons were harvested for luciferase assays using the Dual Luciferase reporter assay system.

Immunocytochemistry

Neurons were injected with the YA13 m29 expression vector or the control empty vector (pSG5) at 20 ng/μl along with 2.5 μg/μl of purified guinea pig IgG (Jackson ImmunoResearch) and fixed and stained as described before. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] In brief, cells were fixed in 4% paraformaldehyde, permeabilised in 0.5% Triton-X-100 and blocked in 50% goat serum (Sigma-Aldrich). After washing, cells were incubated in a primary antibody to detect NF-YA (Santa Cruz Sc-10779:H-209) or Bim (#2819 Cell Signalling, Danvers, MA, USA). Secondary antibodies were a FITC-conjugated antirabbit IgG, and a rhodamine anti-guinea pig IgG if neurons were microinjected (both from Jackson ImmunoResearch). Secondary-only controls were carried out to confirm the specificity of primary antibodies and cells were scored in a blinded manner whenever possible. TUNEL analysis was carried out using an in situ cell death detection kit (Roche, Basel, Switzerland). All slides were viewed using a Zeiss Axioplan 2 fluorescence microscope.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) experiments were performed on nuclear lysates from differentiated PC6-3 cells maintained in medium containing NGF or withdrawn from NGF for 16 hours, and whole cell lysates from sympathetic neurons maintained in medium containing NGF or withdrawn from NGF for 16 hours.

Nuclear lysates were prepared from PC6-3 cells and whole cell extracts were prepared from sympathetic neurons as described previously.

Chromatin immunoprecipitation

Differentiated PC6-3 cells (approximately 4 X 10 6 cells per 9 cm dish) were either maintained in the presence of NGF, or withdrawn from NGF for 16 hours. The cells were fixed in 1% formaldehyde for 3 hours at room temperature and chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit from Upstate (Millipore) with some modifications, as described previously. [START_REF] Towers | The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons[END_REF] The following antibodies from Santa Cruz were used for ChIP: NF-YA (H-209:Sc-10779 X), p300 Samples were analysed between 25 and 30 cycles and the average amount of bim promoter DNA, precipitated by each antibody, was calculated using ImageQuant image analysis software (GE Healthcare, Waukesha, WI, USA).

Co-immunoprecipitation

Co-immunoprecipitation (co-IP) experiments were performed on nuclear lysates from differentiated PC6-3 cells maintained in +NGF medium or withdrawn from NGF for 16 hours. A 50% slurry of protein A/G-agarose (Santa Cruz) was prepared in PBS (-)

and added to a 1.5 ml microfuge tube containing 2 μg of antibody to immunoprecipitate either FOXO3a (Cell Signaling #2497), p300, CBP or NF-Y (NF-Y, p300 and CBP antibodies were the same for co-IP and ChIP), and resuspended in 500 μl of ice-cold PBS (-). This was also repeated for the non-specific control, purified rabbit immunoglobulin in PBS (Jackson ImmunoResearch). The mixtures were incubated at 4ºC for 90 seconds and the beads collected by centrifugation at 15,700 x g for 2 seconds at 4ºC. The supernatant containing the unbound antibody was removed and the beads were resuspended in 1 ml of non-denaturing lysis buffer (1% v/v Triton-X100, 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.02% v/v NaN 3 pH 7.4, 1 mM PMSF) . The tubes were inverted 4 times to mix and the beads were collected by centrifugation as previously. The beads were then washed in 1 ml of non-denaturing lysis buffer and collected by centrifugation. Around 80 μg of nuclear lysate was added to each tube containing specific or control antibody-bound beads. The mixture was incubated at 4ºC for 2 hours with rotation. The beads were then collected by centrifugation and the supernatant was removed. The beads were resuspended in 1 ml of ice-cold wash buffer, the tubes inverted 4 times to mix, and the beads were collected by centrifugation as previously. This wash step was repeated 3 times and the final supernatant was removed to leave the beads in approximately 15 μl of wash buffer (containing the immune complexes). The beads were resuspended in 30 μl of SDS gel sample buffer, boiled for 5 minutes at 100ºC and analysed by immunoblotting as described previously. [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] The NF-YA, FOXO3a, p300 and CBP antibodies used for immunoprecipitation were also used for immunoblots and an ERK1/2 antibody (Cell Signaling #9102) was used to check protein loading.

To prepare nuclear extracts for co-IP, neuronally differentiated PC6-3 cells were washed twice in ice-cold PBS (-) and then scraped off the dishes in 2 ml of PBS (per 9 cm dish). Cells were pelleted by centrifugation at 400 x g for 5 minutes at 4ºC and resuspended in 200 μl of buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) prior to incubation on ice for 15 minutes. 10% NP40 was added to each lysate (to a final concentration of 1%) and the samples were vortexed for 15 seconds prior to incubation for 15 minutes at 4ºC. The lysates were pelleted by centrifugation at 13,000 x g for 30 seconds, resuspended in 200 μl of ice-cold buffer C (20 mM Hepes pH 7.9, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 25% v/v glycerol) and incubated at 4ºC with shaking. After 1 hour lysates were vortexed for 30 seconds and pelleted by centrifugation for 10 minutes at 15,700 x g at 4ºC. The supernatant was removed and this was the nuclear extract.

Statistical analysis

In each set of experiments data is normalised to a control sample (for example, bim-LUC +NGF is set to 1). For microinjection, the relative induction of a DNA construct is calculated by dividing the normalised luciferase activity in the absence of NGF by the normalised luciferase activity in the presence of NGF. All data are presented as the mean ± S.E. of multiple experiments.

The statistical significance of differences between means was evaluated by performing an unpaired Student's T-test (for two-tailed distributions), followed by a Bonferroni correction. To compare normalised data to the control sample, that has no error associated to it (for example, when comparing data to bim-LUC +NGF, which is set to 1), the log10 values of the data were taken and a one sample T-test was used (for two-tailed distributions). Statistical significance is presented as p-values: *** p<0.001, ** p<0.01, * and p<0.05. There is a significant increase in the amount of bim promoter DNA precipitated by the NF-YA antibody in both +NGF (p=0.03) and -NGF (p=0.009) conditions, compared to the negative control antibody. Co-microinjection of CMVp300 does not activate bim-LUC CCAAT (-).

Titles and legends to figures

CTAGCTCCGGCGCGAGCTCACCGCGGG- 3 '

 3 (the mutated bases of the ICB are underlined). Oligonucleotides containing a CCAAT box from the rat GTP cyclohydrolase I gene were used as a positive control: GTP C1 WT 5.[START_REF] Kapatos | Identification and Characterization of Basal and Cyclic AMP Response Elements in the Promoter of the Rat GTP Cyclohydrolase I Gene[END_REF] 20 μl binding reactions were set up as described previously[START_REF] Kapatos | Identification and Characterization of Basal and Cyclic AMP Response Elements in the Promoter of the Rat GTP Cyclohydrolase I Gene[END_REF] , with 5 μg of nuclear extract from PC6-3 cells and 10 μg of whole cell extract from sympathetic neurons. Supershift experiments were carried out by incubating samples on ice with antibodies (4 μg) for 2 hours. The Santa Cruz NF-YA (H-209:Sc-10779 X) antibody was used in EMSA experiments along with c-Jun (H-79:Sc-1694 X) or Fra-2 (L-15:Sc-171 X) antibodies as negative controls (Santa Cruz). Samples were incubated with 0.4 ng of 32 P-labelled oligonucleotide for 15 minutes at room temperature and run on a 5% polyacrylamide gel. After electrophoresis, gels were fixed in 10% acetic acid / 10% methanol, dried and bands were visualised by exposing the gel to MXB X-ray film (Kodak, Rochester, NY, USA).

(C- 20 :

 20 Sc-585 X), CBP (C-20:Sc-583 X) and FOXO3a (H-144:Sc-11351 X). Millipore Bim (AB17003) and Santa Cruz MyoD (M-318:Sc-760 X) antibodies were used as negative controls.ChIP samples were analysed by semi-quantitative PCR. To detect binding of NF-Y to the inverted CCAAT box in the rat bim promoter the following primer . The same primer pair was used to detect binding of p300 and CBP to this region. The following primer pairs were used to detect binding of FOXO3a to FOXO site 1: 5′-CTAAGTTCCGCTCTGCGAGGT-3′ and 5′-TGCAGGCTGCGACAGGTAGT-3′, and FOXO3a to FOXO site 2: 5′-GATTCACACCACCTCCGCT-3′ and 5′-GCCTGTCTTCTATCCATCCC-3′.

Figure 1

 1 Figure 1 Alignment of the proximal promoter sequences for the rat, mouse and

Figure 2

 2 Figure 2 NF-Y binds to the bim ICB in the presence of NGF and following NGF

Figure 3

 3 Figure 3The ICB is critical for basal promoter function and contributes to the

Figure 4

 4 Figure 4 NF-Y activity is critical for endogenous Bim expression in sympathetic

Figure 5

 5 Figure 5 NF-Y activity contributes to cell death following NGF withdrawal in

Figure 6

 6 Figure 6 CBP and p300 are important for activation of the bim promoter in

Figure 7

 7 Figure 7The amount of CBP/p300 and FOXO3a bound to the bim gene increases

Figure 8

 8 Figure 8 The ICB and FOXO sites are critical for the induction of bim transcription

Figure 9

 9 Figure 9 Hypothetical model showing how transcription factors bind to the bim

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9

 123456789 Figure 1

Acknowledgements

We thank Tony Kouzarides for providing the expression vector for CBP, Becky Randall for advice on EMSA assays, and Francesca Menghi and Mike Hubank for critical reading of the manuscript. This work was supported by The Wellcome Trust (Senior Research Fellowship in Basic Biomedical Science 057700 to JH). RM is supported by the Associazione Italiana Ricerca Cancro (AIRC). SD and IL are supported by the Centre National de la Recherche Scientifique (CNSR) and the Institut National de la Santé et de la Recherche Médicale (INSERM).

Conflict of interest

The authors declare that they have no conflict of interest.