Sylvie Rival-Gervier

Mandy Ym

Mandy Ym Lo

Shahryar S Khattak

Peter P Pasceri

Matthew C M C Lorincz

James J Ellis
email: jellis@sickkids.ca

Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Retroviral vectors are transcriptionally silenced in pluripotent stem (PS) cells. This feature facilitated the discovery of induced PS (iPS) cells because delivery of exogenous pluripotency factors in retroviral vectors allowed the transgenes to be silenced as the somatic cells reprogrammed. In virtually all other contexts, silenc ing of retroviral vectors is considered deleterious for their use in stem cells. To overcome silencing, retroviral vector designs mutate or delete known silencer elements in or adjacent to the long term inal repeats (LTRs). [START_REF] Barklis | Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells[END_REF][START_REF] Pannell | Silencing of gene expression: implications for design of retrovirus vectors[END_REF] However, even selfinactivating (SIN) ret roviral vectors with a strong internal promoter are subject to silencing in embryonic stem (ES) cells. [START_REF] Osborne | Amelioration of retroviral vector silencing in locus control region beta-globintransgenic mice and transduced F9 embryonic cells[END_REF] Thus, further improve ments rely on removing additional unknown silencer elements, or on better defining the mechanisms of silencing and discovering how they can be blocked.

Retrovirus silencing occurs via epigenetic mechanisms in ES cells. For example, some retroviral sequences recruit the ZFP809 DNAbinding factor which interacts with repressive complexes including Kap1 (Trim28), the histone methyltransferase ESET (SETDB1), heterochromatin protein 1 (HP1), the nucleosome remodeling and histone deacetylase (NuRD) complex, and the nuclear receptor corepressor complex 1 (NcoR1). [START_REF] Ryan | KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing[END_REF][START_REF] Schultz | SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins[END_REF][START_REF] Schultz | Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD[END_REF][START_REF] Wolf | TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells[END_REF][START_REF] Wolf | Embryonic stem cells use ZFP809 to silence retroviral DNAs[END_REF] The binding of this complex results in the deposition of H3K9me3 marks on the sequences nearby and in transcriptional repression. Moreover, DNA methylation is targeted by the de novo methyltransferases Dnmt3a and 3b [START_REF] Dodge | De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation[END_REF] to CpGrich sequences in LTRs, [START_REF] Challita | Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo[END_REF] enhanced green fluorescent protein (EGFP) or other nonmammalian reporter genes. [START_REF] Dalle | eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides[END_REF] This hypermethylated DNA is bound by MeCP2 [START_REF] Lorincz | Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation[END_REF] and recruits histone deacetylases. [START_REF] Fuks | The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation[END_REF] However, deacetylated histone H3 and H1 can still be associated with silent retrovirus in Dnmt3a and 3b null ES cells, [START_REF] Pannell | Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code[END_REF] and H3K9me3 marks established by SetDB1 in ES cells are also independent of DNA methylation. [START_REF] Karimi | DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs[END_REF] The enzymes G9a/GLP write H3K9me2 marks but also promote DNA methyla tion of LTR elements and other genomic regions independently from their histone methyltransferase activity. [START_REF] Dong | DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity[END_REF][START_REF] Leung | Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing[END_REF] Inhibiting G9a/ GLP activity with a chemical probe (UNC0638) can reactivate the silent internal promoter of a retroviral vector and trigger DNA demethylation. [START_REF] Vedadi | A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells[END_REF] Unfortunately, the kinetics of these epigenetic events is poorly understood, and would be more easily defined by developing a system to synchronize retrovirus silencing in ES cells.

After integration of a SIN retroviral vector, the internal promoter can be subject to residual silencing emanating from cryptic silencer elements in the vector backbone or nonmam malian CpGrich reporter gene sequences. In addition, despite the propensity of murine leukemia virusbased retroviral vectors to integrate near the promoters of active genes, [START_REF] Roth | Gammaretroviral integration into nucleosomal target DNA in vivo[END_REF][START_REF] Wu | Transcription start regions in the human genome are favored targets for MLV integration[END_REF] the chroma tin environment surrounding the provirus may exert position effects that repress expression. [START_REF] Senigl | Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site[END_REF][START_REF] Dickson | VEZF1 elements mediate protection from DNA methylation[END_REF] Insulator elements can pro tect transgenes from position effects by: (i) blocking enhancerpromoter communication when positioned between them and (ii) acting as a barrier to prevent transgene silencing by block ing the spread of heterochromatin. [START_REF] Bell | Insulators and boundaries: versatile regulatory elements in the eukaryotic genome[END_REF] The chicken βglobin HS4 insulator is the most characterized insulator in vertebrates. [START_REF] Chung | A 5' element of the chicken betaglobin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila[END_REF] Its 250bp core has two physically separable and mechanisti cally distinct insulator properties. Enhancer blocking is medi ated by CTCF which is necessary to establish chromosomal loop domains. [START_REF] Ghirlando | Chromatin domains, insulators, and the regulation of gene expression[END_REF][START_REF] Phillips | CTCF: master weaver of the genome[END_REF] Barrier activity is attributed to VEZF1, which limits DNA methylation and USF1/USF2 which recruits enzymes that write activating modifications on histones. [START_REF] Dickson | VEZF1 elements mediate protection from DNA methylation[END_REF][START_REF] Bell | The protein CTCF is required for the enhancer blocking activity of vertebrate insulators[END_REF][START_REF] West | Recruitment of histone modifications by USF proteins at a vertebrate barrier element[END_REF] The HS4 core or larger fragments protect transgenes against silencing, [START_REF] Emery | The use of chromatin insulators to improve the expression and safety of integrating gene transfer vectors[END_REF][START_REF] Giraldo | The potential benefits of insulators on heterologous constructs in transgenic animals[END_REF] but this barrier activity is suboptimal in some contexts including retro viral transduction of ES cells. [START_REF] Aker | Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects[END_REF][START_REF] Moltó | Boundaries in vertebrate genomes: different solutions to adequately insulate gene expression domains[END_REF][START_REF] Truffinet | The chicken beta-globin HS4 insulator is not a silver bullet to obtain copy-number dependent expression of transgenes in stable B cell transfectants[END_REF][START_REF] Yao | Retrovirus silencer blocking by the cHS4 insulator is CTCF independent[END_REF] The D4Z4 element is a 3.3 kb macrosatellite sequence pre sent in 11-150 copies in the subtelomeric region of human chro mosome 4. Reduction of D4Z4 copy number is associated with human facioscapulohumeral dystrophy. [START_REF] Wijmenga | Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy[END_REF] Expression of an open reading frame (ORF) called DUX4 from the last D4Z4 repeat in patients is associated with the disease phenotype. [START_REF] Lemmers | A unifying genetic model for facioscapulohumeral muscular dystrophy[END_REF] A single D4Z4 monomer element can act as an insulator [START_REF] Ottaviani | The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy[END_REF] with enhancer block ing activity in its 5′ region that is dependent on CTCF binding. Unusually, D4Z4 can block position effects when inserted on just one side of the transgene, and therefore is not a classical barrier element like HS4 which must flank the transgene. [START_REF] Ottaviani | The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy[END_REF] These findings indicate that D4Z4 functions differently from HS4, and thus D4Z4 may protect transgenes from heterochromatin effects that are not shielded by HS4. Here, we investigate the utility of the D4Z4 insu lator in preventing retrovirus silencing in ES cells. Our findings demonstrate that 3′D4Z4 cooperates with HS4 barrier activity to drive unprecedented longterm retrovirus expression in ES cells. In situ deletion of 3′D4Z4 induces silencing, providing a system to identify kinetic classes of silencing and the epigenetic marks associated with them.

RESULTS

HSC1 retroviral vector is silenced over time even in the presence of the HS4 core

To characterize the kinetics of retrovirus silencing, we analyzed two different silencing events that occur at different timepoints. First, we examined the frequency of silencing that occurs rap idly after proviral integration and without any cell selection. Second, we tracked the kinetics of silencing that extinguishes transgene expression over prolonged periods of cell culture in cells that initially express the virus, isolated either by sorting for EGFPpositive cells or by drug selection with puromycin. For this purpose, we modified the HSC1 SIN retroviral vector to incorporate an internal human EF1α long promoter driving the expression of a bicistronic EGFP irespuromycin (GiP) cas sette (Figure 1a).

To quantify rapid silencing after infection, we isolated single cell ES clones in the absence of selection. Briefly, we infected ES cells at low multiplicity of infection (Figure 1b) and 1 day after infection performed a limitdilution assay to deposit only one cell per well. These cells were expanded without selection to identify transduced cell clones in which the EGFP transgene is silenced. The remainder of the bulk population was maintained without selection and at 6 days postinfection 2-9% of cells were EGFPpositive after infection with 5 or 25 μl of virus, respec tively (Figure 1c). To isolate clonal lines, wells with more than one colony were eliminated. About 260-280 clones from two independent transductions were replated on day 6 and assayed for EGFP expression by microscopy on day 8. To gather suffi cient genomic DNA (gDNA) for Southern analysis, the clones were cultured until day 17. gDNA used for replicate dot blots was hybridized with an EGFP probe to detect provirus and a mouse endogenous Thy1 probe as a loading control. These data show that about 10-15% of cells contain the proviral DNA, con sistent with single copy integration in the infected clones. We found that 76-84% of provirus positive clones express EGFP at day 8 postinfection (Figure 1d). These results show that about 20% of integrated HSC1GiP proviruses are rapidly silenced in the absence of selection.

To examine longterm silencing of the HSC1 retroviral vec tor, ES cells were infected with HSC1GiP at low multiplicity of infection to produce cells with single copy integrations. Two days after infection, 10-20% of cells were EGFPpositive consis tent with single copy transduction. These expressing cells were immediately selected with 1 μg/ml of puromycin for 4 days and then cell populations were maintained with or without selec tion for up to 150 days (Figure 2a). All viable cells were EGFP positive after 4 days of drug selection, and as expected EGFP expression persisted over time when cells were cultured with puromycin (Figure 2b). However, upon puromycin withdrawal, frequency of retroviral transgene expression decreased over time. At 60 days postinfection, 80% of cells expressed EGFP and by 150 days postinfection only 40% of the cells are EGFP positive (Figure 2b). This retrovirus silencing is detectable in ES cells, as well as in iPS cells (Supplementary Figure S1a-c), but not in NIH3T3 fibroblasts (Supplementary Figure S1d). To ensure that the HSC1GiP population is still undifferentiated, we assessed the pluripotency level of cells by analyzing the presence of SSEA1 protein, an ES cell surface marker, by flow cytometry and the presence of the Nanog ES cell nuclear marker by immu nocytochemistry. We show that 99% of the cells are SSEA1 positive (Supplementary Figure S2a) and Nanog protein is detectable in most nuclei (Supplementary Figure S2b). These results show that silencing and variability of EGFP expression is not a consequence of ES cell differentiation. Overall, these results show that the HSC1GiP retroviral vector is frequently silenced in undifferentiated PS cells over longterm culture, even when integration sites favorable for transgene expression are preselected shortly after infection.

To attempt to overcome this silencing, we used the well characterized dimer HS4 core insulator present in the HSC1 GiP LTRs (Figure 2c). Under puromycin selection, all cells were EGFPpositive (Figure 2d) but without drug selection, at 60 and 150 days postinfection, 90 and 70% of cells expressed EGFP, respectively. Thus, the dimer HS4 core located in the LTRs improves the frequency of transgene expression in ES cells but a significant percentage of cells still show silencing.

3′D4Z4 insulator increases the frequency of long-term transgene expression

Given that the HSC1 retroviral vector is strongly silenced in ES cells, we tested the novel strong D4Z4 element, first on its own at an internal location and subsequently combined with the dimer HS4 core in the LTRs. We cloned the fulllength D4Z4 mono mer upstream of the EF1α promoter (Figure 3a). Unfortunately, no reporter expression was detected after infection, indicating that the full D4Z4 sequence is incompatible with retroviral vector transduction. As only a monomer of D4Z4 is present and there are no endogenous D4Z4 elements in mouse cells, the inability to detect transduced virus is not due to any repetitive sequence of D4Z4. To identify D4Z4 sequences that can transmit at high titer, we subdivided this element into four DNA fragments derived from 5′D4Z4 or 3′D4Z4 regions and cloned them upstream of the EF1α promoter (Figure 3b). The 5′ fragment called D4Z4A contains a putative enhancer that was reported to activate an SV40 viral pro moter [START_REF] Petrov | A nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking activity in vivo: implications for the facio-scapulo-humeral dystrophy[END_REF] but not a mammalian promoter, [START_REF] Ottaviani | The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy[END_REF] an enhancer blocking region with a known CTCFbinding site and a barrier activity. [START_REF] Ottaviani | The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy[END_REF][START_REF] Petrov | A nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking activity in vivo: implications for the facio-scapulo-humeral dystrophy[END_REF] The D4Z4B fragment contains the promoter and 5′ end of the DUX4 ORF, but because this would introduce a second internal promoter, we excluded this vector from further analysis. To evaluate the 3′ end of D4Z4, we generated two fragments. D4Z4C contains only 36 bp of the DUX4 promoter and lacks its TATA box and the 3′ end of the DUX4 ORF. [START_REF] Ding | Characterization of a double homeodomain protein (DUX1) encoded by a cDNA homologous to 3.3 kb dispersed repeated elements[END_REF][START_REF] Dixit | DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1[END_REF][START_REF] Gabriëls | Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element[END_REF] To independently confirm that any activ ity that blocks retroviral silencing found in 3′D4Z4 does not require DUX4 expression, we also used D4Z4D that does not include the promoter nor the 5′ end of the DUX4 ORF. To investigate the pos sibility of spacing effects, we inserted a lambda phage spacer at the same position as a control. None of these fragments have enhancer activity on the EF1α promoter in transient transfection assays in ES cells (Supplementary Figure S3). Therefore, any influence they have on retroviral expression after vector integration likely involves epigenetic effects on chromatin.

Viruses with the spacer fragment and the D4Z4A and D4Z4C fragments were successfully produced and ES cells were infected at low multiplicity of infection resulting in 2-8% of cells expressing EGFP 3 days postinfection. In this experi ment, instead of using puromycin selection, we enriched for the expressing cell population by sorting for EGFPpositive cells 3 days postinfection. Cells were cultured for 2 months and EGFP expression assayed over time by flow cytometry. As expected, the vector with the spacer element was already subject to silencing at 64 days postinfection, with 70% EGFPpositive cells (Figure 3c). This result shows that a spacer element does not prevent silencing. Surprisingly, the D4Z4A virus infection also displays a promi nent peak of nonexpressing cells by 64 days postinfection with only 54% of cells still EGFPpositive. We show that transgene DNA in the D4Z4A population is only detected by Southern blot and quantitative PCR (qPCR) in EGFPpositive and not EGFP negative sorted cells (Supplementary Figure S4). These results show that the D4Z4A construct is unstable in this retroviral vec tor. In contrast, 95% of cells are EGFPpositive in the population containing the D4Z4C fragment (Figure 3c) and the transgene is easily detected by Southern blot and qPCR (Supplementary Figure S4). Therefore, 3′D4Z4 transmits through retroviral vec tors, is stable over extended passage time and ensures a very high frequency of retrovirus expression. These 3′D4Z4 cells display a mean fluorescence intensity (MFI) of 311 (compared with 250 in EGFPpositive cells with the spacer element) but exhibit some celltocell variability of expression (coefficient of variation (CV) = 65) (Figure 3d). To design an optimized retroviral vector for PS cells, it may be important to combine 3′D4Z4 with additional insulator activities that are compatible with transduction.

3′D4Z4 and HS4 elements cooperate to optimize long-term expression

While 3′D4Z4 can block silencing when located upstream of the EF1α promoter, it may be insufficient to fully protect the adjacent promoter from 3′ position effects that reduce the MFI. Because HS4 appeared to have partial barrier activity when located in the LTRs, we tested whether 3′D4Z4 can cooperate with HS4 in the LTRs to minimize transgene silencing in ES cells. We inserted 3′D4Z4 (fragments C or D) upstream of the EF1α promoter in the HSC1HS4GiP virus that contains the HS4 core dimer in the LTRs (Figure 4a). We infected ES cells and briefly selected for expressing cells with puromycin before culture with or with out selection for up to 5 months. After longterm culture, none of these populations shows any evidence of differentiation (Supplementary Figure S2). Strikingly, the expression frequency approaches 95-100% EGFPpositive cells without selection over 150 days after infection with D4Z4C or D4Z4D vectors (Figure 4b), indicating that vectors containing both HS4 and 3′D4Z4 are stable over extended passages. These data confirm that the 3′D4Z4 activity is functional in the presence of HS4 and does To determine whether the HS4 element improves the level (MFI) and consistency (CV) of expression in cooperation with 3′D4Z4, we analyzed the flow cytometry data at day 60 (for com parison to the results shown after EGFP sorting in Figure 3) and day 148 postinfection (Figure 4c). In cells grown without selection, the vector without any insulators has a low frequency of expression with increasing numbers of EGFPnegative cells over time, and the cells that do express have a high CV at the day 148 timepoint (Figure 4c and Supplementary Figure S6). Addition of HS4 improves the frequency of expression, but the MFI is reduced and the CV is almost unchanged, suggest ing that the barrier activity of HS4 may protect the transgene from surrounding genomic sequences but may not block silenc ing directly recruited by the provirus itself. When D4Z4C is present without HS4, the frequency of expression is very high at both timepoints, but the MFI is reduced and the CV remains at the same level consistent with a residual position effect. The shoulder of lowexpressing D4Z4C cells at day 60 is very simi lar to the behavior of these cells after EGFPpositive cell sorting and growth for 60 days as shown in Figure 3d, indicating that the two selection protocols generate similar results. Only in the presence of D4Z4C and HS4 does the frequency stay high and the CV is very low producing a very sharp peak with an MFI of around 300 at both timepoints. This finding is corroborated by virtually identical results from combining D4Z4D with HS4.

Overall, these data demonstrate that 3′D4Z4 cooperates with HS4 barrier activity to promote persistent transgene expression with unprecedented consistency in ES cells.

3′D4Z4 and HS4 cooperate to open chromatin structure on the provirus

To determine whether the 3′D4Z4 insulator protects retroviral transgene expression from silencing through formation of an open chromatin structure, we analyzed the DNA methylation and histone modification profiles on the provirus. We harvested gDNA from transduced ES cells cultured for more than 5 months without selection for bisulfite sequencing of the GAG, EF1α moter, and EGFP regions upstream and downstream of 3′D4Z4. In all but one population, the three regions have a similar level of CpG methylation (Figure 5a). In the population without insulators that have only 35% EGFPpositive cells, all regions are hypermethylated (72-87% CpG methylation). In contrast, the proviruses containing HS4 in both LTRs are partially methylated (16-29% CpG methylation), whereas the proviruses containing D4Z4C insulator display 4 and 2% CpG methylation in GAG and EF1α promoter regions, respectively. Partial methylation of 20% in EGFP may predispose the cells to the variation in expression observed over time. Finally, in the presence of 3′D4Z4 and HS4, the proviral DNA is hypomethylated (1-4% CpG methylation). Thus, the methylation pattern of proviral DNA is in complete concordance with the expression profile of each population. These findings suggest that 3′D4Z4 and HS4 alone are partially effective at blocking DNA methylation, but when present together coop erate to maintain hypomethylated retroviral transgenes that are persistently expressed.

To analyze whether the presence of both insulators alters the histone modification pattern on the provirus compared with the same retroviral vector without insulators, we performed ChIP qPCR on the EF1α promoter after 150 days of culture without puromycin. H3K9 dimethylation (H3K9me2) and trimethylation (H3K9me3) modifications were examined as repressive chro matin marks, and H3 panacetylation (H3Ac) as an active chro matin mark. It has been shown that H3K9me2 and H3K9me3 correlate with silencing of some retroviral vectors. [START_REF] Wolf | TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells[END_REF][START_REF] Karimi | DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs[END_REF] With the HSC1GiP retroviral vector, we found comparable enrichment of H3K9me2 on the EF1α promoter and the expressed endog enous Nanog region with no difference between the two popu lations (Figure 5b). This result reveals that H3K9me2 does not correlate with silencing of a SIN retroviral vector. However, we cannot exclude that it promotes DNA methylation in some silent genomic contexts but not in more active contexts, or that it plays a role in a subset of viral integrations that are not detected in the bulk ChIP data on cell populations. On the other hand, we found a sixfold enrichment in H3K9me3 marks on provirus without insu lators compared with provirus containing both insulators. The presence of the H3K9me3 mark may reflect spreading of repres sive chromatin over the provirus, which is blocked by cooperat ing 3′D4Z4 and HS4 elements. Finally, we detected a marginally greater enrichment in H3Ac histone marks on provirus contain ing both insulators. Overall, these data suggest that 3′D4Z4 and HS4 insulators together reduce H3K9me3 repressive marks to the same level as the expressed endogenous Nanog locus and support active chromatin marks on the provirus to promote high fre quency longterm transgene expression.

Deletion of 3′D4Z4 induces retroviral vector silencing

3′D4Z4 may promote stable transgene expression by direct ing retroviral vector integration away from hostile genomic environments that would otherwise impose a silent chromatin state on neighboring proviral integrants. If redirection to more favorable sites were the case, then deletion of 3′D4Z4 after inte gration would presumably have no effect on EGFP expression. Furthermore, if 3′D4Z4 acts as an enhancer, its deletion in the context of the HS4 construct would be expected to reduce the expression level at all integration sites but to not cause trans gene silencing. In contrast, if 3′D4Z4 activity is responsible for preventing heterochromatin spread at hostile integration sites, then deletion of 3′D4Z4 after integration would induce silencing.

To distinguish these models of 3′D4Z4 function, we cloned the D4Z4C fragment flanked by two FRT sites upstream of the EF1α promoter in vectors with or without HS4 in the LTR (Figure 6a). First for a proofofconcept, we infected ES cells and sorted EGFP positive cell populations (Figure 6b), before moving to individual cell clones in subsequent experiments described below. After expansion, FLP recombinasemCherry was transiently expressed and mCherrypositive cells were sorted 2 days later to enrich the population of cells which expressed FLP recombinase and flipped out D4Z4C (Figure 6b). We maintained transfected and untrans fected cells in parallel. These populations have proviruses at the same integration sites that differ solely by the presence or absence of the D4Z4 element. Southern blot and qPCR analyses show that 3′D4Z4 was deleted in ~80% of cells (Figure 6c,d). In both populations with and without the HS4 core insulator in the LTRs, EGFP expression is silenced when D4Z4C is deleted as shown in Figure 6e. As expected, expression frequency is enhanced when HS4 remains in the LTRs (83 versus 67% of EGFPpositive cells). It is not possible to determine the role of 3′D4Z4 at each inte gration site using these cell populations. However, these results agree with the earlier finding that 3′D4Z4 is not an enhancer of the EF1α promoter in the vector. These data also demonstrate that 3′D4Z4 does not direct integration into favorable sites, but are consistent with a model in which the transgene is silenced at a subset of integration sites when 3′D4Z4 is deleted.

Deletion of 3′D4Z4 in cell clones reveals rapid and gradual kinetics of silencing

To identify the kinetics of retrovirus silencing, we generated single cell clones where proviruses with and without 3′D4Z4 are inte grated at the same integration site. As outlined in Figure 6b, we infected ES cells and 24 hours after infection sorted single EGFP positive cells into 96well plates. This single cell sorting method was chosen as it is more efficient at generating unique cell clones than puromycin selection. Eleven clones with D4Z4C and 10 clones with D4Z4C and HS4 in the LTRs were expanded. All clones expressed the transgene consistently when grown without puromycin. To delete 3′D4Z4 in each clone, we transfected FLP recombinasemCherry plasmid and mCherrypositive cells were sorted to increase the probability of D4Z4C removal. The 3′D4Z4 deletion frequency ranged from 70 to 98% (Supplementary Figure S7). We verified by Southern blot that 19 out of 21 clones have a single copy provirus (Supplementary Figure S8) further support ing that our infection conditions primarily generate cells infected with a single provirus. Each clone with and without 3′D4Z4 was cultured in parallel, and EGFP expression was monitored for more than 60 days (Supplementary Figure S9 for HSC1FRT D4Z4GiP clones and Supplementary Figure S10 for HSC1 HS4FRTD4Z4GiP clones). These data show that clones can be grouped into three distinct classes based on the pattern of trans gene expression: rapid, gradual or no silencing. Representative single copy clones were chosen for further investigations. deletion of 3′D4Z4 (Supplementary Figure S9). Clone 1B10 (Figure 7a) is representative of this group, demonstrating that 3′D4Z4 is required for high frequency persistent expression at some integration sites. This observation provides strong evidence that 3′D4Z4 does not function by redirecting retrovirus integra tion to favouable locations but rather acts by preventing silencing spread over the provirus. It is possible that these rapidly silenced clones have never been studied in cells transduced with nonin sulated vectors. They may correspond to the 20% of silent clones shown in Figure 1 after infection with HSC1GiP. However, any selection with puromycin would eliminate such rapidly silenced clones and only select for integration sites that exhibit gradual or no silencing of the transgene. These clones are the first definitive evidence for very rapid silencing of a retroviral vector at a specific integration site. Interestingly, none of the 10 HSC1HS4FRT D4Z4GiP clones displays rapid silencing. One potential explana tion for this is that continued presence of HS4 in the LTRs may prevent rapid spread of epigenetic silencing into the provirus, allowing only gradual silencing to proceed.

Gradual silencing. Three of 11 HSC1FRTD4Z4GiP and 3 of 10 HSC1HS4FRTD4Z4GiP cell clones are gradually silenced andS10). Clone 1A2, which is progressively silenced over the course of 50 days, is rep resentative of this group (Figure 7a). These six clones demon strate that 3′D4Z4 is required to prevent silencing at a substantial proportion of integration sites, even when HS4 is present. This finding implies that 3′D4Z4 blocks gradual silencing that occurs even in the presence of HS4.

No silencing. Six of 11 HSC1FRTD4Z4GiP and 7 of 10 HSC1 HS4FRTD4Z4GiP clones do not silence after 3′D4Z4 deletion. A representative example is clone 2H9 (Figure 7a). These data in dicate that more than half of the clones contain retroviral vectors at favorable genomic locations that are not subject to silencing. The frequency of this class was likely enriched in the initial sort ing for EGFPexpressing cells, which would exclude any cells that never expressed the provirus, or are silenced immediately after infection.

3′D4Z4 does not redirect transgenes to perinuclear positions

The 5′D4Z4 element has been shown to be capable of relocating a telomere to the nuclear periphery. [START_REF] Ottaviani | The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy[END_REF] We were interested in whether the D4Z4C fragment can also relocalize a retroviral transgene to the nuclear periphery. DNA immunoFISH experiments to label a 3 kb transgene are technically challenging and were unsuccessful; hence, we aimed to determine the nuclear localization of the inte gration sites. For this purpose, we first determined the integration sites of the three classes of HSC1FRTD4Z4GiP by performing ligationmediated PCR and DNA sequencing of the provirus genome junction fragments. BLAT analyses (http://www.genome. ucsc.edu) of the ligationmediated PCR products identify the genomic integration sites (Supplementary Table S2) and bacterial artificial chromosome fluorescence in situ hybridization (FISH) probes were chosen accordingly for one representative clone from each class. Since the precise integration site of clone 1B10 could not be narrowed down to <3 potential sites, clone 1C1 was chosen as the rapidly silenced clone; clones 1A2 and 2H9 were chosen as representative of gradual and no silencing classes, respectively. Association of the integration site with the nuclear periphery was defined by colocalization of FISH signal with lamin B1. The rapidly silenced clone 1C1 is integrated far from any transcription unit (590 kb from Tox and 650 kb from Car8) (Supplementary Figure S11) in a laminassociated domain. [START_REF] Peric-Hupkes | Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation[END_REF] Analysis of published ChIPseq data in ES cells [START_REF] Karimi | DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs[END_REF][START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Meissner | Genome-scale DNA methylation maps of pluripotent and differentiated cells[END_REF][START_REF] Mikkelsen | Genome-wide maps of chromatin state in pluripotent and lineage-committed cells[END_REF][START_REF] Stadler | DNAbinding factors shape the mouse methylome at distal regulatory regions[END_REF] reveals that active chromatin marks (H3K4me, H3K9Ac, and DNase hypersensitive sites) are absent and repressive marks, including H3K9me2 and H3K9me3, are present (Supplementary Figure S11). DNA FISH analysis shows 40% of these alleles are found at the nuclear periphery in noninfected ES cells or infected cells with or without 3′D4Z4 (Supplementary Figure S14). This sug gests that 3′D4Z4 does not modify the perinuclear localization at this integration site.

The provirus in the gradual silenced clone 1A2 is integrated in a gene desert with the nearest genes being 151 kb upstream or 2.7 Mb downstream (Supplementary Figure S12). This integra tion site has not been associated with a laminassociated domain in ES cells, but is close (within 2 kb) to a genomic region enriched in ES cells for H3K9me3 histone marks, which are often associ ated with a repressive chromatin state. Our DNA FISH analysis in noninfected ES cells shows that the site is found at the nuclear periphery, with 50% of the alleles found to be associated with lamin B staining (Supplementary Figure S14). After integration of the expressing provirus containing the 3′D4Z4 insulator, the integration site displays moderate, but significant movement away from the periphery with 40% of the alleles found at the periph ery. After the removal of 3′D4Z4, the allele moves back towards the nuclear periphery, suggesting that silencing of the transgene correlates with movement of the transgene towards the nuclear periphery.

Finally, the provirus in the no silencing clone 2H9 is located in an intragenic region 41 kb upstream of Tcf7 and 36 kb down stream of Vdac1 genes (Supplementary Figure S13). Analysis of published ChIPseq data reveals several regions in ES cells marked by H3K4me1, H3K4me2, and H3K27ac, including two regions about 15.5 and 21.5 kb away from the integration site. These regions correspond to DNase hypersensitive sites and may be active enhancers of nearby genes. This locus is not associated with a laminassociated domain and DNA FISH shows that only 4% of alleles are found at the nuclear periphery (Supplementary Figure S14). Insertion of a transgene with D4Z4C does not alter its localization, although a very small but significant move ment towards the periphery can be observed after the removal of D4Z4C. Overall, our data from three clones suggest that 3′D4Z4 is unlikely to play a consistent role in the perinuclear localization of retroviral transgenes, and that silencing does not correlate with a dramatic movement of the retroviral transgene into the nucleus.

3′D4Z4 protects transgenes from heterochromatin spread

To determine which epigenetic modifications might be blocked by 3′D4Z4 and explain the difference in proviral expression between clones from each silencing kinetic class, we analyzed proviral DNA methylation and histone modifications 36 or 37 passages (80-90 days) after 3′D4Z4 removal. At that stage of cell culture, only 1-2% of cells are EGFPpositive in clones 1B10 and 1A2, respectively when 3′D4Z4 is deleted, but 75-98% of cells are EGFPpositive in the no silencing 2H9 clone and in all the clones when 3′D4Z4 is present (Supplementary Figure S15). All cell cultures are undif ferentiated because 96% of the cells are SSEA1positive. Bisulfite sequencing (Figure 7b and Supplementary Figure S16b) reveals that the rapidsilencing clones (1B10 and 1C1) have a partially methylated EF1α promoter in the presence of 3′D4Z4, but are hypermethylated following deletion. This demonstrates that 3′D4Z4 is capable of partially protecting the transgene from DNA methylation at this site, and the preexisting partial DNA meth ylation may promote the rapid silencing observed after 3′D4Z4 deletion.

In contrast, the gradualsilencing clone 1A2 is hypomethyl ated at the promoter in the presence of 3′D4Z4 and is fully hyper methylated following deletion (Figure 7b). Clone 1A5, which has slower gradual silencing than clone 1A2, has only 60% EGFP positive cells by day 60 (Supplementary Figure S16a). In this case, EF1α is hypomethylated in the presence of 3′D4Z4, but is only partially methylated in the absence of 3′D4Z4 (Supplementary Figure S16b). This shows that 3′D4Z4 can also fully protect the promoter from DNA methylation at some sites, and silencing may be more gradual at the sites that lack preexisting partial DNA methylation. Not surprisingly, the no silencing clone 2H9 is hypo methylated with or without 3′D4Z4, indicating that these sites may be in locations that are not susceptible to DNA methylation.

Finally, we analyzed histone modifications on the provirus by ChIPqPCR for repressive chromatin or open chromatin marks on the retroviral EF1α promoter in comparison to endogenous loci. In the two rapid clones, the H3K9me2 mark shows low enrichment compared with the positive control MageA2 (0.2-0.5) (Figure 7c, top panel) but are 2-5 times greater than the nega tive control Nanog (Figure 7c, bottom panel). Deletion of 3′D4Z4 increases this enrichment by up to 2.6fold. In these two clones, the H3K9me3 enrichment levels are similar to the positive control (Polmrt, ref. 45) but increased after 3′D4Z4 deletion only in clone 1B10. Both clones have a low enrichment of active chromatin marks in the presence or absence of 3′D4Z4, consistent with the EGFP expression level in these clones.

In the clones 1A2 and 1A5 which are gradually silenced, the promoter gains modest H3K9me2 enrichment only in clone 1A2 and the constitutive repressive chromatin mark (H3K9me3) does not change after 3′D4Z4 removal in both the clones. In contrast, the greatest fold difference in 1A2 and 1A5 cells is the loss of active chromatin marks when the insulator is deleted. Indeed, in cells containing provirus without 3′D4Z4, enrichment of H3Ac on the EF1α promoter is about fivefold lower than enrichment in provirus with 3′D4Z4. It is noteworthy that enrichment on the EF1α promoter in clone 1A5 in the presence of 3′D4Z4 is fourfold higher than the endogenous Nanog gene and drops to a lower level of enrichment when 3′D4Z4 is deleted (0.8fold). Finally, clone 2H9, which is not silenced after 3′D4Z4 deletion, does not display any significant chromatin alterations. Overall, we conclude that different repressive histone marks are associated with each silent integration site, indicating that multiple pathways may influence transcription status, depending upon the proviral integration site. However, proviruses within each kinetic class display similar pat terns. Rapid silenced clones are characterized by partial DNA methylation of the provirus in the presence of 3′D4Z4 and the highest enrichment in repressive marks combined with the lowest enrichment in active chromatin marks. Gradualsilencing clones are characterized by a dramatic loss in an active chromatin mark when 3′D4Z4 is deleted. Despite the heterogeneity in repressive marks near or on the provirus, 3′D4Z4 protects transgenes from the spread of repressive chromatin marks by blocking DNA meth ylation and minimizing the intrusion of repressive histone marks.

DISCUSSION

Our findings provide direct evidence that 3′D4Z4 protects retro viral vectors from DNA hypermethylation and deleterious his tone modifications that are dependent on the integration site. This activity allows 3′D4Z4 to direct high frequency and longterm expression from HSC1 retroviral vectors in ES cells. When com bined with HS4 in the LTRs, cooperation between the two insu lators directs unprecedented frequencies of persistent transgene expression in all preselected genomic integration sites that may have important practical applications for genetically manipulating PS cells. Deletion of 3′D4Z4 by FLP recombination induces trans gene silencing which can be classified into three distinct profiles defined by different silencing kinetics. This approach allows us to initiate silencing at defined integration sites as a tool to decipher mechanisms that are distinct for each kinetic class.

High frequency retrovirus expression directed by 3′D4Z4

We tested the ability of D4Z4 to improve the frequency of long term expression of a SIN retroviral vector in ES cells. We found that 3′D4Z4 prevents retroviral vector transgene silencing. This activity was not due to a spacing effect and did not require the DUX4 promoter or its complete ORF. However, cell populations containing 3′D4Z4 exhibited high celltocell variability, indicat ing that additional insulator activities are important to obtain consistent expression levels. In contrast, 5′D4Z4 did not pre vent the generation of a substantial proportion of EGFPnegative cells in which the vector DNA had been lost. This suggests that 5′D4Z4 sequences are unstable in this retroviral vector over time. However, we noted that the proportion of cells that do express D4Z4A virus produced high EGFP intensities with very minimal celltocell variability even in the absence of selection. Thus when selection is maintained, 5′D4Z4 may have valuable properties that enhance expression levels. For our purposes of maintaining high frequency and persistent expression from retroviral vectors in the absence of selection, the very efficient activity present in 3′D4Z4 and its stability in unselected cells are more appropriate.

3′D4Z4 and HS4 cooperate to block retrovirus silencing

Since 3′D4Z4 was unable to prevent variable expression, we rea soned that the location upstream of the internal promoter may be insufficient to prevent downstream genomic position effects. To address this concern, we combined a dimer HS4 core in the LTRs with 3′D4Z4 upstream of the EF1α gene promoter. After selection, the transduced cell population harboring this vector had high frequency and persistent levels of expression coupled with mini mal variability in transgene expression. We conclude that the two insulators cooperate to maintain retroviral transgene expression over time. Since 3′D4Z4 lacks the CTCF site present in 5′D4Z4, it is possible that a more complete silencing activity is produced by combining 3′D4Z4 with HS4 which retains its CTCF site. To our knowledge, this is the first evidence for HS4 cooperation with another insulator resulting in a stronger combined activity that completely neutralizes longterm retrovirus silencing.

Epigenetic analysis of transduced cells confirmed that the silent uninsulated virus is DNA hypermethylated. In contrast, the HS4 virus had partial methylation while 3′D4Z4 virus was hypo methylated on the viral GAG and EF1α sequences but had partial methylation on the EGFP sequences. Cooperation between HS4 and 3′D4Z4 directed hypomethylation on GAG, EF1α, and EGFP, consistent with full barrier or other activities that can block the spread of DNA methylation. In addition, ChIPqPCR on the EF1α promoter demonstrated that H3K9me3 was enriched and H3Ac was depleted on silent uninsulated virus relative to active 3′D4Z4 containing virus in cell populations. Together these data indicate that 3′D4Z4 cooperates with HS4 barrier to block DNA methyla tion and H3K9me3 spread.

We interpret the ability of 3′D4Z4 and HS4 to cooperate as an indication that they insulate the vector from different sequences or epigenetic marks. We propose that both insulators function to counteract genomic position effects, and the internally located 3′D4Z4 also acts to block cryptic silencer elements that remain in the SIN LTR, GAG or reporter gene sequences. Given that HS4 has an enhancer blocking activity mediated by DNA looping between CTCFbinding sites, [START_REF] Gaszner | Insulators: exploiting transcriptional and epigenetic mechanisms[END_REF] we infer that either 3′D4Z4 does not interfere with the presumed looping between the two LTRs, or enhancer blocking activity by HS4 is not required for its coop erativity with D4Z4. In contrast, HS4 barrier activity is mediated by USF and VEZF1 binding which would not be affected by an internally located 3′D4Z4 element. Overall, our novel retroviral vector design employing both 3′D4Z4 and HS4 is the first to fully escape longterm silencing in ES cells. In situations where selec tion can be applied, these vectors should ensure persistent long term expression in PS cells, which is an important criterion for any reporter transgenes designed to mark these stem cells or for potential suicide vectors intended to kill undifferentiated PS cells after cell transplantation. Moreover, expression from the EF1α promoter is maintained after spontaneous in vitro differentia tion into embryoid bodies, although it is not clearly established that D4Z4 function is ubiquitous in all differentiated cell types. Further experiments are required to test the ability of D4Z4 to shield other promoters in differentiated cell types that may be applicable for disease modeling, recombinant protein production, and gene therapy.

Deletion of 3′D4Z4 identifies three kinetic classes of silencing

To determine whether 3′D4Z4 prevents silencing at hostile inte gration sites or merely acts by redirecting integration into favor able integration sites, we developed an FRTbased system to delete the insulator after integration. Our results show that deletion of 3′D4Z4 allows silencing to occur, and that the continued pres ence of HS4 also allows silencing to occur but to a lesser degree. These findings are not consistent with the preferred integration site model but rather support a model for 3′D4Z4 function via effects on the surrounding chromatin landscape.

To evaluate silencing kinetics, we isolated ES cell clones that express a single copy provirus in the presence of 3′D4Z4 but are silenced after its deletion. This approach revealed three silenc ing classes distinguished by their kinetics. Rapidsilencing clones were detected after deletion of 3′D4Z4 in the absence of HS4, gradualsilencing clones were found after deletion of 3′D4Z4 in the presence or absence of HS4, and roughly half of all clones were not subject to silencing after 3′D4Z4 deletion. The discov ery of these three classes shows that integration sites silence ret roviral transgenes with different kinetics, and demonstrate that some integration sites are completely deleterious for expression unless 3′D4Z4 is present to protect the transgene from these rapidly silenced neighborhoods. The different kinetics observed agree well with involvement of multiplesilencing pathways that could be dependent on the integration site. [START_REF] Pannell | Silencing of gene expression: implications for design of retrovirus vectors[END_REF][START_REF] Senigl | Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site[END_REF] Identification of representative integration sites for each class provide examples suggesting that rapid silencing can occur in gene deserts, gradual silencing can occur at an intragenic site near regions with het erochromatin marks, and no silencing can occur at an integra tion site near expressing endogenous genes enriched in an active chromatin mark. While no general associations of genomic posi tion effects on proviral expression can be made from our limited integration site analysis, bacterial artificial chromosome probes for the integration sites permitted 3D DNA FISH to be performed before and after 3′D4Z4 deletion to examine association with the nuclear periphery. These experiments suggest that the rapid and gradualsilencing integration sites had preferential localization to the periphery, whereas the no silencing integration site was preferentially excluded from the periphery. Our results indicate that 3′D4Z4 did not consistently alter this localization, and there fore 3′D4Z4 functions differently from 5′D4Z4 that preferentially directs transfected transgenes toward the periphery.

To examine the 3′D4Z4 activity, we performed epigenetic analyses on the provirus in representative clones of the three silencing classes. The rapidsilencing clones exhibited partial DNA methylation on the EF1α promoter that becomes hyper methylated following 3′D4Z4 deletion. The gradualsilencing clones were hypomethylated and also became hypermethylated following D4Z4 deletion. In contrast, the no silencing clone was hypomethylated in the presence or absence of 3′D4Z4. These data unambiguously demonstrate that 3′D4Z4 blocks DNA methyla tion. This may correlate well with hypomethylation on endoge nous D4Z4 elements in facioscapulohumeral dystrophy patients compared with the nonaffected population, [START_REF] Tsien | Methylation of the FSHD syndrome-linked subtelomeric repeat in normal and FSHD cell cultures and tissues[END_REF][START_REF] Van Overveld | Hypomethylation of D4Z4 in 4q-linked and non-4q-linked facioscapulohumeral muscular dystrophy[END_REF] and our data is the first evidence that D4Z4 itself is capable of preventing DNA methylation of a transgene. The ability of D4Z4 on one side of the transgene to create a DNA methylationfree area that extends into the linked promoter is similar to the activity of the UCOE element on the EF1α short promoter after lentiviral transduction of iPS and ES cells. [START_REF] Pfaff | A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny[END_REF] In addition, rapidsilencing clones have low levels of H3K9me2 and high levels of H3K9me3 enrichment which cor relates with a low level of expression. Deletion of 3′D4Z4 modestly increases H3K9me2 levels in both the clones, but H3K9me3 is only raised in the clone 1B10. In contrast, gradualsilencing clones lost H3Ac marks upon deletion of 3′D4Z4. These results suggest that 3′D4Z4 also contributes to blocking H3K9 methylation marks and enhancing H3 acetylation marks. Our data demonstrate that 3′D4Z4 blocks the spread of repressive epigenetic marks, but the precise factors that mediate this activity are unknown, and took many years to identify with regard to HS4 barrier activity. It is possible they recruit different but complementary factors, and this information may also help to distinguish whether 3′D4Z4 and UCOE are functionally equivalent or distinct. Given that epigen etic marks differ at each integration site and silencing occurs with different kinetics, our results are consistent with the involvement of multiple epigenetic pathways that are dependent at least in part on the surrounding epigenome.

In summary, our use of FLP to delete 3′D4Z4 is a definitive method to identify elements that block transgene silencing and is the first application of this approach in mammalian cells. This tool revealed three classes of silencing kinetics and a diversity of epigenetic alterations on the silent transgene that are governed by multiplesilencing pathways. Better definition of the very earli est epigenetic events that occur on the rapidsilencing clones will require development of a synchronous system for inducing FLP to obtain sufficient cells for epigenetic analyses after 3′D4Z4 dele tion. Such a system will not only identify the kinetics of epigenetic events during rapid retrovirus silencing, but also provide a tool for screening chemicals that can inhibit these pathways.

MATERIALS AND METHODS

Cell culture and retroviral infection. Mouse ES cells were cultured in mouse ES medium: Dulbecco's modified Eagle's medium with 15% fetal bovine serum qualified for ES cell culture supplemented with 4 mmol/l lglutamine, 0.1 mmol/l minimum essential medium nonessential amino acids, 1 mmol/l sodium pyruvate, 0.55 mmol/l 2mercaptoethanol (all from Invitrogen, Carlsbad, CA) and purified recombinant leukemia inhibitory factor. [START_REF] Vedadi | A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells[END_REF] J1 mouse ES cells were cultured on gelatine (0.1%)coated plates; R1 ES cells and mouse iPS cells were grown on feeders. Retroviral produc tion was performed with the PlatE packaging cell line. Retroviral vector constructions are described in Supplementary Materials and Methods. Plasmids described in the paper will be available from Addgene (http:// www.addgene.org). All are derived from the HSC1 SIN retroviral vector. One day before infection, ES cells were seeded at 1 × 10 4 cells per well of a gelatinecoated 24well plate. For infection, virus was added to the tar get cells in the presence of 8 μg/ml Polybrene (hexadimethrine bromide; Sigma, St Louis, MO).

Flow cytometry. Single cell suspensions were fixed with 2% formaldehyde in phosphatebuffered saline supplemented by 2% fetal bovine serum for 10 minutes at room temperature. Cells were then suspended in phosphate buffered saline with 2% fetal bovine serum and filtered through 70 μm nylon membranes. EGFP expression analyses were performed by LSRII flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest Pro software (Becton Dickinson). Cell debris were excluded from analysis by using forward and side scatter gating. Noninfected cells were used as a negative control to adjust EGFP fluorescence. Slot blot. gDNA was extracted directly from 96well plates. Cells were digested overnight with proteinase K at 100 μg/ml in lysis buffer (100 mmol/l TrisHCl, pH 8; 5 mmol/l EDTA, 0.2% SDS, 200 mmol/l NaCl) at 55 °C. gDNA was denaturated with 0.4 N NaOH for 5 minutes at room temperature, neutralized with cold NH 4 Ac (final concentration, 1 mol/l), and then transferred onto nitrocellulose membrane. These membranes were processed following standard procedures and hybridized with an EGFP probe to detect integrated provirus and mThy1, to detect the endog enous mouse Thy1 gene. Southern blot. gDNA were extracted by conventional proteinase K diges tion and phenol/chloroform extraction. They were digested with BglII or SpeIPvuI, separated on a 1% agarose gel and transferred onto a mem brane (Protran BA85 pure nitrocellulose; Schleicher & Schuell, Dassel, Germany). Blots were probed with a fragment of EGFP labeled with P 32 by random priming (Megaprime DNA Labelling System, RPN1606; GE Healthcare, Buckinghamshire, UK). gDNA methylation analysis. gDNA was isolated from ES cells using phe nolchloroform extraction. The DNA (400 ng) was then bisulfitetreated with the EZ DNA methylationgold kit (Zymo Research, Irvine, CA) to convert unmethylated cytosines into uracil, whereas methylated cyto sines remain intact. OSG13/OSG14, BisPGKEFGP2f/BisPGKEFGP2r, and OSG19/OSG20 primers sets (Supplementary Table S1) were used to amplify the GAG, EGFP, and the EF1α gene promoter regions, respectively using Platinum Taq DNA polymerase high fidelity (#11304 011; Invitrogen). PCR products were subcloned in pGEMT easy vec tor (A13160; Promega, Madison, WI) and sequenced by The Centre for Applied Genomics facility (Hospital for Sick Children, Toronto, Ontario, Canada).

Chromatin immunoprecipitation assay and qPCR. ChIP was performed using the magna ChIP G kit (#17611; Millipore, Billerica, MA) following the manufacturer's instructions. Briefly, 10 7 cells were crosslinked in 1% formaldehyde; cells were washed, scraped into a conical tube, and soni cated for 25 minutes in a Bioruptor XL at the high setting with cycles of 30 seconds ON, 30 seconds OFF. Chromatin was subdivided into aliquots containing 10 6 cell equivalents of lysate for each immunoprecipitation. This chromatin was immunoprecipitated with 2 mg of antibodies specific to H3K9me2 (Ab1220; Abcam, Cambridge, MA), H3K9me3 (Ab8898; Abcam), H3Ac (06599; Millipore), mouse IgG control (Ab18413; Abcam), and rabbit IgG control (ab37415; Abcam). In all, 0.9 μl of purified DNA was analyzed by qPCR using SYBR green (#4309155; Applied Biosystems, Foster City, CA) and ABI7900HT fast real time PCR system (Applied Biosystems). The primer sequences are given in Supplementary Table S1.

Figure 1 HSC1

 1 Figure 1 HSC1 retroviral vector is rapidly silenced after infection. (a) Structure of HSC1-GiP provirus. The internal human EF1α promoter drives expression of the bicistronic gene encoding enhanced green fluorescent protein (EGFP) and puromycin resistance. (b) Experimental system. At day 6, wells with more than one colony (circles with a multiplication symbol) were eliminated and wells containing only one colony (closed circles) were passed into duplicate 96-well plates. At day 8, empty wells (open circles), wells with non-expressing cells (closed circles), and wells with expressing cells (green circles) were identified. Genomic DNAs (gDNAs) were harvested at day 17 from both plates and used for slot blot analysis. Membranes were hybridized with an EGFP probe to detect provirus or mThy1 probe to detect endogenous DNA. Interpretation of expression and genotype data are shown on the right. Green circles represent wells with expressing cells containing EGFP proviral DNA; red circles are non-expressing cells containing the proviral vector; yellow circle is a colony detected as expressing where no provirus was detected. (c) EGFP expression in populations of non-selected cells detected by flow cytometry. This graph displays the percentage of EGFP-positive cells at day 6 in the population obtained after infection with 5 or 25 μl of virus (264 and 288 clones picked, respectively; producing 7 and 27 EGFP-expressing clones). (d) Percentage of expressing cells among infected cells. The percentage of expressing clones represents the number of EGFP-expressing clones among the infected (proviral DNA detected) and viable clones. This result is the average of two independent experiments with two independent viral productions. Total numbers of dot blot positive clones are 10 and 33, respectively; for the 5 and 25 μl virus infections. Error bars represent the SD. IRES, internal ribosome entry site; LTR, long terminal repeat; SIN, self-inactivating.

Figure 2 HSC1

 2 Figure 2 HSC1 retroviral vectors with or without HS4 are silenced over time. (a) Experimental system. J1 embryonic stem cells were infected at low multiplicity of infection by HSC1 retroviral vectors. Two days after infection, expressing cells were selected with puromycin for 4 days and maintained as a population in the presence (dashed line) or absence (solid line) of puromycin for up to 5 months. (b,d) HSC1-GiP and HSC1-HS4-GiP retroviral vector expression over time. Populations were maintained without (black marker, solid line) or with (open marker, dashed line) selection over time. Graphs represent the percent of enhanced green fluorescent protein (EGFP)-positive cells over time in the HSC1-GiP (open and closed diamonds in b) and HSC1-HS4-GiP (open and closed circles in d) populations. (c) Structure of HSC1-HS4-GiP provirus. Two copies of the core HS4 are in the 3′ LTR of the HSC1-GiP retroviral vector. After viral replication, this insulator is present in both LTRs. IRES, internal ribosome entry site; LTR, long terminal repeat.

Figure 3

 3 Figure 3 3′D4Z4 protects HSC1 retroviral vector from silencing. (a) Structure of HSC1-D4Z4-GiP provirus. D4Z4 elements or a spacer DNA were cloned upstream of the EF1α promoter in the HSC1-GiP retroviral vector. (b) D4Z4 fragments cloned in HSC1 retroviral vector. The D4Z4 element is 3.3 kb long. It contains an enhancer, an enhancer blocker, and part of the DUX4 open-reading frame (ORF). D4Z4-A is the KpnI-BamHI 5′D4Z4 fragment (1,380 bp); D4Z4-C is the StuI 3′D4Z4 fragment (1,310 bp); and D4Z4-D corresponds to the BamHI 3′D4Z4 fragment (1,172 bp). K, KpnI; B, BamHI; S, StuI; P, Dux4 promoter; E.B., enhancer blocker with the CTCF-binding site; Dux4 gray arrow, Dux4 partial ORF. (c) Enhanced green fluorescent protein (EGFP) expression from provirus containing the spacer element, D4Z4-A and D4Z4-C in embryonic stem (ES) cells. Expressing ES cells were sorted and cultured without further selection. (d) Flow cytometry plots 64 days post-infection. X-axis represents the level of EGFP fluorescence, Y-axis represents the cell numbers. CV, coefficient of variation; IRES, internal ribosome entry site; LTR, long terminal repeat; SIN, self-inactivating.

Figure 4

 4 Figure 4 3′D4Z4 and HS4 insulators cooperate to improve frequency, level, and reproducibility of enhanced green fluorescent protein (EGFP) expression. (a) Structure of HSC1-HS4-D4Z4-GiP proviruses. 3′D4Z4 fragments (D4Z4-C or D4Z4-D) were cloned upstream of the EF1α promoter in the HSC1-HS4-GiP retroviral vector. (b) HSC1-HS4-D4Z4-C-GiP and HSC1-HS4-D4Z4-D-GiP retroviral vector expression over time. J1 embryonic stem (ES) cells were infected and selected with puromycin. Cell populations were maintained without (black marker, solid line) or with (open marker, dashed line) further selection. (c) EGFP expression at 60 (top panel graphs) and 148 days (bottom panel graphs) post-infection in all populations. Flow plots in populations cultured in presence (gray line) or absence (black line) of selection. X-axis shows the level of EGFP fluorescence, Y-axis shows the percentage of maximum cell numbers. Levels (the mean fluorescence intensity (MFI)) and variability (the coefficient of variation (CV)) of EGFP expression at 148 days are displayed below. IRES, internal ribosome entry site.

  HSC1-HS4-Gip HSC1-D4Z4-C-Gip HSC1-HS4-D4Z4-C-Gip HSC1-HS4-D4Z4-D-Gip 10 Molecular Therapy vol. 21 no. 8 aug. 2013

Figure 5

 5 Figure 5 Epigenetic profiles of silent and expressing proviruses. (a) Bisulfite sequencing of proviral DNA at GAG, EF1α promoter, and EGFP regions from HSC1-GiP, HSC1-HS4-GiP, HSC1-D4Z4-C-GiP, and HSC1-HS4-D4Z4-C-GiP proviruses 148 days post-infection. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. Percentages below each methylation profile represent the percentage of total CpG methylation. (b) Analysis of repressive and active chromatin marks on the EF1α promoter by ChIP. ChIP experiments were performed with anti-H3K9me2, H3K9me3, and H3Ac antibodies. DNA isolated from immunoprecipitated chromatin was subjected to quantitative PCR to amplify the EF1α promoter from the retroviral vector and endogenous positive controls (MageA2 for H3K9me2, Polmrt for H3K9me3, and Nanog for H3Ac). Relative enrichments correspond to the enrichment of the studied chromatin mark on the EF1α promoter normalized to endogenous positive control (closed bars). Endogenous negative controls (open bars, Nanog for H3K9me2 and H3K9me3, and MageA2 for H3ac) relative to the enrichment on the corresponding endogenous positive control are shown below. EGFP, enhanced green fluorescent protein.

  (HSC1-D4Z4-C-GiP) D4Z4-C and HS4 (HSC1-HS4-D4Z4-C-GiP)

  Rapid silencing. Two of 11 HSC1FRTD4Z4GiP clones (clones 1B10 and clone 1C1) were silenced by day 7, the first analysis after Molecular Therapy vol. 21 no. 8 aug. 2013

Figure 6

 6 Figure 6 Induction of provirus silencing by FLP recombination. (a) Structure of HSC1-FRT-D4Z4-C-GiP and HSC1-HS4-FRT-D4Z4-C-GiP provirus. D4Z4-C fragment flanked by two FRT sites was inserted upstream of the EF1α promoter, in HSC1-GiP and HSC1-HS4-GiP retroviral vector. The black line indicates the enhanced green fluorescent protein (EGFP) probe used in the Southern blot analysis. SpeI and PvuI restriction sites are shown. (b) Experimental system. R1 embryonic stem cells were infected with HSC1-FRT-D4Z4-C-GiP and HSC1-HS4-FRT-D4Z4-C-GiP viral vectors. One day after infection, EGFP-positive cells were sorted as cell populations or as single cell clones and expanded. A portion was transiently transfected with FLP recombinase-mCherry plasmid and 2 days later mCherry-positive cells were sorted and maintained in culture. (c) Deletion of 3′D4Z4 by FLP recombinase was verified by Southern blot. gDNA was digested by SpeI and PvuI and provirus was detected by 32 P-dCTP-labeled EGFP probe. In the presence of 3′D4Z4, the SpeI-PvuI fragment is about 4,100 bp and about 2,790 bp when 3′D4Z4 is flipped out. (d) Estimation of the deletion frequency by quantitative PCR. D4Z4-C and EF1α promoter regions were amplified and the deletion frequency calculated as 1-2 -(ΔΔCt) between the flipped out D4Z4-C fragment and the remaining EF1α promoter relative to non-flipped cell populations. Each bar represents the mean of three PCRs each performed in triplicate. Error bars represent the SD. (e) EGFP expression. Fluorescence was analyzed by flow cytometry 20 days after 3′D4Z4 removal in the HSC1-FRT-D4Z4-GiP population (top chart) and HSC1-HS4-FRT-D4Z4-GiP population (bottom chart) where D4Z4 was flipped (black line) or not flipped (gray line). Top figures are the percentage of EGFP-positive cells in flipped population and bottom figures in non-flipped population. gDNA, genomic DNA; IRES, internal ribosome entry site; LTR, long terminal repeat; SIN, self-inactivating.

Figure 7 3′D4Z4

 7 Figure 7 3′D4Z4 protects retrovirus expression from silencing independent of position effects. (a) Enhanced green fluorescent protein (EGFP) time course expression in three representative single cell clones with HSC1-FRT-D4Z4-C-GiP provirus. Cells were transfected with FLP recombinase-mCherry plasmid and those containing the provirus with (gray line) and without (black line) 3′D4Z4 were maintained in culture in parallel. (b) DNA methylation status of 27 CpG sites in the human EF1α promoter obtained from bisulfite sequencing of cells containing provirus with (non-flipped) or without (flipped) 3′D4Z4 36 or 37 passages after FLP recombinase-mCherry plasmid transfection and mCherry sorting. Closed circles represent methylated cytosines and open circles represent unmethylated cytosines. Percentages below each methylation profile represent the percentage of total CpG methylation. (c) Analysis of H3K9me2, H3K9me3, and H3Ac histone modifications on the EF1α promoter. ChIP-qPCR was performed on representative clones in presence (gray bars) or absence (black bars) of 3′D4Z4 36 or 37 passages after 3′D4Z4 removal. Two rapidly silenced clones (clones 1C1 and 1B10), two gradually silenced clones (clones 1A2 and 1A5), and one not silenced clone (clone 2H9) were analyzed. qPCR on the EF1α promoter (top) was used to evaluate fold enrichment relative to input and normalized to the endogenous positive controls MageA2 for H3K9me2, Polmrt for H3K9me3, and Nanog for H3Ac. Chip-qPCRs were performed in triplicate. The negative controls (below) are the endogenous Nanog region for H3K9me2 and H3K9me3, and MageA2 for H3Ac. qPCR, quantitative PCR.

Molecular

  Therapy vol. 21 no. 8 aug. 2013

Molecular Therapy vol. 21 no. 8 aug. 2013

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© The American Society of Gene & Cell Therapy D4Z4 Element Prevents Retroviral Vector Silencing

www.moleculartherapy.org vol. 21 no. 8 aug. 2013 © The American Society of Gene & Cell Therapy D4Z4 Element Prevents Retroviral Vector Silencing

www.moleculartherapy.org vol. 21 no. 8 aug. 2013 © The American Society of Gene & Cell Therapy

ACKNOWLEDGMENTS

We thank Mehdi Karimi (University of British Columbia, Vancouver, British Columbia, Canada) for help with generating the tracks for bioinformatics analysis, Oliver Tam for helpful discussions on ChIP-seq data analysis, and Akitsu Hotta (CiRA, Kyoto, Japan) for critical comments on the manuscript. We acknowledge the assistance of Sherry Zhao for fluorescence-activated cell sorting in the SickKids-UHN Flow Cytometry Facility, Jodi Garner in the SickKids ES Cell Facility, Mike Woodside and Paul Paroutis in the SickKids Imaging Facility and the SickKids TCAG Sequencing Facility. We thank Eric Gilson and Frederique Magdinier (LBMC, ENS, Lyon, France) for providing the C1X plasmid and Elly Tanaka (CRTD, Dresden, Germany) for SKSce-CAGGs-PA plasmid. This work was funded by grants from the Canadian Institutes of Health Research (CIHR MOP-111065 and IG1-102956 to J.E. and RMF-92090 to M.C.L. and J.E.) and a CIHR Frederick Banting and Charles Best Graduate Scholarship to M.Y.M.L. The authors declared no conflict of interest.

SUPPLEMENTARY MATERIAL

 S1. List of primers. Table S2. Integration site of provirus. Materials and Methods.


J Peydecastaing 
email: jerome.peydecastaing@ensiacet.fr

C Vaca-Garcia

E Borredon

Bi-acylation of cellulose: determining the relative reactivities of the acetyl and fatty-acyl moieties

Keywords: mixed cellulose esters, mixed anhydrides, acetic-fatty esters

The global reaction between acetic anhydride and a fatty acid yields, at equilibrium, an asymmetric acetic-aliphatic anhydride in a medium containing finally: acetic-fatty anhydride, acetic anhydride, fatty acid, acetic acid and fatty anhydride. No solvent or catalyst was used to evaluate the impact of the actual reactivity of the anhydrides. The competition between the formation of acetyl and fatty acyl ester functions was evaluated by determining the ratio of acetyl/fatty acyl groups grafted on solid cellulose. The influence of temperature, reaction time, and length of fatty chain on the total degree of substitution and on the ratio of acetyl/fatty acyl ester functions was investigated. For the first time, a correlation has been established between esterification and the length of the aliphatic chain of the fatty acid.

Reactivity of the medium decreased with the number of carbons in the fatty acid, raised to the power 2.37.

Introduction

Mixed cellulose esters (MCE), i.e. substituted by different aliphatic chains, are polymers with interesting properties. In particular, cellulose acetate-propionate (CAP) and cellulose acetate-butyrate (CAB), are very important industrially. Research on other mixed acetic-aliphatic cellulose esters has led to the discovery of polyfunctional materials combining both good mechanical properties and high hydrophobicity [START_REF] Vaca-Garcia | Cellulose esterification with fatty acids and acetic anhydride in lithium chloride/N,N-dimethylacetamide medium[END_REF].

Two step preparations of MCE, with acetylation usually first, followed by the other substituent, or with two different anhydrides, are common, even in industrial processes. Alternatively, a reaction mixture can be used comprising one anhydride and carboxylic acids. [START_REF] Malm | Mixed organic acid esters of cellulose such as mixed cellulose acetate-oleate, -linoleate or -stearate suitable for precipitation and washing with water[END_REF] proposed chloroacetic anhydride, plus a fatty acid with at least 6 C atoms, such as stearic acid, in the presence of an inert, lowboiling solvent such as methylene chloride and an esterification catalyst such as ptoluenesulfonic acid.

Another procedure uses trifluoroacetic anhydride as co-reagent, with a mixture of two different carboxylic acids to form mixed anhydrides in situ (trifluoroacetic-aliphatic or aliphatic-aliphatic). The former permit fatty acylation without reacting the fluorinated moiety, whereas the latter can graft either of the aliphatic moieties [START_REF] Matsuzaki | Synthesis of mixed cellulose esters of a,b-unsaturated carboxylic and acetic acids and their utilization. II. Reaction of mixtures of a,bunsaturated carboxylic acid and acetic anhydride with cellulose[END_REF]. The same technique has also been used for the synthesis of various mixed esters of starch [START_REF] Yang | Preparation and physical properties of starch mixed esters[END_REF].

The procedure we will consider in this paper consists of the acylation of cellulose with mixed acetic-aliphatic anhydrides produced in situ by the reaction between acetic anhydride and a carboxylic acid [START_REF] Vaca-Garcia | Cellulose esterification with fatty acids and acetic anhydride in lithium chloride/N,N-dimethylacetamide medium[END_REF][START_REF] Vaca-Garcia | Solvent-free fatty acylation of cellulose and lignocellulosic wastes. Part 2. Reactions with fatty acids[END_REF][START_REF] Chemeris | Synthesis of cellulose esters in a trifluoroacetic acid medium[END_REF]. In this way, either of the moieties of the asymmetric anhydride is grafted on the cellulose (Fig. 1).

The esterification of cellulose using this mixture depends on three factors: the relative proportion of acetylium and acylium ions, the effect of steric hindrance and the relative activation energies of each reaction.

The aim of this paper is to study the overall reactivity of this mixture on cellulose, and to compare the impact of the aliphatic chain length (n), of the different fatty acids used, on cellulose acylation.

Experimental

Chemicals

Acetic anhydride and the carboxylic acids: propionic (C2:0), octanoic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), and oleic (C18:1) of 99%+ purity were purchased from Sigma-Aldrich France. Pentadecanoic acid (C15:0) was used as internal standard (I.S.) in gas chromatography and was obtained from Fluka France (99% purity). Phosphoric acid, 85% solution in water, was purchased from Acros France. Alpha-cellulose from Sigma-Aldrich France was the initial biopolymer (this cellulose was characterized and gave: degree of polymerization = 960, 4% pentosans, 7% moisture content).

Trimethylsulphonium hydroxide (TMSH) was obtained from Macherey-Nagel France as a 0.2 mol/L solution in methanol. Tert-butyl methyl ether (MTBE) HPLC grade was purchased from Scharlau Spain. All chemicals were stored at 4°C prior to use.

Preparation of the mixed acetic-fatty anhydrides

The appropriate amounts (molar ratio 3:2) of fatty acid and acetic anhydride were added to a glass reactor fitted with a condenser. Reactions were carried out at 100°C with mechanical stirring at 500 rpm for 1 hour. The reaction media were analyzed by reversed-phase HPLC [START_REF] Peydecastaing | Quantitative Analysis of Mixtures of Various Linear Anhydrides and Carboxylic Acids[END_REF]) and used without purification for the synthesis of MCE.

Synthesis of MCE

Reactions were performed in 50 mL reactors fitted with a condenser. 1 g of undried cellulose (7% moisture) was stirred at 350 rpm into 15 mL of reaction medium without catalyst at the chosen temperature for the chosen duration. After cooling down to about 80°C, 20 mL of ethanol were added to precipitate the soluble fraction. Cellulose esters were separated by filtration over fritted glass and purified by Soxhlet extraction with ethanol for 8 h. The purified product was then vacuum dried to constant weight at 70°C for at least 24 h.

DS determination

To our knowledge, there is no single analytical technique that allows the simultaneous and accurate determination of the DS of long and short substituents in MCE when the DS is below 0.1. Consequently, we have employed two different techniques. The first was used to determine the fatty acyl content and the second, to quantify the acetyl groups. Both analyses are required to evaluate the individual and total DS-values.

Determination of the fatty acyl content

The method based on the reaction of cellulose esters with TMSH followed by gas chromatography (GC) analysis, was used. The conditions were those recommended for low DS-values according to a previous work (Peydecastaing et al. 2009 a).

Basically, the MCE sample is reacted in a vial with TMSH for 1 hr at 75°C with MTBE as solvent. Once the sample has cooled down and the solid decanted, the fatty acid methyl esters (FAME) are quantified by GC and, from this, the concentration of fatty acyl groups C f in solution can be determined.

Determination of the acetyl content

This method is based on saponification of the MCE followed by GC analysis.

A precise amount (about 20 mg) of MCE was weighed and introduced into a 2 mL vial, to which 1 mL of aqueous sodium hydroxide (0.5 N) and 0.5 L of a 0.5 mmol/L propionic acid (internal standard) solution in water, were added. The vial was stirred/shaken for 5 hours in a shaking incubator, set at 1200 rpm and 100°C. The sample was then cooled down, 45 µL of H 3 PO 4 added and the vial shaken again at 1200 rpm for 10 min at room temperature. Once the solid had decanted, a small sample of the reaction mixture supernatant, was analyzed by GC. Separation was achieved in a CP-Select CB for FFAP fused silica capillary column (CP7845, Varian) 25 m, 0.32 mm i.d., 0.25 µm film thickness. Helium was used as carrier gas at a flow rate of 2.6 mL/min. The temperature of the injector was set at 250°C, the FID detector at 270°C, and the split ratio at 1:100. Oven temperature was programmed as follows:

80°C for 1 minute rising to 145°C at a rate of 20°C/min, and then to 250°C, at a rate of 50°C/min, for 2 minutes. This allowed the separation of the acetic and propionic acids in less than 12 min. From the GC analysis the concentration of acetyl groups C 2 in solution can be determined.

DS calculation

The DS values of the grafted fatty chain (DS f ) and of the acetyl chain (DS 2 ) are calculated as follows:

where:

€ DS f = 162.14 × C f × V f m f - m f m 2 × C f × V f × (M f -18.02) -C 2 × V 2 × 42.03 € DS 2 = 162.14 × C 2 × V 2 m 2 -C f × V f × (M f -18.02) - m 2 m f × C 2 × V 2 × 42.03
3. Results and discussion

Acylating agent

The mixed anhydrides are reactive molecules that allow the simultaneous reaction of two acyl moieties onto cellulose. Their reactivity is related to the asymmetry of the molecule.

In acidic medium, the anhydride is protonated and readily cleaved to form an acylium ion and a carboxylic acid (Fig. 1). In the case of an acetic-fatty anhydride, both types of acylium ions can be formed.

Moreover, an asymmetric anhydride coexists with its symmetric anhydrides regardless of the synthetic pathway used to prepare it. This phenomenon has been thoroughly described by Peydecastaing et al. (2009 b). At the end of the reaction, a mixture of five compounds: acetic acid, fatty acid, acetic anhydride, fatty anhydride, and acetic-fatty anhydride is obtained. Under non-catalyzed conditions, only the anhydrides are reactive to cellulose (Fig. 2). Acetic acid provides the proton to cleave the mixed anhydride and this can be considered as an auto-catalytic reaction. In order to study the reactivity phenomena on solid cellulose, a large excess of liquid reaction medium was used to maintain the concentration of the reactive species relatively constant. In addition, following the study of Peydecastaing et al. (2009 b), a ratio of 1.5 mol of fatty acid per 1 mol of acetic anhydride was chosen to make the asymmetric anhydride the most abundant reactive entity (58%).

Synthesis of cellulose acetate-oleate

As expected, the substitution yield of acetyl and oleoyl moieties increases with temperature. And because this system has no external catalyst, the DS-values are relatively low compared to traditional setups using H 2 SO 4 for cellulose esterification in heterogeneous systems. However, this choice allows the real reactivity of the molecules to be focused on. For the maximum tested temperature (175°C), the values for the degree of substitution of oleates (DS 18:1 ) and acetates (DS 2 ) were 3.9x10 -4 and 7.1x10 -2 respectively.

In other words, 2% of the hydroxyl groups in cellulose have been esterified.

The individual acetyl and oleoyl DS-values obtained at 160°C are around ten and twenty times greater respectively than those obtained at 80°C. These results highlight the fact that the acylation reaction is strongly temperature dependent, and we found a linear correlation between DS-values and 1/T (Fig. 3). The value of the slope is directly related to the intrinsic reactivity of the moiety concerned. The gradient for acetylation is 170 times greater than that of oleoylation, and the temperature at which DS=0, can be found by extrapolation. These points (72°C and 89°C for acetylation and acylation respectively)

represent the temperature at which the reaction, carried out for 1 hr with no external catalyst, becomes insignificant.

In order to confirm that these DS-values do not originate from residual reagents trapped in the structure of cellulose, we performed FTIR analysis of the solvent-extracted MCE. No characteristic carboxylic acid band was observed, however the characteristic ester band was clearly seen. In addition, the purification method was also validated by impregnating a sample of cellulose with the acylation mixture at 20°C for 1 hr followed by the same purification steps. The DS of this sample, determined by the very accurate GC method, was null (accuracy of the method for the fatty chains is up to 10 -5 values of DS, see Peydecastaing et al. 2009 a).

In the range of temperature studied, the proportion of grafted acetyl moieties to acyl moieties (DS 2 /DS 18:1 ) decreases from 389 to 187 as temperature increases from 80 to 175°C as shown in Fig. 4, thus at lower temperatures fatty acylation is inhibited. A change in the composition of the reaction medium cannot account for this since in a previous article (Peydecastaing et al. 2009 b), it was demonstrated that it remains practically constant with temperature.

The most likely hypothesis is that the activation energy for the acetylation is lower than for the acylation by oleoyl moities. At high temperatures (175°C), there is the necessary energy for acetylation and fatty acylation and none of the reactions is inhibited. The ratio then reaches a plateau (linearization of the function DS=f(1/T), and here, esterification by acetyl groups is 200 times higher than by the fatty acyl groups. Such a difference can be accounted for by steric hindrance of the fatty chain, with the acetyl group more able to reach hydroxyl functions in the cellulose microfibrils. Moreover, in Fig. 1 the proportion of acylium ions (path A or B) will depend on the ease of forming the associated carboxylic acid. Since the pK of fatty acids is slightly higher than that of acetic acid, the acetylium ion will be more abundant than the fatty acylium ion.

The reaction time, between 1 and 4 hours, has a typical influence on the reaction. The total DS-value increases rapidly and levels off after 3 hours (at 140°C). The weak reactivity of cellulose and the low accessibility of OH groups accounts for this.

It is interesting to note that the DS 2 /DS 18:1 ratio remains constant at about 200 over the whole reaction time range studied (Fig. 5). This means that acetyl and fatty acyl have different reaction rates but are not interdependent, confirming the hypothesis formulated above concerning the activation energies. In addition, as the reaction medium is in a state of equilibrium and the consumption of acetyl groups is greater than oleoyl ones, we would have expected a change in composition with time and then an effect on the ratio of grafting. However, this does not happen, and the explanation could be that the molar ratio of the acetyl/fatty acyl groups present in the reaction medium, has only a small impact on the concentration of the mixed anhydride in the reaction medium. This observation was made in a previous paper concerning the study of the synthesis of acetic-oleic anhydride mixtures (Peydecastaing et al. 2009 b).

Synthesis of cellulose acetate-alkanoates

In the following series of experiments, the temperature and reaction time were kept constant (140°C, 1 h) and only the nature of the fatty chain was varied, by preparing mixtures using acetic anhydride and different fatty acids with saturated aliphatic chains from C8 to C16, at a molar ratio of 1.5 (Table 1).

The steric hindrance evidently diminishes the fatty acylation (Fig. 6), and logically the DS2/DS f ratio also decreases as the fatty acid chain length increases. This is well illustrated by the plot of this ratio as a function of the chain length on a logarithmic scale (Fig. 7). The regression trend is given by the equation DS 2 /DS f = 5.1/n 2.37 for the conditions studied. Reactivity decreases with the number of carbon atoms, not in a linear way, nor to the square but to the power 2.37. This trend is valid for short (C2) to long (C8-16) and even mono-unsaturated (C18:1) side-chains. Other mono-and poly-unsaturated chains (linoleic, linolenic) must be tested to extend the validity of this equation to other cellulose esters.

Conclusions

•

The reaction medium obtained from acetic anhydride and a fatty acid contains aceticfatty anhydride as the most abundant reactive compound. On reaction with cellulose, it leads to the formation of mixed cellulose esters bearing acetyl and fatty acyl groups. In the absence of any external catalyst or cellulose solvent, low DS-values are obtained (DS 2 ≥ 0.072; DS f ≥ 1.8x10 -3 with 8≤f≤18) but this allows the impact of the actual reactivity of the molecules to be evaluated.

•

Precise DS determination was employed to accurately characterize these mixed, low substituted cellulose esters.

• Straight reactivity revealed an acetyl to fatty acyl ratio varying from 27 to 659. Steric hindrance and the low pK of acetic acid account for preferential acetylation.

• This ratio is dependent on the nature of the fatty acid and on the reaction temperature.

However, it is not dependent on reaction time, being constant throughout the reaction.

• Reactivity decreases with the number of carbon atoms to the power 2.37. This equation is valid for short and long and even mono-unsaturated (C18:1) side-chains.

Acknowledgments: The authors would like to thank the LAPEYRE Company (France) for financial support.

  C f : concentration of the FAME in MTBE determined by GC (in mol/L) V f : volume of internal standard (pentadecanoic acid) solution added to the analyzed sample (in mL) m f : mass of the MCE sample analyzed in MTBE with TMSH (in g) M f : molar mass of the RCOOH fatty acid C 2 : concentration of the acetic acid after saponification and acidification determined by GC (in mol/L) V 2 : volume of internal standard (propionic acid) solution added to the analyzed sample (in mL) m 2 : mass of the MCE sample analyzed in water (in g)

Fig. 1 FigFig. 5 Fig. 7

 157 Fig. 1 Acylation of cellulose treated with a mixed anhydride