Changwei Yu

Nevena Cvetesic

Kapil Gupta

Tao Ye

Emese Gazdag

Vincent Hisler

Luc Negroni

Petra Hajkova

Boris Lenhard

Ferenc Müller

Imre Berger

Stéphane D Vincent
email: vincent@igbmc.fr

László Tora

TBPL2/TFIIA complex overhauls oocyte transcriptome during oocyte growth

The first steps of oocyte development from primordial follicle are characterised by a growth phase, when unique RNA and protein reserves are created to achieve oocyte competence. During this growth, oocytes do not divide and the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (also called TBP2 or TRF3), which is essential for RNA polymerase II transcription (Pol II) 1,2 . However, the composition and function of transcription machinery and the regulatory mechanisms mediating Pol II transcription during this developmental stage remain unknown. In somatic cells, the general transcription factor TFIID, which contains TBP and 13 TBPassociated factors, is the first to bind gene promoters to nucleate Pol II transcription initiation 3 . Here, we show that in oocytes TBPL2 does not assemble into a canonical TFIID complex, while it stably associates with TFIIA via distinct TFIIA interactions when compared to TBP. Our transcript analyses in wild type and Tbpl2 -/-oocytes demonstrates that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA destabilisation factors genes, as well as specific endogenous retroviral elements (ERVs).

Transcription start site (TSS) mapping from wild-type and Tbpl2 -/-growing oocytes demonstrates that TBPL2 has a strong preference for TATA-like motif in gene core promoters driving specific sharp TSS selection. This is in marked contrast with TBP/TFIID-driven TATA-less gene promoters in preceding stages that have broad TSS architecture. We anticipate that our findings describing oocyte-specific transcription regulation will help to understand the mechanisms associated with primary ovarian insufficiency, which constitutes a frequent cause of infertility among women.

Pol II transcription requires the stepwise assembly of multi-protein complexes called general transcription factors (GTFs) and Pol II [START_REF] Roeder | 50+ years of eukaryotic transcription: an expanding universe of factors and mechanisms[END_REF] . The evolutionary conserved TFIID complex plays a major role in transcription initiation as it is the first GTF to initiate the assembly of the pre-initiation complex (PIC) by recognizing the core promoter [START_REF] Vo Ngoc | The punctilious RNA polymerase II core promoter[END_REF][START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] . TFIID is a large multi-protein complex composed of the TATA box-binding protein (TBP) and 13 TBP-associated factors (TAFs) in metazoa [START_REF] Tora | A unified nomenclature for TATA box binding protein (TBP)-associated factors (TAFs) involved in RNA polymerase II transcription[END_REF] . The model suggesting that transcription is always regulated by the same transcription complexes has been challenged in metazoans by the discovery of cell-type specific complexes containing specialized GTF-, TBP-or TAF-paralogs 3,[START_REF] Müller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF][START_REF] Akhtar | TBP-related factors: a paradigm of diversity in transcription initiation[END_REF] . However, how alternative initiation complexes form and how they regulate cell type-specific transcription remains unknown. During oocyte growth TBP is absent and replaced by a vertebrate-specific TBPrelated factor, TBPL2, required for female fertility 1,2,[START_REF] Malecova | TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells[END_REF] . During this phase the oocyte transcriptome is overhauled in a TBPL2-dependent manner. Importantly, Pol II transcription is blocked during oocyte growth in the absence of TBPL2 2 .

TBPL2 forms an oocyte-specific complex with TFIIA different from TFIID

To characterize TBPL2-containing transcription complexes we prepared whole cell extracts (WCE) from 14 days post-natal (P14) mouse ovaries and analysed TBPL2-associated proteins by anti-mTBPL2 immunoprecipitation (IP) coupled to label free mass spectrometry (Fig. 1a, Extended Data Fig. 1a,b). We identified TFIIA-ab and TFIIA-g subunits as unique GTF subunits associated with TBPL2 (Fig. 1a, Supplementary Table 1). In parallel, anti-TBP IP from the same extracts showed that TBP assembles into the canonical TFIID complex in non-oocyte cells present in large excess in the ovary (Extended Data Fig. 1c, Supplementary Table 2). To determine the stoichiometry of the composition of the immunoprecipitated complexes, normalized spectral abundance factor (NSAF) values were calculated [START_REF] Zybailov | Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae[END_REF] . As growing oocytes represent only a tiny minority of ovary cells, we further tested the TBPL2-TFIIA interaction by a triple IP strategy: first, we depleted TAF7-containing TFIID complexes with an anti-TAF7 IP, second, the remaining TFIID and SAGA complexes, which contain also some shared TAFs [START_REF] Helmlinger | Sharing the SAGA[END_REF] , were depleted with an anti-TAF10 IP using the anti-TAF7 IP flow-through as an input, third we performed an anti-TBPL2 IP on the anti-TAF7/anti-TAF10 flow-through fraction (Fig. 1b-d, Extended Data Fig. 1d, Supplementary Table 3). The analysis of this third consecutive IP further demonstrated that TBPL2 forms an unique complex with TFIIA-ab, and TFIIFA-g, but without any TFIID subunits. To further analyse the requirement of TFIID during oocyte growth, we carried out a conditional deletion of TFIID-specific Taf7 gene during oocyte growth using the Zp3-Cre transgenic line [START_REF] Lewandoski | Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line[END_REF] . The oocyte-specific deletion of Taf7 did not affect the presence of secondary and antral follicles and the numbers of collected mature oocytes after superovulation (Fig. 1e, f, Extended Data Fig. 1e). Thus, the TBPL2-containing transcription complex does not require TFIID TAFs for its function during oocyte growth. These results together show that during oocyte growth a stable TBPL2-TFIIA complex forms, and may function differently from TBP/TFIID.

Structure/function characterization of the TBPL2/TFIIA complex

Next, we determined the X-ray crystal structure of the mouse TBPL2 core domain (TBPL2 C ) (Fig. 2a, Supplementary Table 4). The 2Å structure reveals a TBPL2 C dimer reminiscent of the dimer observed in TBP core (TBP C ) crystals [START_REF] Chasman | Crystal structure of yeast TATA-binding protein and model for interaction with DNA[END_REF][START_REF] Nikolov | 2.1 A resolution refined structure of a TATA boxbinding protein (TBP)[END_REF] . TBPL2 C forms a pseudosymmetric bi-lobal fold similar to TBP C (Extended Data Fig. 2a). The respective electrostatic surface charge distributions show that while TBPL2 C and TBP C exhibit similarly basic charges of their N-terminal lobes and DNA binding concave surfaces, their central regions and C terminal lobes are markedly different, with TBPL2 C shifted towards acidic charge (Fig. 2b,c, Extended Data Fig. 2b,c). Based on the crystal structure of TBP C in complex with TATA box DNA and a crystallisable core of TFIIA (TFIIA C ) [START_REF] Bleichenbacher | Novel interactions between the components of human and yeast TFIIA/TBP/DNA complexes[END_REF] , we modelled the TBPL2 C -TFIIA C interactions (Fig. 2d). The resulting model suggests that TBPL2 C has a conserved, but not identical interaction interface with the TFIIA-g chain when compared with TBP C . We next analysed the functional interactions of TBPL2 C with full-length processed TFIIA and adenovirus major late promoter TATA box DNA by electrophoretic mobility shift assay (EMSA) (Fig. 2e, f, Extended Data. Fig. 2d,e). Preformed TBPL2/DNA complex was supplemented with increasing amounts of processed TFIIA-α, -β and TFIIA-g (Fig. 2e). Unexpectedly, we observed two shifted complexes in EMSA (labelled complex 1 and 2 in Fig. 2e), but complex 2 was barely noticeable when TBP C was used (Fig. 2f). Intriguingly, when only low amounts of TFIIA were added, a prominent band corresponding to free DNA reproducibly re-emerged in the TBPL2 C EMSA experiment, disappearing again when increasingly more TFIIA was supplied (Fig. 2e). No such re-emergence of free DNA was observed with TBP C (Fig. 2f). Our analyses together suggest that TBPL2 C can bind differently to TFIIA in the presence of canonical TATA box DNA, as compared to TBP C .

TBPL2-dependent oocyte transcriptome

To characterize the growing oocyte-specific transcriptome and its dependence on TBPL2, we have performed a transcriptomic analysis of wild-type (WT) and Tbpl2 -/-oocytes isolated from primary and secondary follicles [post-natal day (P) 7 and P14] (Fig. 3, Supplementary Table 5). We observed down-regulation of a number of oocyte-specific genes, such as Bmp15 and Gdf9 [START_REF] Dong | Growth differentiation factor-9 is required during early ovarian folliculogenesis[END_REF][START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF] (Fig. 3a, b and Extended Fig. 3a). Principal component analysis showed that the four distinct RNA samples clustered in individual groups and that the main explanation for the variance is the genotype, and then the stage (Extended Data Fig. 3b). Comparison of the RNA level fold changes between mutant and WT oocytes showed that in Tbpl2 -/-, there is a massive down-regulation of the most highly expressed transcripts, both at P7 and P14 (Extended Data Fig. 3c). The Pearson correlation between the P7 and P14 fold change data sets for transcripts expressed above 100 normalized reads was close to 0.8, indicating that Tbpl2 loss of function similarly altered RNA levels at P7 and P14 stages. We therefore focused on the P14 stage for the rest of the study.

In WT P14 oocytes transcripts corresponding to 10791 genes were detected. Importantly, many of these detected transcripts have been transcribed at earlier stages and are stored in growing oocytes [START_REF] Bettegowda | Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development[END_REF] . As there is no Pol II transcription in Tbpl2 -/-growing oocytes 2 , RNAs detected in the Tbpl2 -/-mutant oocytes represent mRNAs transcribed by a TBP/TFIIDdependent mechanism and deposited into the growing oocytes independently of TBPL2 activity at earlier stages. The proportion of genes (1396) upregulated following Tbpl2 deletion (Fig. 3c) can be explained by two mutually not exclusive ways: i) the consequence of the normalization to the library size resulting in the over-estimation of up-regulated transcripts, and underestimation of down-regulated transcripts and/or by transcript buffering mechanisms due to mRNA stabilization [START_REF] Timmers | Transcript Buffering: A Balancing Act between mRNA Synthesis and mRNA Degradation[END_REF] . Validation of the up-regulation of some candidate transcripts levels (Extended Data Fig. 3d,e) strongly supports the latter hypothesis.

Nevertheless, we detected 1802 significantly downregulated transcripts in the Tbpl2 -/- oocytes (Fig. 3c). Key genes known to be expressed during oocyte growth, such as Bmp15, Eloc, Fgf8, Gdf9 and Zar1 [START_REF] Dong | Growth differentiation factor-9 is required during early ovarian folliculogenesis[END_REF][START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF][START_REF] Yu | BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes[END_REF] , were confirmed to be down-regulated (Extended Data Fig. 3f,g). These results suggest that TBPL2 has an important role in gene expression in the growing oocytes. Gene Ontology (GO) analyses of biological process of the identified down regulated categories of genes (Supplementary Table 6) indicated that many genes involved in meiosis II and distinct cell cycle processes were significantly down-regulated (Extended Data. Fig. 3h).

The most enriched molecular function GO category was "poly(A)-specific ribonuclease activity" containing many genes coding for factors or subunits of complexes contributing to deadenylation/decapping/decay activity in eukaryotes (Fig. 3d) (i.e. CCR4-NOT, PAN2/PAN3 22 ; DCP1A/DCP2 [START_REF] Ma | Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse[END_REF] , or BTG4 [START_REF] Yu | BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes[END_REF] ). Transcripts coding for these "poly(A)-specific ribonuclease activity" factors were significantly down regulated in Tbpl2 -/-mutant P14 oocytes (Fig. 3e, Extended Data. Fig. 3i). Thus, in P14 oocytes TBPL2 is regulating the transcription of many genes coding for factors, which are crucial in regulating the stability and translation of the mRNA stock deposited during early oogenesis, as well as transcription of meiosis II-and cell cycle-related genes to prepare the growing oocytes for the upcoming meiotic cell division.

A remarkable feature of oocyte is the very high expression of retrotransposons driven by Pol II transcription. There are three major classes of retrotransposons in mammals: long interspersed nuclear elements, short interspersed nuclear elements and ERVs. There are three main sub-classes of ERVs: ERV1, ERVK and endogenous retrovirus like ERVL-MaLR [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF] . As expected, in WT P14 oocytes the expression of ERVs was found to be the most abundant [START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF][START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF] (Extended Data Fig. 3j,k). Importantly, the transcription of the vast majority of MaLR elements was the most affected in Tbpl2 -/-mutant oocytes (Fig. 3f-i). Among them, three highly expressed members, MT-int, MTA_Mm, and MTA_Mm-int, were dramatically down-regulated in P14 Tbpl2 -/-mutant oocytes (Extended Data Fig. 3l). As in P14 oocytes TBPL2 depletion is reducing transcription more than 4-fold from MaLR ERVs, which often serve as promoters for neighbouring genes [START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF][START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF] , TBPL2 could seriously deregulate oocyte-specific transcription and consequent genome activation. Therefore, TBPL2 is important for the de novo restructuration of the oocyte transcriptome and indirectly for silencing the translation of the TBP-dependent earlier deposited transcripts.

TBPL2-driven promoters are TATA box-containing with sharp TSS selection

The promoter usage changes during zebrafish maternal to zygotic transition revealed different rules of transcriptional initiation in oocyte and in embryo, driven by independent and often overlapping sets of promoter "codes" [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF] . This switch has not yet been demonstrated in mammals and the role of TBPL2 during oogenesis remained to be investigated. To this end, we mapped the TSS usage by carrying out super-low input carrier-CAGE (SLIC-CAGE) [START_REF] Cvetesic | SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA[END_REF] from WT and Tbpl2 -/-P14 oocytes. To characterize only the TBPL2-driven promoters we removed the CAGE tags present in the Tbpl2 -/-dataset from the WT P14 dataset, to eliminate transcripts that have been deposited at earlier stages (hereafter called "TBPL2-specific-only"). Conversely, the Tbpl2 -/-dataset corresponds to the TBP/TFIID or TBPL2-independent-only TSSs (hereafter called "TBPL2-independent-only").

Next, we analysed the genome-wide enrichment of T-and/or A-rich (WW) dinucleotide motifs within the -250/+250 region centred on the dominant TSSs of the TBPL2-specific-only and TBPL2-independent-only oocyte TSS clusters (Fig. 4a,b). TBPL2-specific-only TSS clusters are strongly enriched in a well-defined WW motif around their -30 bp region (Fig. 4a, read arrowhead) (see also Cvetesic et al., in submission). In contrast, only about 1/3 rd of the TBPL2-independent-only TSS clusters contained WW-enriched motifs at a similar position (Fig. 4b, read arrowhead), as would be expected from promoters that lack maternal promoter code determinants [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF] (Cvetesic et al., in submission). As canonical TATA boxes are often associated with tissue-specific gene promoters, we investigated whether the above observed WW motif densities correspond to TATA boxes using the TBP position weight matrix (PWM) from the JASPAR database as a reference. To this end the presence of TATA boxes was analysed in the TSS clusters of the two data sets and revealed that TBPL2-specific-only TSS clusters were enriched in high quality TATA boxes, including a clear increase in the proportion of canonical TATA boxes, when compared to TBPL2-independent-only TSS clusters (Fig. 4c).

Depending on promoter sequence motifs, transcription initiation can occur within a narrow region with a major dominant TSS, or within a less defined region containing a wider distribution of TSSs, leading to the distinction of "sharp" and "broad" TSS-type initiation, respectively [START_REF] Carninci | Genome-wide analysis of mammalian promoter architecture and evolution[END_REF] . Genome browser view snapshots indicate that TSS clusters in P14 WT oocytes tend to be sharp and are associated with TATA-like motifs (Extended Data. Fig. 4a,b). Analysis of the global distribution of the number of TSSs and of the width of the TSS clusters in the (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

above defined two categories confirmed that TBPL2-specific-only TSS are sharper compared to the TBPL2-independent-only TSS clusters (Extended Data Fig. 4c,d).

In order to test whether TBPL2 controls transcription initiation from maternal promoter code determinants, we grouped the expression profiles corresponding to each consensus TSS clusters, to characterise promoter activity profiles among datasets by performing selforganizing maps (SOM) (Extended Data Fig. 4e). We then focussed on the two most distinct SOM groups: the down-regulated promoters (blue group, containing 9442 consensus TSS clusters) (Fig. 4d) and the up-regulated promoters (red group, with 6900 consensus TSS clusters) (Fig. 4e). Motif analyses of these two categories of promoters in their -35/+5 regions relative to the different dominant TSSs indicated that only the core promoters associated to TBPL2-specific-only dominant TSSs belonging to the down-regulated gene promoters contain a well-defined 7 bp long TATA box-like motif (W-box) in their -31 to -24 regions (Fig. 4f,g, Extended Data Fig. 4f-i). Importantly, W-box associated TSSs architecture usage distribution for these TBPL2-specific-only dominant TSSs was sharp (Extended data Fig. 4j,l), as expected for motif-dependent transcriptional initiation [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF] (Cvetesic et al., in submission). In contrast, TBPL2-independent-only TSSs belonging to the up-regulated promoters exert a much broader TSS pattern (Extended Data Fig. 4k,m). Interestingly, GO analyses of the genes associated with the down-regulated promoters revealed a strong association with deadenylation/decapping/decay activity (Extended Data Fig. 4n-p, Supplementary Table 7), further confirming our initial RNA-seq analysis observations (Fig. 3).

Importantly, TSS architecture analyses of the TBPL2-specific-only MaLR ERV TSSs indicated that the majority of MaLR core promoters contain high quality TATA box motif (median of the TATA box PWM match is 85%, Fig. 4i,j). These observations together demonstrate that the TBPL2-TFIIA complex drives transcription initiation primarily from core promoters that contain a TATA box-like motif in their core promoter and directs sharp transcription initiation from the corresponding promoter regions to overhaul the growing oocyte transcriptome.

In addition, we observed that TSS usage can shift within the promoter of individual genes depending on the genetic background (Extended Data Fig. 4b). To get more insights in these promoter architecture differences, we identified genome-wide 6429 shifting promoters by comparing either TBPL2-specific-only to TBPL2-independent-only TSS data. These results are consistent with TSS shifts between somatic and maternal promoter codes (Cvetesic et al., in submission) occurring either in 5', or 3' directions (Fig. 4k, Extended Data Fig. 4q). WW motif analysis indicated that on each shifting promoter, TBPL2-specific-only dominant TSSs are associated with WW motifs, while TBPL2-independent-only dominant TSSs are not (Fig. 4l).

In addition, the TATA box PWM match analyses indicated that these WW motifs are enriched in TATA box like elements compared to the corresponding TBPL2-independent-only shifting TSSs (Fig. 4m). Thus, our experiments provide a direct demonstration that TBP/TFIID and TBPL2/TFIIA machineries recognize two distinct sequences co-existing in promoters of the same genes with TBPL2 directing a stronger WW/TATA box-dependent TSS selection in them.

Discussion

In conclusion, our results demonstrate that in growing oocytes TFIID (TBP/TAFs) is replaced by a non-canonical TBPL2/TFIIA complex that uses canonical TATA box-containing core promoters to drive oocyte-specific sharp TSS-transcription initiation. Thus, the TBPL2/TFIIA complex is a key regulator of growing oocyte-specific transcription, including the high expression of MaLR ERVs, by creating a novel transcriptome pool, consisting of new TBPL2-dependent transcripts and TBP/TFIID-dependent transcripts deposited earlier during the primordial follicle stage (Fig. 5). Importantly, to block the translation of mRNAs deposited at the earlier developmental stage, TBPL2 is regulating the activity of several deadenylation/decapping/decay complexes, which further contributes to establishing the novel TBPL2-dependent growing oocyte transcriptome and consequent proteome required for further development and oocyte competence for fertilization (Fig. 5). The indirect regulation of previously deposited mRNAs by a global transcription regulator resembles the well characterised maternal to zygotic transition (MZT), during which a clearance of inherited transcriptome is mediated by de novo gene products generated by newly activated transcription machinery [START_REF] Ferg | The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish[END_REF][START_REF] Giraldez | Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs[END_REF][START_REF] Vastenhouw | The maternal-to-zygotic transition revisited[END_REF] . At hundreds of gene promoters, two distinct TSS-defining "grammars" co-exist in close proximity genome-wide and are differentially utilised in either TBPL2/TFIIA in primary/secondary follicular oocytes or by TBP/TFIID in primordial follicular oocytes. This again shows a striking parallel to MZT [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF] , where multiple layers of information are embedded in the same promoter sequence, each representing a different type of regulatory grammar interpreted by dedicated transcription machinery depending on the cellular environment.

Main references

1.

Gazdag, E., Rajkovic, A.

Methods

Cell lines and cell culture

The NIH3T3-II10 line overexpressing TBPL2 and the control NIH3T3-K2 have already been described 1 and were maintained in high glucose DMEM supplemented with 10% of new-born calf serum at 37°C in 5% CO2.

Whole cell extracts

NIH3T3-II10 and NIH3T3-K2 cells cultured in 15 cm dish were washed twice with 1x PBS, subsequently harvested by scrapping on ice. Harvested cells were centrifuged 1000 rcf at 4°C for 5 min and then resuspended in 1 packed cell volume of whole cell extraction buffer (20 mM Tris HCl pH7.5, 2 mM DTT, 20% Glycerol, 400 mM KCl, 1x Protease inhibitor cocktail (PIC, Roche)). Cell lysates were frozen in liquid nitrogen and thawed on ice for 3 times, followed by centrifugation at 20817 rcf, at 4°C for 15 min. The supernatant was collected and protein concentration was measured by Bradford protein assay (Bio-Rad). The cell extracts were used directly for immunoprecipitation and western blot, or stored at -80°C.

Ovaries collected from post-natal day 14 (P14) CD1 and C57BL/6N female mice were homogenized in whole cell extraction buffer (20 mM Tris HCl pH7.5, 2 mM DTT, 20%

Glycerol, 400 mM KCl, 5x PIC (Roche)). Cell lysates were frozen in liquid nitrogen and thawed on ice for 3 times, followed by centrifugation at 20817 rcf, at 4°C for 15 min. The supernatant extracts were used directly for immunoprecipitation.

Antibodies and antibody purification

The 2B12 anti-TBPL2, the 3TF13G3 anti-TBP and the 15TF21D10 anti-GST mouse monoclonal antibodies have already been described 1,33,34 . The J7 rabbit polyclonal anti TFIIA

Western blot

Protein samples (15-25 µg of cell extracts or 15 µL of IP elution) were mixed with 1/4 th volume of loading buffer (100 mM Tris-HCl pH 6.8, 30% glycerol, 4% SDS, 0.2% bromophenol blue and freshly added 100 mM DTT) and boiled for 10 min. Samples were then resolved on a 10 % SDS-PAGE and transferred to nitrocellulose membrane (Protran, Amersham). Membranes were blocked in 3% non-fat milk in 1x PBS at room temperature (RT) for 30 min, and subsequently incubated with the primary antibody overnight at 4°C. Membranes were washed three times (10 min each) with 1x PBS -0.05% Tween20. Membranes were then incubated with HRP-coupled secondary antibodies (Jackson ImmunoResearch) for 1 hour at RT, followed by ECL detection (Thermo Fisher). The signal was acquired with the ChemiDoc imaging system (Bio-Rad). filtered with a false discovery rate (FDR) at 5 %, rank 1 and proteins were identified with 1 unique peptide. Normalized spectral abundance factor (NSAF) [START_REF] Zybailov | Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae[END_REF] were calculated using custom R scripts (R software version 3.5.3). Only proteins detected in at least 2 out of 3 of the technical or biological replicates were considered for further analyses.

Mass spectrometry analyzes and NSAF calculations

Animal experimentation

Animal experimentations were carried out according to animal welfare regulations and guidelines of the French Ministry of Agriculture and procedures were approved by the French Ministry for Higher Education and Research ethical committee C2EA-17 (project n°2018031209153651). The Tg(Zp3-Cre), Taf7 flox and Tbpl2 -lines have already been described 2, [START_REF] Lewandoski | Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line[END_REF]35 .

Histology analyses of ovaries

Ovaries were collected from 6 weeks-old Tg(Zp3-Cre/+);Taf7 flox/+ and Tg(Zp3-Cre/+);Taf7 flox/∆ oocyte specific mutant females, fixed in 4% paraformaldehyde (Electron Microscopy Sciences)

over-night at 4°C, washed 3 times in PBS at room temperature and embedded in paraffin. Five µm-thick sections were stained with hematoxylin and eosin and images were acquired using a slide scanner Nanozoomer 2.0HT (Hamamatsu Photonics).

Supervovulation

Five units of pregnant mare serum (PMS) was injected intraperitoneally in 4-week-old female mice between 2-4 pm. After 44-46 hours, GV oocytes were collected from the ovaries by puncturing with needles.

Expression and purification of TBPL2 C and TBP C

Mouse TBPL2 C (amino acids 170 to 349) fused to an N-terminal hexa-histidine tag was expressed in Sf21 insect cells using the MultiBac system 36 . Cells were infected with composite baculovirus, harvested and pelleted following published protocols 37 . Cell pellets were resuspended and lysed by freeze-thaw in liquid nitrogen in Talon Buffer A (25 mM Tris pH

Crystallization and structure determination of TBPL2 C

Purified TBPL2 C was screened for crystallization in a range of conditions using the sitting drop method. Crystals obtained in 26% PEG3350, 0.2 M magnesium formate were harvested flashfrozen in liquid nitrogen and used for data collection at Diamond Light Source, (beamline I04-1). Diffraction data were processed and scaled using software XDS 38 and the structure solved by molecular replacement using software Phenix 39 , with TBP C (from PDBID 1NVP) as a starting model. The structure was iteratively rebuilt and refined. Data and refinement statistics are provided (Supplementary Table 5). Structure images were prepared using software PyMOL.

Expression and purification of TFIIA

The gene encoding for mouse TFIIA-γ was cloned in the pIDC plasmid from the MultiBac suite 37 . The gene encoding for mouse TFIIA-αβ fused to an N-terminal hexa-histidine tag and a TEV protease site was cloned in the pACEBac1 plasmid of the MultiBac system. A second TEV protease site was also introduced in TFIIA-αβ at the TASPASE 1 cleavage site, to prepare processed TFIIA comprising native-like TFIIA-a and TFIIA-b polypeptides (Extended Data

Electrophoretic mobility shift assay (EMSA)

Cy3 labelled Adenovirus major late promoter (AdMLP, coding strand sequence 5'CTGCTATAAAAGGCTG3') was purchased from Eurofins Genomics. Double-stranded DNA (dsDNA) substrate was generated by mixing coding and complimentary non-coding DNA oligonucleotides at equimolar ratio, followed by heating to 90°C and annealing by slowly cooling to room temperature (RT). Samples for EMSA experiments were prepared by mixing annealed Cy3 labelled dsDNA (0.5 µM) with either TBP C or TBPL2 C (1 µM) in EMSA Reaction Buffer (10 mM Tris pH 8.0, 60 mM KCl, 10 mM MgCl2, 10% glycerol, 2.5 mM DTT). Purified processed TFIIA was added to aliquots of this mix at increasing concentrations (0.25 µM to 3 µM) followed by 90 min incubation on ice. Samples were analysed by nondenaturing 6% Tris-glycine gels (Invitrogen) using 1x Tris-Glycine native running buffer. Gels were imaged by recording fluorescence signal for Cy3 using a Typhoon FLA 9000 Gel Imager (GE Healthcare).

Oocytes collection

After dissection, ovaries are freed from adhering tissues in 1x PBS. Series of 6 ovaries were digested in 500 µL of 2 mg/mL Collagenase (SIGMA), 0.025% Trypsin (SIGMA) and 0.5 mg/mL type IV-S hyaluronidase (SIGMA), on a thermomixer at 600 rpm for 20 minutes. The digestion was then stopped by the addition of 1 mL of 37°C pre-warmed αMEM -5% FBS.

The oocytes were then size-selected under a binocular.

RNA preparation

Pool of 100-200 oocytes collected were washed through several M2 drops, and total RNA was isolated using NucleoSpin RNAXS kit (Macherey-Nagel) according to the user manual. RNA quality and quantity were evaluated using a Bioanalyzer. Between 5-10 ng of RNA was obtained from each pool of oocytes.

RNA-seq analyses

PolyA+ RNA seq libraries were prepared using the SMART-Seq v4 UltraLow Input RNA kit (Clonetch) followed by the Nextera XT DNA library Prep kit (Illumina) according to the manufacturer recommendations from 3 biological replicates for each condition (P7 wild-type (WT), P7 Tbpl2 -/-mutant, P14 WT and P14 Tbpl2 -/-mutant oocytes) and sequenced 50 pb single end using an Illumina HiSeq 4000 (GenomEast platform, IGBMC).

Reads were preprocessed in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20). After this preprocessing, reads shorter than 40 bases were discarded for further analysis. These preprocessing steps were performed using cutadapt version 1.10 40 .

Reads were mapped to spike sequences using bowtie version 2.2.8 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] , and reads mapping to spike sequences were removed for further analysis. Reads were then mapped onto the mm10 assembly of Mus musculus genome using STAR version 2.7.0f [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF] . Gene expression quantification was performed from uniquely aligned reads using htseq-count version 0.9.1 [START_REF] Anders | HTSeq--a Python framework to work with highthroughput sequencing data[END_REF] , with annotations from Ensembl version 96 and "union" mode. Read counts were normalized across samples with the median-of-ratios method [START_REF] Anders | Count-based differential expression analysis of RNA sequencing data using R and Bioconductor[END_REF] , to make these counts comparable between samples and differential gene analysis were performed using the DESeq2 version 1.22.2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF] . All the figures were generated using R software version 3.5.3.

RT-qPCR

Complementary DNA was prepared using random hexamer oligonucleotides and SuperScript IV Reverse Transcriptase (Invitrogen) and amplified using LightCycler® 480 SYBR Green I Master (Roche) on a LightCycler® 480 II (Roche). Primers used for qPCR analysis are listed in Supplementary Table 8.

Repeat element analyses

Data were processed as already described [START_REF] Fadloun | Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA[END_REF] using Bowtie1 47 instead of Maq. The repeatMasker annotation was used to identified the different types of repeat elements (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-4.0. 2013-2015 http://www.repeatmasker.org). Differential expression analyses were performed using DESeq2 (version 1.22.2) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF] . All the figures were generated using R custom scripts (version 3.5.3).

SLIC-CAGE analyses

Twenty-eight and 13 ng of total RNA isolated from P14 oocytes (biological replicate 1 and replicate 2, approximately 500-1000 oocytes pooled for each replicate) and 15 ng of total RNA isolated from P14 Tbpl2 -/-mutant oocytes (approximately 550 pooled oocytes) were used for SLIC-CAGE TSS mapping [START_REF] Cvetesic | SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA[END_REF] . Briefly, 5 µg of the carrier RNA mix were added to each sample prior to reverse transcription, followed by the cap-trapping steps designed to isolate capped RNA polymerase II transcripts. The carrier was degraded from the final library prior to sequencing using homing endonucleases. The target library derived from the oocyte RNA polymerase II transcripts was PCR-amplified (15 cycles for P14 WT, 16 cycles for P14 Tbpl2 - /-mutant) and purified using AMPure beads (Beckman Coulter) to remove short PCR artifacts (< 200bp, size selection using 0.8 x AMPure beads to sample ratio). The libraries were sequenced using HiSeq2500 Illumina platform in single-end, 50 bp mode (Genomics Facility, MRC, LMS).

Sequenced SLIC-CAGE reads were mapped to the reference M. musculus genome (mm10 assembly) using the Bowtie2 41 with parameters that allow zero mismatches per seed sequence (22 nucleotides). Uniquely mapped reads were kept for downstream analyses using CAGEr

Bioconductor package (version 1.20.0) [START_REF] Haberle | CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses. -PubMed -NCBI[END_REF] and custom R/Bioconductor scripts. Bam files were imported into R using the CAGEr package, where the mismatching additional G, if added through the template-free activity of the reverse transcriptase, was removed. Same samples sequenced on different lanes and biological replicates were merged prior to final analyses.

Promoter analyses

In order to consider only the CAGE TSS dependent only on TBPL2, we removed all the P14

WT CAGE tags at position where CAGE tags were also present in the P14 Tbpl2 -/-mutant CAGE tags dataset: for the rest of the analysis, this dataset was called "TBPL2-specific-only" and we compared it to the P14 Tbpl2 -/-mutant CAGE data (hereafter called "TBPL2independent-only"). Briefly, a CAGE set object was created from the TBPL2-specific-only and TBPL2-independent-only CTSS files using CAGEr Bioconductor package (version 1.20.0) [START_REF] Haberle | CAGEr: precise TSS data retrieval and high-resolution promoterome mining for integrative analyses. -PubMed -NCBI[END_REF] , data were normalized using normalizeTagCount (fitInRange = c(5,1000), alpha = 1.53, T = 1e6)

and the powerLaw option. Cluster of CTSS were collected using clusterCTSS (threshold = 1, thresholdIsTpm = TRUE, nrPassThreshold = 1, method = "distclu", maxDist = 20, removeSingletons = TRUE, keepSingletonsAbove = 5). Width of the TSS regions was calculated using cumulativeCTSSdistribution and quantilePositions (clusters = "tagClusters", qLow = 0.1, qUp = 0.9): interquantile width corresponds to the 10 th -90 th percentile of the total tag cluster signal. In order to compare the different samples, consensus promoters were computed using aggregateTagCluster (tpmThreshold = 3, qLow = 0.1, qUp = 0.9, maxDist = 100). Self-organizing map (SOM) expression profiling was performed using getExpressionProfiles using a tpmThrshold of 3, the method "som", xDim = 3 and yDim = 2.

Shifting TSS were obtained after calculation of the cumulative distribution along the consensus clusters using cumulativeCTSSdistribution and calculation of the shift score using scoreShift with the Kolomogorov-Smirnov test. Shifting promoters were extracted using getShiftingPromoters (tpmThreshold = 3, scoreThreshold = -Inf, fdrThreshold = 0.01).

TSSs corresponding to the MaLR-ERVS were identified after annotation using HOMER (version 4.10) [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF] .

Sequences analyses were performed using seqPattern (version 1.14){Haberle:co} and R custom scripts. WW dinucleotides enrichment was computed using plotPatternDensityMap on -250/+250 regions centered on the dominant TSSs. TATA box position weight matrix (PWM) matches analyses was performed using the MotifScanScores function applied on the -35/-20 sequences centered on the dominant TSSs, using the TBP PWM provided in the SeqPattern package (derived from the JASPAR data base). Distribution of the best match for each sequence was then plotted. Sequence Logo were created using SeqLogo (version 1.48.0) [START_REF]seqLogo: Sequence logos for DNA sequence alignments[END_REF] .

Methods references

  , Torres-Padilla, M.-E. & Tora, L. Analysis of TATA-binding protein 2 (TBP2) and TBP expression suggests different roles for the two proteins in regulation of gene expression during oogenesis and early mouse development. Reproduction 134, 51-62 (2007). 2. Gazdag, E. et al. TBP2 is essential for germ cell development by regulating transcription and chromatin condensation in the oocyte. Genes Dev 23, 2210-2223 (2009). 3. Goodrich, J. A. & Tjian, R. Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nat Rev Genet 11, 549-558 (2010).

  was a gift of H.G. Stunnenberg. The IGBMC antibody facility raised the anti-TBPL2 polyclonal 3024 serum against the CPDEHGSELNLNSNSSPDPQ peptide (amino acids 111-129) coupled to ovalbumin and injected into one rabbit. The resulting serum was affinity purified by using the Sulfolink Coupling Gel (Pierce) following the manufacturer's recommendations.ImmunoprecipitationOvary extract were incubated with anti-GST, anti-TBP, anti-TBPL2, anti-TAF7 or anti-TAF10 coated Dynabeads (Invitrogen) at 4°C overnight. After incubation, beads were washed 3 x 5min at 4°C with 500 mM KCl buffer [25 mM Tris-HCl (pH 7.9), 5 mM MgCl2, 10% glycerol, 0.1% NP40, 2 mM DTT, 500 mM KCl and 1x PIC (Roche)], then washed 3 x 5min at 4°C with 100 mM KCl buffer (25 mM Tris-HCl pH 7.9, 5 mM MgCl2, 10% glycerol, 0.1% NP40, 2 mM DTT, 100 mM KCl and 1x). Immunoprecipitated proteins were eluted with 0.1 M glycine pH 2.8 and neutralized with 1.5 M Tris-HCl pH 8.8. Immunoprecipitation performed from whole cell extracts of NIH3T3-II10 and NIH3T3-K2 cells were following the same procedures with protein G Sepharose beads (GE Healthcare).

  Samples were TCA precipitated, reduced, alkylated and digested with LysC and Trypsin at 37°C overnight. After C18 desalting, samples were analyzed using an Ultimate 3000 nano-RSLC (Thermo Scientific, San Jose, California) coupled in line with a linear trap Quadrupole (LTQ)-Orbitrap ELITE mass spectrometer via a nano-electrospray ionization source (Thermo Scientific). Peptide mixtures were loaded on a C18 Acclaim PepMap100 trap column (75 µm inner diameter × 2 cm, 3 µm, 100 Å; Thermo Fisher Scientific) for 3.5 min at 5 µL/min with 2% acetonitrile (ACN), 0.1% formic acid in H2O and then separated on a C18 Accucore nanocolumn (75 µm inner diameter × 50 cm, 2.6 µm, 150 Å; Thermo Fisher Scientific) with a 240 minutes linear gradient from 5% to 50% buffer B (A: 0.1% FA in H2O / B: 80% ACN, 0.08% FA in H2O) followed with 10 min at 99% B. The total duration was set to 280 minutes at a flow rate of 200 nL/min. Proteins were identified by database searching using SequestHT (Thermo Fisher Scientific) with Proteome Discoverer 1.4 software (Thermo Fisher Scientific) a combined Mus musculus database (Swissprot, release 2015_11, 16730 entries) where 5 interesting proteins sequences (TrEMBL entries) were added. Precursor and fragment mass tolerances were set at 7 ppm and 0.5 Da respectively, and up to 2 missed cleavages were allowed. Oxidation (M) was set as variable modification, and Carbamidomethylation© as fixed modification. Peptides were

7. 5 ,

 5 1000 mM NaCl, 10 mM imidazole and compete protease inhibitor tablet (Roche)). Lysate was cleared by centrifugation at 40,000 rcf for 60 min. Cleared lysate was incubated with Talon Resin (Thermo Fisher) and unbound protein washed with 20 column volumes (CVs) of Talon Buffer A. Protein was eluted with Talon Buffer B (25 mM Tris pH 7.5, 1000 mM NaCl, 600 mM Imidazole) using a step gradient. The tag was removed by TEV protease cleavage during dialysis overnight into Dialysis Buffer (25 mM Tris pH 8.0, 300 mM NaCl, 2 mM β-MeSH) followed by a reverse IMAC step to remove uncleaved protein. TBP was then subjected to size exclusion chromatography (SEC) using a SuperdexS200 16/600 column equilibrated in SEC Buffer (25 mM Tris pH 7.5, 300 mM NaCl, 5 mM β-MeSH). Mouse TBP C (amino acid residues 136 to 316) was expressed in E. coli Rosetta (DE3) cells at 18°C and purified as described for TBPL2 C .

Fig. 1n )

 1n Fig.1n). Plasmids were fused by Cre recombinase and expression cassettes inserted into the

Fig. 1 |Fig. 2 |Fig. 3 |

 123 Fig. 1 | TBPL2 does not assemble in a TFIID-like complex during oocyte growth. a, anti TBPL2 immunoprecipitation followed by mass spectrometry (IP-MS) analysis from three biological replicates of mouse ovarian whole cell extracts (WCE). The colour code for the different proteins or complexes is indicated on the right. NSAF; normalized spectral abundance factor. b-d, Sequential IP-MS experiment from ovarian WCE (technical triplicates). Anti TAF7 IP-MS (b), followed by an anti TAF10 IP-MS (c) and then an anti TBPL2 IP-MS (d). The colour code is the same as in (a). e, f, Hematoxylin and eosin stained ovaries section from control (e) and oocyte-specific Taf7 mutant (f) ovaries. The presence of antral follicles is indicated by an asterisk. Scale bars: 500 µm.

Tbpl2Fig. 4 |

 4 Fig. 4 | Core promoter regions of TBPL2 specific transcription units in P14 oocyte are enriched in TATA-like elements and are sharp. a, b, Genome-wide A/T-rich dinucleotide (WW) motif analyses of -250/+250 sequences centred on the dominant transcription start sites (TSS, position +1, dashed red line) of TBPL2-specific-only (a) and TBPL2-indendent-only (b) TSS clusters. The sequences have been ordered by increasing size of the width of each cluster (corresponding to the 10 th -90 th percentile of the total tag cluster signal). The red arrowheads indicate the WW enrichment at position -30 in the TBPL2-specific-only TSS clusters (a) and the equivalent position in the TBPL2-independent-only TSS clusters (b). The number of TSS clusters is indicated in brackets. c, Distribution of the best TATA box position weight matrix (PWM) matches within a -35 to -20 region upstream of the dominant TSSs (+1) of TBPL2-specific-only (blue) compared to the TBPL2-independent-only (orange) TSS clusters. The dashed lines indicate the median of the TATA box PWM matches for the TBPL2-specific-only (blue) and the TBPL2-independent-only (orange) TSS clusters (p value after a two-tailed Wilcoxon rank-sum test). d, e, Two selected self-organizing map (SOM) groups of the consensus TSS clusters: the down-regulated promoters (blue, d) and the up-regulated promoters (red, e) groups. f, g, Sequence logos of the -35/+5 sequence of the TBPL2-specific-only dominant TSSs from the down-regulated promoters (f) and of the TBPL2-independent-only dominant TSSs from the up-regulated promoters (g). h, Distribution of the best TATA box PWM matches within a -35 to -20 region upstream of the TBPL2-specific-only (blue) and TBPL2-independent-only (orange) MaLR ERVs dominant TSS. The dashed lines indicate the median of the TATA box PWM matches for the TBPL2-specific-only (blue) and the TBPL2-independent-only (orange) TSS clusters (p value after a two-tailed Wilcoxon rank-sum test). i, j, Sequence logo of the -35/+5 sequence of the MaLR ERVs TBPL2-specific only (i) and TBPL2-independent-only (j) dominant TSSs. k, l, Analysis of the TBPL2-specific-only versus TBPL2-independent-only shifting promoters within a -250/+250 region centred on the position of the TBPL2-independent-only TSS clusters (position +1 in (k, l) and red dashed line in (l)). Position of the TBPL2-specific-only (blue) and of the TBPL2-independent-only (orange) dominant TSSs for each shifting promoter sequence (k) and WW dinucleotide enrichment heat map (l) from the same set of sequences ordered as in (k). m, Distribution of the best TATA box PWM matches within a -35 to -20 region upstream of the TBPL2-specific-only (blue) and TBPL2-independent-only (orange) dominant TSS of the shifting promoters. The dashed lines indicate the median of the TATA box PWM matches for the TBPL2-specific-only (blue) and the TBPL2-independent-only (orange) shifting TSS clusters (p value after a two-tailed Wilcoxon rank-sum test).

Fig. 5 |

 5 Fig. 5 | Transcriptome overhaul controlled by TBPL2/TFIIA during oocyte growth.

  33. Brou, C. et al. Distinct TFIID complexes mediate the effect of different transcriptional activators. EMBO J 12, 489-499 (1993). 34. Nagy, Z. et al. The metazoan ATAC and SAGA coactivator HAT complexes regulate different sets of inducible target genes. Cell Mol Life Sci 67, 611-628 (2010). 35. Gegonne, A. et al. The general transcription factor TAF7 is essential for embryonic development but not essential for the survival or differentiation of mature T cells. Mol

	Cell Biol 32, 1984-1997 (2012).
	36. Sari, D. et al. The MultiBac Baculovirus/Insect Cell Expression Vector System for
	Producing Complex Protein Biologics. Advances in Experimental Medicine and
	Biology 896, 199-215 (2016).
	37. Bieniossek, C., Richmond, T. J. & Berger, I. MultiBac: multigene baculovirus-based
	eukaryotic protein complex production. Curr Protoc Protein Sci Chapter 5, Unit 5.20-
	5.20.26 (2008).
	38. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132 (2010).
	39. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons
	and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861-
	877 (2019).
	40. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing
	reads. EMBnet.journal 17, 10-12 (2011).

Acknowledgements

We thank D. Singer and A. Gegonne for the gift of the Taf7 flox mouse line and H. Stunnenberg for TFIIA antibodies. We would also like to thank D. Devys for critically reading the manuscript, all members of the Tora lab for thoughtful discussions and suggestions throughout the course of the work. We are grateful to I. Kukhtevich, M. Borsos, M.E. Torres Padilla, T. Gupta and L. Casini for advice and preliminary experiments. We thank C. Hérouard and M. Jung from the GenomEAST platform for library preparation and preliminary analyses, P. Eberling for peptide synthsis, F. Ruffenach for proteomic analyses, G. Duval for polyclonal antibody generation, the histology platform, the IGBMC cell culture facility and S. Falcone, M. Poirot and F. Memedov of the IGBMC animal facility for animal care taking. This work was supported by funds from CNRS, INSERM, and Strasbourg University. This study was also supported by the European Research Council (ERC) Advanced grant (ERC-2013-340551, Birtoaction) and grant ANR-10-LABX-0030-INRT (to LT) and a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02. IB and FM acknowledge support by Wellcome Trust Senior Investigator awards (106115/Z/14/Z and 106955/Z/15/Z, respectively).

Data availability

The datasets generated during the current study are available in different repositories: structure factors and coordinates for TBPL2 C ; Protein Data Bank (PDB ID 6TH9), proteomic data; ProteomeXchange PRIDE database (PXD0316347), RNA-seq data; Gene Expression Omnibus database (GSE140090) and SLIC-CAGE data; ArrayExpress (submission in progress).

Authors' contribution

CY, SDV and LT designed the study; SDV and LT supervised the project; CY performed all molecular lab and mouse experiments, EG generated the anti-TBP2 polyclonal antibodies, LN carried out the proteomic analyses, PH organized the SLIC-CAGE, NC carried out SLIC-CAGE analyses, NC and BL analysed the SLIC-CAGE data, and SDV analysed the proteomic, RNA-seq and SLIC-CAGE data. FM oriented the promoter analyses. KG performed X-ray crystallography and biochemical experiments, supervised by IB. CY, SDV, KG, IB, FM, and LT wrote the manuscript with contributions to manuscript text and figure legends from all authors. All authors gave final approval for publication.

Competing interests

The authors declare that they have no competing interests.

Additional Information

Supplementary information is available for this paper.


Xiaoning Wang

Marcello Brigante

Wenbo Dong

Zhangxiong Wu

Gilles Mailhot
email: gilles.mailhot@uca.fr

Degradation of Acetaminophen via UVA-induced advanced oxidation processes (AOPs). Involvement of different radical species: HO • , SO 4 •-and HO 2 • /O 2 •

Keywords: advanced oxidation process, photocatalysis, UVA, radicals, synergistic effects

In this work, UVA radiation that is part of solar light is taken as the irradiation source and radicals (HO • , SO 4

•-and HO 2 • /O 2 •-) are generated through activation of hydrogen peroxide (H 2 O 2 ), sodium persulfate (Na 2 S 2 O 8 ) and Bismuth catalyst (BiOCl), respectively. The distinguished performance in removing acetaminophen (ACTP), a model pharmaceutical pollutant, by these three radicals was compared for the first time. Effect of pH, halide ions concentration and interfacial mechanism have been investigated in detail. Interestingly, results show that heterogeneous UVA/BiOCl process has higher degradation efficiency than homogeneous UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 systems whatever the solution's pH. To explain these results, second order reaction rate constant (k radical,ACTP ) have been determined with laser flash photolysis (LFP) or radical scavenging experiments. The strongly interfacial-depended HO 2 • /O 2 •-radicals have the lowest second order rate constant with ACTP but highest steady state concentration. BiOCl was much easier activated by UVA, and outstanding ACTP mineralization can be achieved. Combination of BiOCl and Na 2 S 2 O 8 exhibits synergistic effects rather than antagonism effects with H 2 O 2 . This study highlights the relative effective utilization of solar light through interfacial directed BiOCl photocatalysis and its synergistic effects with traditional oxidants.

Introduction

In recent years, advanced oxidation processes (AOPs) have been proposed as alternative methods to effectively remove persistent organic pollutants (POPs) in environment. Normally, AOPs have always proved to achieve the best yields in pollutant destruction when biological treatments are unfeasible, like for endocrine disrupting chemicals (EDCs) [START_REF] Pera-Titus | Degradation of chlorophenols by means of advanced oxidation processes: a general review[END_REF][START_REF] Rosenfeldt | Degradation of endocrine disrupting chemicals bisphenol A, ethinyl estradiol, and estradiol during UV photolysis and advanced oxidation processes[END_REF], pharmaceuticals or personal care products (PPCPs) [START_REF] Huber | Oxidation of pharmaceuticals during ozonation and advanced oxidation processes[END_REF][START_REF] Abdelmelek | Removal of pharmaceutical and personal care products from reverse osmosis retentate using advanced oxidation processes[END_REF]. The predominant oxidation processes are usually based on H 2 O 2 or persulfate activation by in situ production of hydroxyl radical (HO • ) or sulfate radical (SO 4

•-), followed by reaction on the targets pollutants in aqueous solutions [START_REF] Andreozzi | Advanced oxidation processes (AOP) for water purification and recovery[END_REF]Esplugas et al., 2002). Especially, traditional Fenton reaction (Fe(II)/H 2 O 2 ) (Neyens and Baeyens, 2003;[START_REF] Huang | Assessment of the Fe (III)-EDDS complex in Fenton-like processes: from the radical formation to the degradation of bisphenol A[END_REF] and UV/H 2 O 2 are predominant strategies to generate HO • . Similarly, SO 4 •-are always produced through activation of persulfate by heat [START_REF] Nie | Degradation of chloramphenicol by thermally activated persulfate in aqueous solution[END_REF], UV, alkalinity [START_REF] Nie | Enhanced removal of organic contaminants in water by the combination of peroxymonosulfate and carbonate[END_REF] and transition metals (Wu et al., 2015a). However, Fenton reactions always suffer from several limitations like strict acidic solution (pH < 4.0) and iron sludge disposal.

At the same time, interfacial based heterogeneous photocatalysis is also one of the most promising oxidation processes through producing electron (e -) and hole (h + ) pairs under illumination. HO • or hydroperoxyl radicals/superoxide radical anion (HO 2 • /O 2 •-, pKa 4.88) will be subsequently produced by reaction of photogenerated charge carriers with O 2 and H 2 O [START_REF] Shaban | Photocatalytic degradation of phenol in natural seawater using visible light active carbon modified (CM)-n-TiO 2 nanoparticles under UV light and natural sunlight illuminations[END_REF]. Among these photocatalysts, TiO 2 and its doped products have received a great deal of attention due to its efficient and abundant production of HO • (Yang et al., 2008a). Different to TiO 2 , in bismuth-related photocatalytic process, especially layered BiOX (X = Cl, Br, I) [START_REF] Zhang | Generalized one-pot synthesis, characterization, and photocatalytic activity of hierarchical BiOX (X= Cl, Br, I) nanoplate microspheres[END_REF], quenching experiments and theoretical calculation have proved that they are featured with HO 2

• /O 2 •-generation for organic compound degradation [START_REF] Tian | Residual Fe enhances the activity of BiOCl hierarchical nanostructure for hydrogen peroxide activation[END_REF]. Taking BiOCl as catalyst, the photocatalytic process can supply a simple route to produce HO 2 • /O 2 •-effectively. In our last studies, a series of BiOCl related catalysts have been synthesized by precipitation method successfully followed with detailed characterization [START_REF] Wang | Low crystallized BiOCl 0.75 I 0.25 synthesized in mixed solvent and its photocatalytic properties under simulated solar irradiation[END_REF](Wang et al., , 2016a(Wang et al., , 2016b [START_REF] Andreozzi | Paracetamol oxidation from aqueous solutions by means of ozonation and H 2 O 2 /UV system[END_REF]), Fenton (de Luna et al., 2013), TiO 2 photocatalyst (Yang et al., 2008b) and persulfate activation [START_REF] Noorisepehr | Photo-assisted catalytic degradation of acetaminophen using peroxymonosulfate decomposed by magnetic carbon heterojunction catalyst[END_REF][START_REF] Yun | Enhanced degradation of paracetamol in water using sulfate radical-based advanced oxidation processes catalyzed by 3-dimensional Co 3 O 4 nanoflower[END_REF]. Its degradation pathway by HO [START_REF] Li | Mechanism of Photodecomposition of H 2 O 2 on TiO 2 Surfaces under Visible Light Irradiation[END_REF] or persulfate activation [START_REF] Xu | Persulfate activation towards organic decomposition and Cr(VI) reduction achieved by a novel CQDs-TiO 2-x /rGO nanocomposite[END_REF] has been widely investigated. However, rare studies focus on the synergistic effects between BiOCl with H 2 O or persulfate [START_REF] Zhang | Efficient Degradation of Aqueous Carbamazepine by Bismuth Oxybromide-Activated Peroxide Oxidation[END_REF].

In brief, the main goal of this paper is to compare, for the first time, three different active species HO

Materials and methods

Chemicals

BiOCl catalyst was synthesized as in our previous papers (Wang et al., 2016a

Irradiation setup and experimental procedure

The photocatalytic degradation experiments were performed in a homemade photoreactor placed in a cylindrical stainless steel container. Four fluorescent lightbulb lamps (Philips TL D15W/05) were separately placed in the four different axes while the photoreactor, a water-jacketed Pyrex tube of 2.8 cm internal diameter, was placed in the center of the setup. The lamp emitting from 300 to 500 nm was used to simulate UVA solar light; their emission spectrum is shown in Fig. S1. 100 mL of solutions were magnetically stirred with a magnetic bar during irradiation and experiments were carried out at room temperature (293 ± 2 K) set by a circulating cooling water system. Samples were taken from the reaction tube at fixed interval times and the catalyst solid was removed by 0.22 μm PTFE filters before analysis. In the experiments, the initial concentrations used were: ACTP 50 μM, H 2 O 2 and Na 2 S 2 O 8 1 mM and BiOCl catalyst 0.3 g L -1 (approximately 1.1 mM). All experiments were performed in duplicates, experimental points represent the averages of two values, and the error bars indicate the standard deviation.

Analytical method

The remaining ACTP concentration in the aqueous solution was determined by high performance liquid chromatography (HPLC) (Alliance, Waters, 2695, USA) equipped with a photodiode array detector (Waters 2998). A Nucleodur 100-3 C18 reverse column (150 × 2.0 mm, 3.0 μm) was used to separate the compound in the solution. The flow rate was 0.15 mL min -1 with a mobile phase mixture of methanol and water with 0.1% H 3 PO 4 (30/70, v/v). The UV detection of ACTP was set at 244 nm wavelength, and the retention time was 4.9 min. Mineralization of ACTP solution was followed by total organic carbon (TOC) analysis (5050A, Shimadzu, Japan).

UV-vis spectra were recorded with a Cary 300 UV-visible spectrophotometer. pH values of the solutions were measured using a Cyberscan 510 pH meter. The zeta potential of BiOCl was determined by the potentiometric titration method under N 2 atmosphere at 298 K in 1 mM NaCl solutions. X-ray photoelectron spectroscopy (XPS) spectra were collected by using a VG Scientific ESCALAB 250Xi spectrometer (Thermo Fisher Scientific) with C 1s as the calibration standard line.

The optical properties of BiOCl samples were analyzed through the UV-vis diffuse

Determination of the second order rate constant

For the detection of second order rate constant between ACTP and HO • (k ACTP, HO• ), LFP method was adopted. Experiments were carried out using the fourth harmonic (λ exc = 266 nm) of a Quanta Ray GCR 130-01 Nd: YAG laser system instrument and the energy was set as approximately 45 mJ/pulse. Other conditions were kept the same to those described in previous articles [START_REF] Brigante | Photochemistry of 1-Nitronaphthalene: a potential source of singlet oxygen and radical species in atmospheric waters[END_REF][START_REF] Huang | UVA-UVB activation of hydrogen peroxide and persulfate for advanced oxidation processes: Efficiency, mechanism and effect of various water constituents[END_REF]. High concentrated stock solutions (ACTP, H 2 O 2 and SCN -) were mixed just before each experiment and diluted with Mili-Q water to obtain the desired For the detection of second order rate constant of sulfate radical on ACTP (k ACTP, SO4•-), competition method was adopted by taking BPA as the competitor [START_REF] Ji | Photochemical degradation of sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid in different water matrices[END_REF], because k SO4•-,BPA =4.7×10 9 M -1 s -1 has been detected in previous studies by LFP [START_REF] Huang | UVA-UVB activation of hydrogen peroxide and persulfate for advanced oxidation processes: Efficiency, mechanism and effect of various water constituents[END_REF]. For this experiment, BPA (50 µM) and ACTP (50 µM)

were undergoing competition reactions under Na 2 S 2 O 8 (1 mM)/UVA at pH 5.4 for 240 min. At this pH conditions, sulfate radical prevailed with negligible hydroxyl radical existence (Fang et al., 2013a). The concentrations of BPA and ACTP were followed at different treatment time intervals and the rate constant of ACTP with SO 4 •-can be calculated through Eq.1. blank experiments were firstly conducted in the dark to check its stability. ACTP alone in aqueous solution is stable between pH 3-9.5 within 40 hours (Fig. S2A). In addition, when ACTP was mixed with H 2 O 2 and Na 2 S 2 O 8 (Fig. S2B,C), no detectable degradation can be observed until pH 9.0, while at higher pH a degradation was measured particularly with persulfate.

Degradation of ACTP with H 2 O 2 and Na 2 S 2 O 8 under UVA during irradiation are shown in Fig. 1, and all the reactions follow pseudo first order reaction kinetics (data are shown in Fig. 4C). From the control experiment, direct photolysis of ACTP could be neglected (Fig. 1A). Results of quenching experiments demonstrated that HO In UVA/H 2 O 2 system, slightly acidic solution has the maximum removal percentage rather than acidic and alkaline solutions, although the maximum removal is only approximately 10%. In contrast, degradation rate is increasing with pH further increasing (Fig. 1B) in UVA/Na 2 S 2 O 8 system, and about 57% ACTP can be removed at pH 10. At least two reasons are involved simultaneously to account for this phenomenon. Firstly, Fig. S2B has been taken into consideration, ACTP is easier to be oxidized in anionic form. Furthermore, alkaline solution favors more formation of HO • radicals from SO 4 •-than neutral pH (Eq.4-5) (Fang et al., 2013a), and these two radicals have different second order reaction rate constants with ACTP, which will be explained in the following part.

H O h HO • (2) S O -h SO 4 •- (3) SO 4 •-OH -HO • SO 4 - k = (6.5±1.0)×10 7 M -1 s -1 (4) SO 4 •-H O HO • HSO 4 - k = 11.9 M -1 s -1 (5)

Halide ions (chloride and iodide) effects on ACTP degradation in UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 systems

Effects of halide ions were widely studied in previous research works, especially chloride and bromide [START_REF] Tamtam | New insight into photo-bromination processes in saline surface waters: the case of salicylic acid[END_REF][START_REF] Li | The roles of halides in the acetaminophen degradation by UV/H 2 O 2 treatment: Kinetics, mechanisms, and products analysis[END_REF]. In our study for the first time the comparison of Cl -and I -, which is also commonly detected in costal seawater, saline water or industrial wastewater discharges, were investigated in UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 systems. In the dark and whatever the systems, the addition of Cl -has o impact o ACTP's concentration change (see Fig. S3). Under irradiation, the impact of Cl -is also very weak. The only effect is observed in the UVA/H 2 O 2 system, when NaCl is set as 10 mM, the pseudo first order reaction rate constant increase around 30% (see Fig. S4). In the presence of chloride ions, generated reactive species such as chlorine radical (Cl • ) and dichlorine radical anion (Cl 2 •-) could react more selectively with electron-rich organic compounds [START_REF] Grebel | Effect of halide ions and carbonates on organic contaminant degradation by hydroxyl radical-based advanced oxidation processes in saline waters[END_REF].

On the contrary, when iodide ions at same concentrations were added to the solution, ACTP degradation rates are strongly affected as shown in Fig. 2. In the dark, the addition of KI from 260 µM to 5 mM leads to an increase of the ACTP degradation with H 2 O 2 , which is much less the case with Na 2 S 2 O 8 at pH 5.4. In the presence of H 2 O 2 or Na 2 S 2 O 8 , iodide ions could be oxidized to iodine I 2 (Eq.6). Under UVA irradiation, KI alone in solution has no effect on ACTP (Fig. S5), indicating that iodide ions under UVA cannot transform or generate any active species. However, in the presence of H 2 O 2 or Na 2 S 2 O 8 in the KI solution, the photo-degradation rate is highly enhanced (Fig. 2). In addition, HOI •-/I • reactive halogenated species can be generated from HO • or SO 4 •-as shown in Eq.7-8 [START_REF] Elliot | A pulse radiolysis study of the reaction of OH with I 2 and the decay of I 2[END_REF], indicating that iodide radicals (I • , HOI •-and I 2 •-) have higher reactivity with organic pollutants than chloride radicals (Yang et al., 2018a). [START_REF] Tamtam | New insight into photo-bromination processes in saline surface waters: the case of salicylic acid[END_REF] and [START_REF] Li | The roles of halides in the acetaminophen degradation by UV/H 2 O 2 treatment: Kinetics, mechanisms, and products analysis[END_REF] have demonstrated that reactive halogenated species reacted more selectively than hydroxyl radicals with electron-rich organic pollutants. As ACTP contains hydroxyl and acetamido groups, which are electron donors to the aromatic rings, so it is hypothesized that these electron-rich moieties may selectively react with these iodide related radical reactive species. These selectivity properties of iodide based radicals are more emphasized than chloride ions. However, too much higher concentrated iodide ions (5 mM) will decrease the efficiency of ACTP degradation by self-quenching effects (Eq.9).

-

H O S O - HO-SO 4 - (6) SO 4 •-HO • -→ → HO •-• (Elliot, 1992) (7) • - • - k = 8.8×10 9 M -1 s -1 (Elliot, 1992) (8) • • k = 1.5×10 10 M -1 s -1 (Elliot, 1992) (9)

ACTP's degradation performance in BiOCl/UVA system

Heterogeneous photocatalytic degradation of ACTP has attracted numerous attentions recently, and various degradation pathways have been proposed for SO 4

•and HO • reactivity on ACTP. On the contrary, seldom studies have investigated the degradation of ACTP predominantly by superoxide radicals. In our previous studies (Wang et al., 2016a), BiOCl catalyst has been synthesized following with detailed characterization properties, as shown in Table 1. The XPS characterization is presented in Fig. S6, the valance state of each element is in accordance with previous literatures, suggesting that BiOCl is well crystal organized with high purity. With a relatively wide band-gap (3.2 eV), BiOCl could hardly absorb the light wavelengths longer than 400 nm (Fig. S7A,B). The valance band of BiOCl determined from XPS spectra is 2.2 eV (Fig. S7C), which is not positive enough to oxidize H 2 O to form hydroxyl radicals (E 0 (H 2 O/HO • ) = 2.8 V vs NHE) (Wu et al., 2015b). Accordingly, the conduction band position can be calculated to be -1 eV, that is sufficiently negative enough to combine with dissolve oxygen to generate HO 2 (Wu et al., 2015b). Consequently, UVA/BiOCl is adopted to produce HO 2

• /O 2 •-(E 0 (O 2 /O 2 •- ) = -0.28 V vs NHE)
• /O 2 •-radicals, which is further confirmed by quenching experiments (Fig. 3A). In fact, no inhibition is obtained in the presence of TBA (1 mM) and KI (1 mM), which are traps of hydroxyl radicals and holes. Strong inhibition is noted with BQ (2 mM) which is able to trap also superoxide radicals. Furthermore, the obvious inhibition in N 2 purging condition further verifies that HO 2 • /O 2 •-originate from the reaction between dissolved oxygen and electrons.

Before irradiation, adsorption capacity of the catalyst is tested and the results

show that less than 10% of ACTP are adsorbed whatever the pH. Under irradiation, pH effects are investigated and strong increase of the ACTP degradation is observed at pH 3.0 if we compare with pH 5.8 and 7.5 (Fig. 3B). These results are consistent with previous reports about BiOX used as photocatalyst [START_REF] Gao | Photocatalytic degradation of carbamazepine using hierarchical BiOCl microspheres: Some key operating parameters, degradation intermediates and reaction pathway[END_REF][START_REF] Liang | Bactericidal mechanism of BiOI-AgI under visible light irradiation[END_REF] and the speciation of HO 2 • /O 2 •-(pKa of 4.88) can explain the effect of pH (Li et al., 2017). Indeed, the second order rate constant between O 2 •-with phenol is 5.8×10 2 M -1 s -1 [START_REF] Tsujimoto | Superoxide radical scavenging activity of phenolic compounds[END_REF] and Kozmér et al. have demonstrated that in acidic solutions, HO 2 • shows a higher reactivity with phenol 2.7×10 3 M -1 s -1 [START_REF] Kozmér | Determination of the rate constant of hydroperoxyl radical reaction with phenol[END_REF], which is reasonably similar for phenolic compound ACTP. Furthermore, Zeta-potential of BiOCl is more negative with pH increasing (see Fig. 3C), so less photo-induced electrons can transferred to the surface to combine with oxygen because electrostatic repulsion. While, as discussed above, electrons are crucial to

form HO 2 • /O 2 •-.
In terms of organic compounds mineralization, UVA/BiOCl shows excellent degradation and mineralization abilities compare to H 2 O 2 or Na 2 S 2 O 8 whatever the solution's pH (Fig. 3D).

Heterogeneous interfacial mechanism in UVA/BiOCl

For heterogeneous photocatalytic degradation, interfacial mechanism is essential to be investigated to confirm if the reaction is occurring in the solution or on the surface of catalyst. To clarify this question, NaH 2 PO 4 has been proved to be a perfect desorbent in our experiment. Because NaH 2 PO 4 could occupy almost all the adsorption sites on the surface of catalyst, and the surface was tightly surrounded by NaH 2 PO 4 molecules instead of ACTP [START_REF] Wang | Bismuth catalyst mediated degradation of p-hydroxyphenylacetic acid: Photoactivation, interfacial mechanism, and influence of some critical parameters[END_REF]. With the addition of NaH 2 PO 4 , the degradation rate and removal percentage are both strongly suppressed (Fig. 4A). As we discussed above, the main active species HO 2 • /O 2 •-comes from the combination of dissolved oxygen in water and conduction band electrons. With a very short half-life time, HO 2 • /O 2 •-would easily undergo disproportionation [START_REF] Liang | Bactericidal mechanism of BiOI-AgI under visible light irradiation[END_REF]. Consequently, the fast and direct contact of pollutant and catalyst surface is crucial for the reaction. Furthermore, BiOCl nanosheets prepared in alkaline conditions in this research were usually dominated with high [010] facets exposure.

The [010] facets possessed open channels and abundant unsaturated Lewis/base sites, which was helpful for intimate contact between the interfacial surface and organic molecules [START_REF] Jiang | Synthesis and facet-dependent photoreactivity of BiOCl single-crystalline nanosheets[END_REF]. So in this photocatalytic degradation system, degradation reactions must be occurred on the surface or very close to the surface of the catalyst. 4.63 × 10 -5 s -1 ) and UVA/BiOCl (k = 1.42 × 10 -4 s -1 ) through accelerating ACTP's removal and mineralization rate (Fig. 4B,3D). The increased degradation can be ascribed to the following reasons. Firstly, the direct photolysis of S 2 O 8 2-generating SO 4

Combination effects between H

•-could contribute to the oxidation of ACTP. Secondly, S 2 O 8 2-also can trap the photogenerated conduction band electrons result in the formation of SO 4

•-(Eq.10) [START_REF] Sarwan | Mineralization and toxicity reduction of textile dye neutral red in aqueous phase using BiOCl photocatalysis[END_REF]. For the third reason, previous research has demonstrated the reaction of O 2 •-with persulfate anion to generate SO 4 •-according to Eq.11 (Fang et al., 2013b). However, addition of H 2 O 2 slows down the degradation rate associated with TOC removal (Fig. 4B, C and 3D). The higher O-O bond energy of H 2 O 2 compared with the band in free persulfate ions under natural pH could explain why persulfate is easier to be activated than H 2 O 2 by the same ROS (reactive oxygen species) [START_REF] Zhang | Efficient Degradation of Aqueous Carbamazepine by Bismuth Oxybromide-Activated Peroxide Oxidation[END_REF].

S O -e -SO 4 •-SO 4 k = 1.2×10 10 M -1 s -1 [START_REF] Buxton | Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (•OH/•O-) in aqueous solution[END_REF] (10)

S O -O •- SO 4 •-+ SO 4 -O (11)
ESR spectrum was performed to confirm the active species formed in this system (Fig. 5). No signal was detected in UVA/BiOCl system even repeat for several times or with higher catalyst loading. This result is consistent with the results in paragraph 3. solutions, which all contribute to higher mineralization abilities as shown in Fig. 3D. ) and radical's concentrations. k ACTP,HO• was determined from Eq.S1-S4 and Scheme S1, by following the absorbance of SCN 2 •-at 475 nm using different ACTP concentrations. The absorbance decreased from 0.021 to 0.006 when concentration of ACTP increases from 0 to 2.33×10 -4 M (Fig. S8A), because ACTP compete with SCN -to react with HO • . The slope "a" ca be obtained from the linear regression of Abs 0 /Abs versus different ACTP concentrations, and it was equal to 11276. Thus the second order constant k ACTP,HO• ca be evaluated from the product of "a" concentration of SCN -(1×10 -4 M) and k HO• SCN-=1.2×10 10 M -1 s -1 [START_REF] Motohashi | Competitive measurement of rate constants for hydroxyl radical reactions using radiolytic hydroxylation of benzoate[END_REF], finally it was calculated to be 1.35×10 10 M -1 s -1 .

Determination of second order reaction rate constant between ACTP and HO

From competition method (Fig. S8B), k SO4•-,ACTP =6.44×10 9 M -1 s -1 was determined. For k HO •/O •-,ACTP , reference's results, already mentioned in part 3.3

(5.8×10 2 M -1 s -1 for O 2 •-and 2.7×10 3 M -1 s -1 for HO 2

•

) are took into account and because it has similar oxidation abilities towards phenolic compounds.

The steady state concentration of each radical ( can be estimated from the pseudo first degradation constant of ACTP in three systems determined in Fig. S8C. Considering that the pseudo first-order kinetic constant ( ', s -1 ) is equal to , we can estimate the steady state concentration of each radical as , where is the second order rate constant between ACTP and selected radical. Under adopted experimental conditions, a steady state concentration of HO • and SO 4 •-was determined to be 1.0×10 -15 and 7.2×10 -15 M respectively while for HO 2 • /O 2 •-was in the range 10 -8 -10 -7 M (Table 2).

Although HO

  reflectance (DRS) by Shimadzu UV-3600. The electron spin resonance (ESR) experiments were carried out on a JES-X320 spectrometer to detect the reactive radicals generated over the photocatalytic degradation process. As radical probe, DMPO (100 mM) was used for SO 4 •-, HO • and HO 2 • /O 2 •-. ESR spectrum was recorded at room temperature under the following operating conditions: a modulation frequency of 100 kHz, a sweep width of 0.5 mT, a microwave power of 1 mW, a microwave frequency of 9.15 GHz and a centerfield of 326.0 ± 5 mT.

  mixtures and concentrations in a 3 mL quartz cuvette. The value of k ACTP, HO• can be obtained through mixing SCN -(0.1 mM), H 2 O 2 (5.5 mM) with different concentrations of ACTP in the quartz cell. The detection wavelength was set at 475 nm to detect maximum absorbance of SCN 2 •-. The specific detection calculation was shown in Scheme S1 and Equations S1-S4.

ACTP degradation in UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 systems under different pH

  To explore ACTP's degradatio with H 2 O 2 and Na 2 S 2 O 8 under UVA irradiation,

	ACTP	•-	PA	•-	l	ACTP t ACTP 0	PA t	(1)
							PA 0
	3 Results and Discussion
	3.1

  No direct photolysis of ACTP was observed under UVA radiation. With H 2 O 2 or Na 2 S 2 O 8 addition to the solution, ACTP shows different degradation kinetics under various pH. In general, UVA/Na 2 S 2 O 8 shows a higher degradation efficiency than UVA/H 2 O 2 system whatever the solution's pH. It has been reported that H 2 O 2 only can be activated by the light at λ < 320 nm, which almost have been confirmed in this research for the weak degradation in UVA/H 2 O 2[START_REF] Li | Mechanism of Photodecomposition of H 2 O 2 on TiO 2 Surfaces under Visible Light Irradiation[END_REF]. The obvious degradation of ACTP in UVA/Na 2 S 2 O 8 indicates that Na 2 S 2 O 8 is easier to be activated than H 2 O 2 under UVA irradiation.

		• and
	SO 4	•-are the predominant radicals involved in the UVA/H 2 O 2 and UVA/Na 2 S 2 O 8
	system (Eq.2-3) at pH 5.4 respectively by taking IPA (10 mM) and EtOH (1 M) as
	HO

• (k IPA, HO• =1.9×10 9 M -1 s -1 )

[START_REF] Wu | Activation of persulfate by Fe(III) species: Implications for 4-tert-butylphenol degradation[END_REF]

and SO 4 •-(k EtOH, SO4•-=1.6-7.7×10 7 M -1 s -1 )

[START_REF] Tan | Kinetic study of acetaminophen degradation by UV-based advanced oxidation processes[END_REF]

scavengers, respectively.

2 O 2 , Na 2 S 2 O 8 and BiOCl under UVA Degradation

  of ACTP is investigated in H 2 O 2 /BiOCl/UVA and Na 2 S 2 O 8 /BiOCl/UVA systems to explore the possible synergy effects between H 2 O 2 or Na 2 S 2 O 8 and BiOCl, as shown in Fig.4B, C. It is obvious that UVA/BiOCl/Na 2 S 2 O 8 (k = 7.13 × 10 -4 s -1 ) exhibit strong synergy effects compared with UVA/Na 2 S 2 O 8 (k =

  radical has the weakest oxidation capacities, its concentration is 7-8 orders of magnitude higher than HO• and SO 4 •-radicals, thus directing the fast degradation and mineralization efficiency of ACTP. From the view of economic, H 2 O 2 and Na 2 S 2 O 8 are cheaper than the as-prepared BiOCl. But, it's essential to note that, H 2 O 2 and Na 2 S 2 O 8 are gradually consumed in solution under irradiation and as a contrary, BiOCl catalyst, which is stable and can be preserved after catalytic reaction, has the advantage of easy recovery and so to be recyclable. In summary, heterogeneous photocatalytic process UVA/BiOCl was always more efficient than the homogeneous UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 processes to degrade ACTP whatever the solution's pH. Halide ions such Cl -plays a negligible role in UVA/H 2 O 2 and UVA/Na 2 S 2 O 8 systems, while I -could significantly improve the degradation performance due to the reactive species formation. BiOCl catalyst can be efficiently activated by solar light than traditional oxidant such as H 2 O 2 and Na 2 S 2 O 8 . Addition of Na 2 S 2 O 8 shows synergistic effects in UVA/BiOCl compared to the addition of H 2 O 2 . This research could provide implications in heterogeneous photocatalyst utilization, and shed light on capable combinations between photocatalyst and oxidants in future wastewater treatment under solar light irradiation.

	2 With the same amount of oxidant or catalyst loading, UVA/BiOCl system could • /O 2 produce higher concentration of HO 2 • /O 2 •-radicals, compared to HO • and SO 4 •-. This surface-depended HO 2 • /O 2 •-oxidation process could induce significant mineralization •-Conclusion abilities.

Acknowledgement

Financial support from the National Natural Science Foundation of China (No. 21875153 and 21906111), the China Postdoctoral Science Foundation (2019M661933), Natural Science Research of Jiangsu Higher Education Institutions (19KJB610006), and the Young Thousand Talented Program are greatly appreciated. The authors gratefully acknowledge financial support from China Scholarship Council for Xiaoning Wang to study at the University Clermont Auvergne, France.

Authors acknowledge financial support from the Region Council of Auvergne, from the "Féderation des Recherches en Environnement" through the CPER "Environment" founded by the Region Auvergne, the French government, FEDER from the European Community from PRC program CNRS/NSFC n°270437 and from CAP 20-25 I-site project.


Marco Molinaro
email: marco.molinaro@inaf.it

Mark G Allen

Sara Bertocco

Catherine Boisson

François Bonnarel

Margarida Castro Neves

Markus Demleitner

Françoise Genova

Dave Morris

André Schaaff

Giuliano Taffoni

Stelios Voutsinas

Integrating the VO Framework in the EOSC

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The European Open Science Cloud (EOSC 1 with metadata catalogues to higher level structures. In the astrophysical domain already exists an open approach to science data: the Virtual Observatory view put in place by the International Virtual Observatory Alliance (IVOA 2 ) architecture of standards. The ESCAPE (European Science Cluster of Astronomy & Particle physics ESFRI research infrastructures 3 ) project has, among its tasks, to demonstrate that the VO architecture can be integrated within the EOSC building one and to provide guidelines to ESFRI partners (European Strategy Forum on 1 https://ec.europa.eu/research/openscience/index.cfm?pg=open-science-cloud) is starting to build up, from its portal 2 http://ivoa.net/ 3 https://projectescape.eu/ 1 Research Infrastructures) in doing this. This contribution reports on the progress of this integration after the first months of work inside ESCAPE. After a quick overview of the EOSC goals (Sec. 2) and a brief description of the ESCAPE WP4 Task-4.1 (Sec. 3), the contribution summarises the planned and ongoing activities (Sec. 4) and quickly describes the nect steps (Sec. 5).

The EOSC

Over the past years, numerous policy makers from around the world have articulated a clear and consistent vision of global Open Science as a driver for enabling a new paradigm of transparent, data-driven science. In Europe, this vision is being realised through the European Open Science Cloud (EOSC). The EOSC will offer a virtual environment with open and seamless services for storage, management, analysis and re-use of research data, across borders and scientific disciplines by federating existing scientific data infrastructures, currently dispersed across disciplines and the EU Member States. In To reach its goals the task members will focus on four main points:

• interfacing the VO framework with the EOSC;

• build an Astronomy portfolio of VO services;

• contributing to the EOSC Hybrid Cloud;

• domain-specific services.

Planning and ongoing work for Task-4.1 is detailed in the nect section (Sec. 4, while Fig. 2 VO mapping onto the EOSC data lifecycle.

Planned Activities for CEVO Task 4.1

Activities for Task-4.1 have been planned among the Task participants and reported also in the first deliverable document of the CEVO WP. They can be roughly summarised as:

• Inclusion of the VO Registry into the EOSC service catalogues;

• assessment of the methods for contributing an Astronomy Portfolio to the EOSC Marketplace;

• study of accessing VO-compliant data and services using science analysis platforms (in coordination with WP5);

• Assessment of the possibility to bring existing VO standards for data sharing (VOSpace) within the EOSC services;

• Identification of existing VO services or tools to serve as test-cases for containerisation.

In the first months, that included some ramp up and preparation of the work, a few points have been already worked out, namely:

• VO Resource mapping against EUDAT B2-FIND metadata has been proven possible, first step on Registry inclusion in EOSC;

• contact points have been identified for catalogue integration and service portfolio;

• first steps in identifying a VOSpace back-end storage solution to allow VO standard data sharing;

• preliminary identification of the services and tools to be used in containerisation tests.

Among the latter, the most advanced activity is the one on the IVOA Registry Resources inclusion in the EOSC catalogue. The metadata mapping of VO resources to the DataCite-based5 EUDAT catalogue service has been done and proven working. Give it a try through the EUDAT B2-FIND service itself6 .

Further Steps

The deliverable document for the work plan of the CEVO package includes a list of activities to be brought on. This is an help to cross-check the progress of the activities.

For Task-4.1 next steps to pursue include consolidating the contact points in the EOSC architecture to pave the way to ESFRIs resources inclusion, inclusion of established VO services and technologies within the EOSC Hybrid Cloud and containerisation, but also bringing feedback to the EOSC building projects and stakeholders symposia.

  Fig. 1 a representation of the EOSC Model action lines as available from the Implementation Roadmap for the European Open Science Cloud document 4 .

Figure 1 .

 1 Figure 1.EOSC roadmap: action lines from the implementation document.

  shows a graphical representation of how the VO framework and technologies can be mapped onto the EOSC data lifecycle blocks (the diagram is taken from the ESCAPE's proposal Document of Work for the CEVO WP detailed description).

  Figure 2.VO mapping onto the EOSC data lifecycle.

https://ec.europa.eu/research/openscience/pdf/swd_2018_83_f1_staff_working_ paper_en.pdf

DataCite Metadata Schema: https://schema.datacite.org/

IVOA is listed as one of the communities in it: http://b2find.eudat.eu/group?q=ivoa&sort= title+asc

Acknowledgments. Authors acknowledge support from ESCAPE project (the European Science Cluster of Astronomy & Particle Physics ESFRI Research Infrastructures) that has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement n. 824064.