The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.

ABSTRACTColorectal cancer (CRC) is becoming one of the most prevalent cancers worldwide. Among cancers, it ranks the third place in terms of incidence and the second in terms of mortality. Even though immunological test allows fast and easy diagnostic method, there is no specific and reliable methods for early detection of CRC. Despite different treatments, high risk of re-occurrence is associated with advanced and metastatic CRC stages. An exhaustive knowledge on specific biomarkers or molecular actors involved in CRC could help to eradicate tumors or limit cancer recurrence. In this study, we focused on PAMR1 (Peptidase Domain Containing Associated with Muscle Regeneration 1), which is already considered as a tumor suppressor in breast and cervical cancers.In silicoanalysis of RNASeq data showed that PAMR1 was significantly downregulated in CRC tissues compared to their adjacent normal ones, as well as in cervical cancer. Our analysis showed that this downregulation, probably due to promoter hypermethylation, such as in breast cancer tissues, appeared in the four cancer stages as early as the first stage. In consistency within silicoanalyses, the expression of PAMR1 was found to be lower at the transcript and protein levels in CRC tissue samples compared to normal ones, as well as in different CRC cell lines (HCT116, HT29, and SW620) compared to normal colon cell line (CCD841CoN). To understand the role of PAMR1 in CRC cancer, recombinant purified PAMR1 or concentrated secretome from CHO overexpressing PAMR1 were used to exogenously treat CRC cell lines with a focus on HT-29 cells as well as Hela cervical cancer cell line known to be sensitive to PAMR1. Transient or stable transfections were also performed to determine the impact of PAMR1 overexpression in HT29 and/or HeLa cells. In this study, we finally showed that presence of PAMR1 could reduce both cell proliferation and cell migration with a positive correlation between these biological effects and PAMR1’s quantity. This implies that PAMR1 expresses anti-proliferative and anti-migrative effects in CRC. Further studies to be done in order to confirm the tumor suppressive role of PAMR1 in CRC.

Tumor cells have a modified glycosylation profile that promotes their evolution and/or their maintenance in the tumor. Sialylation is a type of glycosylation that is often altered in cancers. RNA-Seq database analysis revealed that the sialyltransferase gene ST3GAL2 is significantly overexpressed at all stages of colorectal cancer (CRC). ST3GAL2 sialylates both glycoproteins and glycolipids. The aim of this work was to investigate the involvement of ST3GAL2 in CRC. Using the HT29 tumor cell line derived from a stage II of CRC, we decreased the expression of ST3GAL2 by specific shRNA, and then characterized these cells by performing functional tests. We found that ST3GAL2 knock down (KD) significantly decreases tumor cell proliferation, cell migration and invasiveness properties in vitro. The cell cycle of these cells is affected with a change in cell cycle distribution and an increase of cell apoptosis. The effect of ST3GAL2 KD was then studied in vivo, following xenografts into nude mice, in which the tumor progression was significantly reduced. This work demonstrates that ST3GAL2 is a major player in the behavior of colorectal tumor cells, by modifying the sialylation state of glycoproteins and glycolipids which remain to be specifically identified.

The arrival of Neolithic culture in North Africa, especially domestic animals has been essentially documented from archaeological records. As the data relative to sheep are scarce, we studied the genetic relationship between Moroccan sheep breeds and Mediterranean ones using the sequencing of 628 bp of the mitochondrial DNA control region in 193 Moroccan individuals, belonging to six breeds, and 652 sequences from other breeds in Europe and Middle East. Through Network analysis and an original phylogenetically derived method, the connection proportions of each Moroccan breed to foreign ones were estimated, highlighting the strong links between Moroccan and Iberian breeds. The first founders of Moroccan sheep population were issued at 79% from Iberia and 21% from a territory between Middle East and Africa. Their calculated expansion times were respectively 7,100 and 8,600 years B.P. This suggests that Neolithization was introduced by a double influence, from Iberia and from another route, maybe Oriental or Sub-Saharan. The consequence of the environmental changes encountered by founders from Iberia was tested using different neutrality tests. There are significant selection signatures at the level of Moroccan and European breeds settled in elevated altitudes, and an erosion of nucleotide diversity in Moroccan breeds living in arid areas.

The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.

Background: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. Methods: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. Interpretation: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.

A hundred of human proteins have one or more EGF-like domains (EGF-LD) bearing the O-fucosylation consensus motif C2X4(S/T)C3 but to date, only a few of them have been shown to be O-fucosylated. The protein O-fucosyltransferase (POFUT1) specifically recognizes correctly folded EGF-LD of the human EGF (hEGF) type and transfers fucose on serine or threonine residue within the O-fucosylation motif. Here, we propose a strategy for a rapid screening for ability of any EGF-LD to be O-fucosylated, using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By an oligonucleotide hybridization approach, double-stranded fragments encoding any EGF-LD can be first rapidly cloned into the prokaryotic vector pET-25b to promote its targeting to periplasm and formation of the three conserved disulfide bonds. After protein production and purification, an in vitro POFUT1-mediated O-fucosylation can be performed with azido GDP-fucose. Successful transfer of O-fucose is finally revealed by blotting technique after CuAAC. In this study, we specially focused on mouse NOTCH1 EGF12 and EGF26, which are both known to be O-fucosylated although having different binding affinities towards POFUT1. Indeed, we clearly showed here that addition of O-fucose by POFUT1 was much more efficient for EGF26 than for EGF12. This experimental approach is rapid and sufficiently sensitive to reveal propensity of any EGF-LD to be O-fucosylated; it is thus useful prior to perform structure-function studies on target proteins containing one or several EGF-LD.

Myogenesis is a physiological process which involves the proliferation of myoblasts and their differentiation into multinucleated myotubes, which constitute the future muscle fibers. Commitment of myoblasts to differentiation is regulated by the balance between the myogenic factors Pax7 and MyoD. The formation of myotubes requires the presence of glycans, especially N‐glycans, on the cell surface. We examined here the involvement of α2,6 sialylation during murine myoblastic C2C12 cell differentiation by generating a st6gal1‐knockdown C2C12 cell line; these cells exhibit reduced proliferative potential and precocious differentiation due to the low expression of Pax7. The earlier fusion of st6gal1‐knockdown cells leads to a high fusion index and a drop in reserve cells (Pax7+/MyoD−). In st6gal1‐knockdown cells, the Notch pathway is inactivated; consequently, Pax7 expression is virtually abolished, leading to impairment of the proliferation rate. All these results indicate that the decrease in α2,6 sialylation of N‐glycans favors the differentiation of most cells and provokes a significant loss of reserve cells.

Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po –) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po – cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po –, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po –. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.

Colorectal cancer (CRC) is a major health burden that can be well managed if the tumour is detected at an early stage. To identify novel biomarkers for early diagnosis, an increasing number of studies have focused on gene expression dysregulations in the colon and rectal cancers, as in CRC cell lines. Several studies revealed that the 20q11.21 chromosomal region is amplified in at least 65% of CRC cases [[1]Carvalho B. Postma C. Mongera S. Hopmans E. et al.Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression.Gut. 2009; 58: 79-89Google Scholar]. Such a genomic instability leading to amplification is also found in numerous other cancer types, including breast cancer, lung cancer, melanoma, and numerous others [[2]Lefort N. Feyeux M. Bas C. Féraud O. et al.Human embryonic stem cells reveal recurrent genomic instability at 20q11.21.Nat Biotechnol. 2008; 26: 1364-1366Google Scholar]. The shortest amplified portion associated with acute myeloid leukemia only contains four genes including pleomorphic adenoma gene-like 2 (PLAGL2) and protein O-fucosyltransferase 1 (POFUT1) [[3]Mackinnon R.N. Selan C. Wall M. Baker E. Nandurkar H. Campbell L.J. The paradox of 20q11.21 amplification in a subset of cases of myeloid malignancy with chromosome 20 deletion.Genes Chromosomes Cancer. 2010; 49: 998-1013Google Scholar]. Their expressions have been particularly investigated in the context of CRC by the team of Xiaorong Li, published in the July issue of EBioMedicine [[4]Li D. Lin C. Li N. Du Y. et al.PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemness.EBioMedicine. 2019; (pii:S2352–3964(19)30432–3. Available from:)https://doi.org/10.1016/j.ebiom.2019.06.051Google Scholar]. PLAGL2 is a zinc-finger transcription factor located in the nucleus, whose expression shown to be significantly higher in CRC tissues than in adjacent non-tumour tissues [[5]Liu B. Lu C. Song Y.X. Gao P. et al.The role of pleomorphic adenoma gene-like 2 in gastrointestinal cancer development, progression, and prognosis.Int J Clin Exp Pathol. 2014; 7: 3089-3100Google Scholar]. In intestinal organoid cultures, PLAGL2 has shown to drive intestinal epithelial stem cell expression signature, which then activates WNT gene expression and enhances TCF/LEF reporter. Overall, PLAGL2 overexpression in CRC promotes in vitro and in vivo cell proliferation by the Wnt/β-catenin pathway [[6]Li N. Li D. Du Y. Su C. et al.Overexpressed PLAGL2 transcriptionally activates Wnt6 and promotes cancer development in colorectal cancer.Oncol Rep. 2019; 41: 875-884Google Scholar]. POFUT1 is an ER-resident glycosyltransferase that allows fucose addition on S or T included in the C2X4(S/T)C3 consensus motif, where C2 and C3 are the second and third cysteines out of six conserved in EGF-like domain. The major known target of POFUT1 is NOTCH receptor, which contains 14 to 20 O-fucosylation consensus sites according to the four human paralogs; most of the 20 sites on NOTCH1 are O-fucosylated [[7]Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev Cell. 2017; 40: 193-201Summary Full Text Full Text PDF PubMed Scopus (103) Google Scholar]. POFUT1 is overexpressed in colorectal tumours compared to non-tumour adjacent tissues and positively regulates colorectal tumour progression through activation of Notch signaling pathway [[8]Du Y. Li D. Li N. Su C. et al.POFUT1 promotes colorectal cancer development through the activation of Notch1 signaling.Cell Death Dis. 2018; 9: 995Google Scholar,[9]Chabanais J. Labrousse F. Chaunavel A. Germot A. Maftah A. POFUT1 as a promising novel biomarker of colorectal cancer.Cancers. 2018; 10: 411Google Scholar]. Both PLAGL2 and POFUT1 overexpression in CRC can be associated to copy number amplification. In the article by Li and colleagues [[4]Li D. Lin C. Li N. Du Y. et al.PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemness.EBioMedicine. 2019; (pii:S2352–3964(19)30432–3. Available from:)https://doi.org/10.1016/j.ebiom.2019.06.051Google Scholar], several experimental data are given to support the hypothesis that their correlated expression is driven by the activity of a bidirectional promoter. First, the authors showed that the adjacent head-to-head genes are separated by a short promoter sequence that is evolutionarily conserved in mammals. In 2015, the downregulation of Pofut1 in Plagl2−/− mice had already led Chen and colleagues [[10]Chen A.C. Donovan A. Ned-Sykes R. Andrews N.C. Noncanonical role of transferrin receptor 1 is essential for intestinal homeostasis.Proc Natl Acad Sci U S A. 2015; 112: 11714-11719Google Scholar] to consider that the two genes likely share a common promoter region. Interestingly, the conservation of the bidirectional promoter would mean that under physiological conditions, coordinated regulation of both genes' expression is essential, at least in mammals. This opens the possibility to reproduce the amplification of the two genes in CRC animal models i.e. mouse and to test the efficiency of modification of the transcription factor binding sites to attenuate the overexpression of the two genes and thus carcinogenicity, without altering their essential physiological functions [[4]Li D. Lin C. Li N. Du Y. et al.PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemness.EBioMedicine. 2019; (pii:S2352–3964(19)30432–3. Available from:)https://doi.org/10.1016/j.ebiom.2019.06.051Google Scholar]. Next, the demonstration of the bidirectional activity of the promoter was done in vitro using reporter gene constructs and CRC cell lines. Upon silencing PLAGL2, POFUT1 or both in HCT116 and SW480 cells, the authors were able to show that downregulation of the gene pair synergistically reduced colorectal tumourigenesis. Further studies will be required to understand the mechanism leading to this co-expression and biological implications within the tumour environment. Indeed, the tumour tissue is complex because of its cellular heterogeneity compared to a cell line. Tumour is certainly not subjected to the same potential epigenetic regulations, nor to the expression of the same transcription factors compared to cancer cell lines. Another interesting finding is that the correlated overexpression of PLAGL2 and POFUT1 initiates deregulation of the two signaling pathways, Wnt/β-catenin and Notch, and subsequently a deregulation of the cell cycle and tumour progression [[4]Li D. Lin C. Li N. Du Y. et al.PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemness.EBioMedicine. 2019; (pii:S2352–3964(19)30432–3. Available from:)https://doi.org/10.1016/j.ebiom.2019.06.051Google Scholar]. Further studies are needed to specify the respective contributions of Wnt/β-catenin and Notch signaling in promoting CRC and in maintaining the stemness of CRC stem cells. This study also raises the question of whether, for other cancers, the two genes cooperate in the same way as in the CRC. While it is fairly easy to imagine that overexpression of a transcription factor, like PLAGL2, may modulate several gene expressions, it is a bit more complicated to figure out how overexpression of POFUT1 encoding an O-fucosyltransferase, modifies the expression of genes that affect the cell cycle and the cellular proliferation and invasion. It is widely accepted that O-fucosylation of Notch, by POFUT1, affects Notch signaling. However, it remains to be determined what is the O-fucosylation state of NOTCH receptors in the colorectal tumour. Moreover, NOTCH receptors are not the only targets of POFUT1 and it is not excluded that other O-fucosylated actors participate in the consequences of POFUT1overexpression in CRC. Finally, this work contributes to the understanding of the mechanisms involved in tumour progression and to the identification of potential biomarkers. In connection with human health, it is rather conceivable to use this type of data for the purpose of improving diagnostics, but it seems more premature to project on a therapeutic approach focusing on the bidirectional promoter region. The authors declare no conflict of interest. PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer by maintaining stemnessOur team identified for the first time a bidirectional promoter pair oncogene, PLAGL2-POFUT1, in CRC. The two genes synergistically promote the progression of CRC and affect the characteristics of CSCs, which can offer promising intervention targets for clinicians and researchers. Full-Text PDF Open Access

Background: While protein O-fucosyltransferase 1 (POFUT1) overexpression has been recently proposed as a potential biomarker for different cancer types, no study was carried out on POFUT1 implication in colorectal cancer (CRC). Methods: Data from 626 tumors and 51 non-tumor adjacent tissues available in FireBrowse had been used in this study. Statistical analyses on POFUT1 expression and gene copy number, NOTCH receptors (main targets of POFUT1 enzymatic activity) expression and association of POFUT1 and NOTCH1 expressions with clinical parameters were investigated. Data were completed by POFUT1 histological labeling on six tumor tissues from patients with CRC. Results: We found that POFUT1 is overexpressed from the stage I (p < 0.001) and 76.02% of tumors have a 20q11.21 amplification, associated in 90.13% of cases with a POFUT1 overexpression, compared to non-tumor adjacent tissues. The POFUT1 copy number in tumors is mainly between 2 and 3. POFUT1 is positively correlated with NOTCH1 (rs = 0.34, p < 0.001), NOTCH3 (rs = 0.087, p = 0.0297), and NOTCH4 (rs = 0.097, p = 0.0148) expressions, while negatively correlated with NOTCH2 expression (rs = −0.098, p = 0.0142). POFUT1 overexpression is markedly associated with rectal location, non-mucinous adenocarcinoma and cancer stages IV and M1. NOTCH1 overexpression is only associated with rectal location and non-mucinous adenocarcinoma. Conclusion: We conclude that POFUT1 is overexpressed in CRC from stage I, and its high expression is associated with metastatic process, probably through NOTCH pathway activation. Then, POFUT1 could represent a potential novel biomarker for CRC diagnosis.

The protein O-glucosyltransferase 1 (Poglut1) links O-glucose to epidermal growth factor-like repeats harboring the C1XSX(P/A)C2 consensus sequence. Poglut1 is a ubiquitous endoplasmic reticulum-resident protein largely found in metazoans, but only about 50 proteins possess this consensus sequence. Among them, Notch receptors have multiple O-glucosylation sites and their activation depends on this status. In adult skeletal muscle, Notch signaling contributes to the maintenance of satellite cell (SC) quiescence and the proliferation of myoblasts after SC activation. To address the role of Poglut1 in myogenesis, we created two stable C2C12 cell lines where Poglut1 was downexpressed by 42% and 81%, and assessed their ability to differentiate. We showed that Poglut1 knockdown reduced Notch signaling and largely affected the key regulators of myogenic differentiation, with PAX7 decrease and MYOD increase. This perturbed Pax7/MyoD expression balance led to a premature myogenic differentiation and an increase in myotube size, accentuated in case of strong Poglut1 downexpression. Differences observed between myotubes of the two Poglut1 knockdown cell lines could reflect dissimilar fusion defects. We concluded that Poglut1 contributes to myogenesis by regulating Notch signaling and defining, directly or indirectly, the proportion of cells that commit differentiation.

Les ouvrages de la collection « Mini manuels » presentent sous une forme concise et attractive (2 couleurs, nombreux schemas) les notions essentielles. Le cours est illustre par des encarts apportant des complements techniques. En fin de chapitre, un rappel des points clefs, des exercices, des QCM ou des QROC, tous corriges, permettent de tester ses connaissances et de s'entrainer avant l'epreuve. Cet ouvrage presente les connaissances de base sur la structure des acides nucleiques, sur les processus de replication et de reparation de ces molecules, et sur le role de l'ADN et de l'ARN dans le fonctionnement cellulaire. Dans cette nouvelle edition, actualisee, une partie des exercices a ete renouvelee.

There are seven main sheep breeds in Algeria, partly investigated by their polymorphism using microsatellite and SNP markers by recent authors. The aim of this work is to determine the phylogenetic origin of the three breeds endemic to this country, e.g. Ouled Djellal, Rembi and Berbere, using the sequences of the whole Control Region of mitochondrial DNA. These three breeds are thin-tailed: Ouled Djellal or Arabic sheep of the steppe is the most important breed in Algeria in term of population size; Rembi or sheep of the Saharan Atlas differs from the previous one by its larger spiral horns reminding the mouflon; Berbere or sheep of the Tell Atlas mountain has a smaller body size than both previous ones and considered as close to the ancestor of the sheep of North Africa. The 23 sequences of these animals were included in a dataset containing 320 sequences of 28 breeds (haplogroups A, B, C, D, and E) to assess their phylogenic position. It appears that 87% of Algerian individuals belong to haplogroup B, the rest to haplogroup C, only recorded among Ouled Djellal breed. Given the scattered position of Algerian individuals within the tree, Minor Allele Frequencies of each breed were calculated to construct a distance tree and a network through Reduced Median method. These data were used to estimate the divergence dates of the endemic Algerian breeds, indicating that they appeared through three independent migration events and not through local differentiations following only one migration event. Ouled Djellal breed would be the latest to come from Middle-East, a fact supported by the significant percentage of C haplotype in our sampling.

In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-β1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-β1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation.

Bidirectional promoters are regulatory regions co-regulating the expression of two neighbouring genes organized in a head-to-head orientation. In recent years, these regulatory regions have been studied in many organisms; however, no investigation to date has been done to analyse the genetic variation of the activity of this type of promoter regions. In our study, we conducted an investigation to first identify bidirectional promoters sharing genes expressed in bovine Longissimus thoracis and then to find genetic variants affecting the activity of some of these bidirectional promoters. Combining bovine gene information and expression data obtained using RNA-Seq, we identified 120 putative bidirectional promoters active in bovine muscle. We experimentally validated in vitro 16 of these bidirectional promoters. Finally, using gene expression and whole-genome genotyping data, we explored the variability of the activity in muscle of the identified bidirectional promoters and discovered genetic variants affecting their activity. We found that the expression level of 77 genes is correlated with the activity of 12 bidirectional promoters. We also identified 57 single nucleotide polymorphisms associated with the activity of 5 bidirectional promoters. To our knowledge, our study is the first analysis in any species of the genetic variability of the activity of bidirectional promoters.

Algeria represents a reservoir of genetic diversity with local sheep breeds adapted to a large range of environments and showing specific features necessary to deal with harsh conditions. This remarkable diversity results from the traditional management of dryland by pastoralists over centuries. Most of these breeds are poorly productive, and the economic pressure leads farmers to realize anarchic cross-breeding (that is, not carried out in the framework of selection plans) with the hope to increase animal's conformation. In this study, eight of the nine local Algerian sheep breeds (D'men, Hamra, Ouled-Djellal, Rembi, Sidaoun, Tazegzawt, Berber and Barbarine) were investigated for the first time by genome-wide single-nucleotide polymorphism genotyping. At an international scale, Algerian sheep occupied an original position shaped by relations with African and European (particularly Italian) breeds. The strong genetic proximity with Caribbean and Brazilian breeds confirmed that the genetic make-up of these American breeds was largely influenced by the Atlantic slave trade. At a national scale, an alarming genetic dilution of the Berber (a primitive breed) and the Rembi was observed, as a consequence of uncontrolled mating practices with Ouled-Djellal. A similar, though less pronounced, phenomenon was also detected for the Barbarine, another ancestral breed. Genetic originality appeared to be better preserved in Tazegzawt, Hamra, D'men and Sidaoun. These breeds should be given high priority in the establishment of conservation plans to halt their progressive loss. For Berber and Barbarine that also occur in the bordering neighbor countries, urgent concerted transnational actions are needed.

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1cax/cax (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1cax/cax mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1cax/cax SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7+/MYOD− progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.