Multiple Mechanisms for Pitx-1 Transactivation of a Luteinizing Hormone β Subunit Gene

The pituitary homeobox factor-1 (Pitx-1) transactivates a number of pituitary-specific genes through direct interaction with other specific transcription factors. We demonstrate here that Pitx-1 plays a crucial role in the regulation of the Chinook salmon luteinizing hormone β gene promoter through a number of novel mechanisms. On the proximal promoter its action involves a synergistic effect with steroidogenic factor-1 (SF-1) alone or in combination with the estrogen receptor; promoter activity being induced by 9- or 35-fold over controls, respectively. Further upstream, a series of four Pitx-1 response elements (located between 1366 and 1506 bp from the transcriptional start site) is also involved in regulating the promoter activity. The two distal sequences have the greatest effect on the basal activity and are also essential for the gonadotropin-releasing hormone (GnRH) response. Mammalian two-hybrid assays revealed that Pitx-1 can homodimerize. Moreover, circular permutation assays indicate that binding of Pitx-1 to more than one response element induces conformational changes of the target DNA. This constitutes an additional mechanism through which Pitx-1 can mediate transactivation of this gene, allowing the demonstrated interaction of proximal response elements and distal enhancers, thus facilitating the maximal GnRH response that was seen in the longer promoter constructs. Our research also indicates that Pitx-1 is phosphorylated on three residues when bound to the DNA. The pituitary homeobox factor-1 (Pitx-1) transactivates a number of pituitary-specific genes through direct interaction with other specific transcription factors. We demonstrate here that Pitx-1 plays a crucial role in the regulation of the Chinook salmon luteinizing hormone β gene promoter through a number of novel mechanisms. On the proximal promoter its action involves a synergistic effect with steroidogenic factor-1 (SF-1) alone or in combination with the estrogen receptor; promoter activity being induced by 9- or 35-fold over controls, respectively. Further upstream, a series of four Pitx-1 response elements (located between 1366 and 1506 bp from the transcriptional start site) is also involved in regulating the promoter activity. The two distal sequences have the greatest effect on the basal activity and are also essential for the gonadotropin-releasing hormone (GnRH) response. Mammalian two-hybrid assays revealed that Pitx-1 can homodimerize. Moreover, circular permutation assays indicate that binding of Pitx-1 to more than one response element induces conformational changes of the target DNA. This constitutes an additional mechanism through which Pitx-1 can mediate transactivation of this gene, allowing the demonstrated interaction of proximal response elements and distal enhancers, thus facilitating the maximal GnRH response that was seen in the longer promoter constructs. Our research also indicates that Pitx-1 is phosphorylated on three residues when bound to the DNA. pituitary homeobox factor-1 cAMP-response element-binding protein CREB-binding protein steroidogenic factor-1 early growth response factor-1 lutenizing hormone β gonadotropin-releasing hormone mitogen-activated protein kinase response element estrogen receptor estrogen response element chloramphenicol acetyl transferase multiple cloning site DNA binding domain activation domain. The pituitary homeobox factor-1 (Pitx-1)1 is expressed in all pituitary cell types and is essential in the development and synthesis of pituitary hormones such as prolactin, pro-opiomelanocortin, and the gonadotropins (1Lamonerie T. Tremblay J.J. Lanctôt C. Therrien M. Gauthier Y. Drouin J. Genes Dev. 1996; 10: 1284-1295Crossref PubMed Scopus (349) Google Scholar, 2Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu R. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (339) Google Scholar, 3Tremblay J.J. Lanctôt C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar, 4Drouin J. Lamolet B. Lamonerie T. Lanctôt C. Tremblay J.J. Mol. Cell. Endocrinol. 1998; 140: 31-36Crossref PubMed Scopus (93) Google Scholar, 5Lanctôt C. Moreau A. Chamberland M. Tremblay M.L. Drouin J. Development. 1999; 126: 1805-1810Crossref PubMed Google Scholar). Although first discovered in the pituitary, Pitx-1 is also expressed in tissues derived from the first branchial arch, in the lateral mesenchyme, and in the developing hindlimb. Its role in development includes regulating growth, morphogenesis, and hindlimb patterning (2Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu R. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (339) Google Scholar, 6Lanctôt C. Moreau A. Chamberland M. Tremblay M.L. Drouin J. Development. 1997; 124: 2807-2817Crossref PubMed Google Scholar). Previous studies have shown that it interacts with a number of different protein partners including Pit-1, steroidogenic factor-1 (Sf-1), and basic helix-loop-helix factors such as NeuroD/Pan1 (3Tremblay J.J. Lanctôt C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar, 7Poulin G. Lebel M. Chamberland M. Paradis F.W. Drouin J. Mol. Cell. Biol. 2000; 20: 4826-4837Crossref PubMed Scopus (133) Google Scholar). It has further been suggested that the cell-specific actions of this transcription factor are a function of these interacting proteins (2Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu R. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (339) Google Scholar,3Tremblay J.J. Lanctôt C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar, 8Kioussi C. Carriere C. Rosenfeld M.G. Mech. Dev. 1999; 81: 23-35Crossref PubMed Scopus (67) Google Scholar, 9Sheng H.Z. Westphal H. Trends Genet. 1999; 15: 236-240Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). A consensus of research has suggested that Pitx-1 interacts with Sf-1 and early growth response factor-1 (Egr-1) to form a tripartite complex on the mammalian lutenizing hormone β (LHβ) subunit gene promoter, which mediates the gonadotropin-releasing hormone (GnRH)-regulated transcription of this gene (10Tremblay J.J. Drouin J. Mol. Cell. Biol. 1999; 19: 2567-2576Crossref PubMed Google Scholar, 11Dorn C., Ou, Q.L. Svaren J. Crawford P.A. Sadovsky Y. J. Biol. Chem. 1999; 274: 13870-13876Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Halvorson L.M. Kaiser U.B. Chin W.W. Mol. Endocrinol. 1999; 13: 106-116Crossref PubMed Scopus (93) Google Scholar, 13Quirk C.C. Lozada K.L. Keri R. Nilson J.H. Mol. Endocrinol. 2001; 15: 734-746Crossref PubMed Scopus (78) Google Scholar). The very similar structure of the proximal promoter across species further indicates that the role of this tripartite element has been highly conserved through evolution (for example, see Refs. 14Kaiser U.B. Halvorson L.M. Chen M. Mol. Endocrinol. 2000; 14: 1235-1245Crossref PubMed Scopus (112) Google Scholar, 15Wolfe M.W. Call G.B. Mol. Endocrinol. 1999; 13: 752-763PubMed Google Scholar, 16Weck J. Anderson A.C. Jenkins S. Fallest P.C. Shupnik M.A. Mol. Endocrinol. 2000; 14: 472-485Crossref PubMed Google Scholar). Egr-1 appears to be the main activator, because it is transcriptionally up-regulated and probably also phosphorylated within minutes of GnRH exposure (10Tremblay J.J. Drouin J. Mol. Cell. Biol. 1999; 19: 2567-2576Crossref PubMed Google Scholar, 11Dorn C., Ou, Q.L. Svaren J. Crawford P.A. Sadovsky Y. J. Biol. Chem. 1999; 274: 13870-13876Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Sf-1 may also be phosphorylated as a result of activation of the MAPK pathway by GnRH (17Hammer G.D. Krylova I. Zhang Y. Darimont B.D. Simpson K. Weigel N.L. Ingraham H.A. Mol. Cell. 1999; 3: 521-526Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Pitx-1 acts on the LHβ gene in synergy with these two factors, although the molecular mechanism of its actions has yet to be elucidated. The LHβ promoter of a fish, the Chinook salmon (csLHβ or GtHIIβ), shows only partial structural similarity with its mammalian homolog, although it is highly responsive to stimulation by GnRH. It contains putative Pitx-1 REs at 232 and 586 bp from the transcriptional start site but does not contain a functional Egr-1 response element (RE) and is completely unresponsive to Egr-1 (Fig.1 A) (18Le Dréan Y. Liu D. Xiong F. Hew C.L. Mol. Cell. Endocrinol. 1997; 135: 31-40Crossref PubMed Scopus (18) Google Scholar). 2P. Melamed, J. Seah, and C. Hew, unpublished observations.2P. Melamed, J. Seah, and C. Hew, unpublished observations. Previous studies on the csLHβ promoter have demonstrated that Sf-1 binding the proximal RE acts synergistically with ER, binding a proximal ERE at 260 bp (18Le Dréan Y. Liu D. Xiong F. Hew C.L. Mol. Cell. Endocrinol. 1997; 135: 31-40Crossref PubMed Scopus (18) Google Scholar,19Le Dréan Y. Liu D. Wong A.O.L. Xiong F. Hew C.L. Mol. Endocrinol. 1996; 10: 217-229PubMed Google Scholar). Moreover, the ER binding this proximal RE interacts with ER binding a distal RE at 2659 bp (20Liu D. Xiong F. Hew C.L. Endocrinology. 1995; 136: 3486-3493Crossref PubMed Scopus (37) Google Scholar). Our recent studies have indicated that both of these EREs are involved in mediating the GnRH effect.2 Apart from the potentially important role of the proximal promoter Pitx-1 REs, further upstream on the csLHβ promoter there is a series of four Pitx-1 REs located within 150 bp, between nucleotides 1366 and 1506. The aim of this study was to verify which of these proximal or distal Pitx-1 REs are responsible for regulating basal or GnRH-mediated transcription of this gene, and to understand some of the molecular mechanisms. The csLHβ (sGtHIIβ)-chloramphenicol acetyl transferase (CAT) constructs have been previously described (21Xiong F. Liu D. Elsholtz H.P. Hew C.L. Mol. Endocrinol. 1994; 8: 771-781PubMed Google Scholar). Additional constructs were created by introducing aSalI restriction site upstream of the desired cutting site, and an Xho restriction site on the CAT 3 vector multiple cloning site (MCS) that was removed in the original cloning of the 3.3 construct. This SalI-Xho fragment was then cloned into CAT 3 linearized with the same enzymes. Site-directed deletions were carried out using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the 3.3-kbp construct as a template, according to the manufacturer's instructions. Primers used for these deletions were comprised of 12–22 bp of the region flanking the RE on both sides and its complementary sequence. All constructs were sequenced to verify correct deletions. Expression vectors for Pitx-1, Sf-1, ER, CBP, and P-300 were gifts from J. Drouin (Montreal), K. Parker (Durham, NC), Y. Valotaire (Rennes, France), and H. Zimmermann and H.U. Bernard (Singapore), respectively. Constructs for the two-hybrid assay were prepared using the Gal4 activation domain (AD) and Gal4 DNA binding domain (DBD) vectors, pM and pVP16 (CLONTECH, Palo Alto, CA). Sequence-specific primers containing restriction enzyme recognition sites at the 5′-ends were used to PCR amplify the DNA fragment from Pitx-1, CBP, and P300 1–5 expression vectors. This CBP expression vector contains the transcriptional adaptor motif (TRAM) and the entire cysteine-rich region (C/H3 region; amino acids 1765–1852). The p300 cDNA was divided into five fragments spanning amino acid residues 1–438, 439–1038, 1034–1452, 1453–1882, and 1883–2378, named p300 1–5, respectively (22Zimmermann H. Koh C.H. Degenkolbe R. O'Conner M.J. Muller A. Steger G. Chen J.J. Lui Y. Androphy E. Bernard H.U. J. Gen. Virol. 2000; 81: 2617-2623Crossref PubMed Scopus (38) Google Scholar). The reporter gene used was pG5CAT (CLONTECH). COS1 cells were cultured in modified Eagle's medium supplemented with 10% fetal calf serum, 10 mm HEPES, 0.1 mm modified Eagle's medium non-essential amino acids, 1 mm sodium pyruvate solution, 100 units/ml penicillin, and 100 μg/ml streptomycin, (Invitrogen). LβT2 cells were cultured in Dulbeco's modified Eagle's medium containing 4.5 g/l glucose with 10% certified fetal calf serum, 10 mm HEPES, and the same antibiotics (Invitrogen). Cells (5 × 105 in 2 were at for or with the two-hybrid the pVP16 and pG5CAT were with the reporter gene in the of was as an for cells were for a further or (for two-hybrid cells to this was to the medium and cells were of the which was at a of of to a of 10 cells were with ER, was to the to a of 10 CAT and assays were as described previously Dréan Y. Liu D. Wong A.O.L. Xiong F. Hew C.L. Mol. Endocrinol. 1996; 10: 217-229PubMed Google Scholar). The activity was as a to the basal of activity in the and construct for or the for the two-hybrid DNA (csLHβ or a Pitx-1 response element was with of with LβT2 cell 3 in at The DNA was then to through The bound DNA were with acid in and the bound proteins were with 1 at The of the bound proteins was by the to an of The of the bound proteins was used as a for its in the protein through the of the bound protein was 4.5 100 at then 100 at with 100 at The bound were acid in and and as LβT2 were prepared according to E. Muller PubMed Scopus Google and and at to the putative Pitx-1 REs, with the of a were with using fragment (Invitrogen). were removed using The at × was to 2 of the mm HEPES, mm 10% 1 mm mm 10 with 2 and for on The were an and with 1 mm at at The was and to at assays were carried out according to the described by and PubMed Scopus Google Scholar). Sequence-specific one of which a Pitx-1 binding site at the were used to PCR amplify the of vector The fragment was to a linearized vector to a construct with the Pitx-1 RE by two in the same additional vector containing three Pitx-1 response elements was by a fragment from 1 the Pitx-1 RE and one an additional and a fragment of the csLHβ promoter containing three Pitx-1 REs All constructs were by 1 and 3 were with a number of restriction to a series of and linearized DNA containing Pitx-1 REs at from the to the of the fragment by fragments of the The of the were and assays were carried out as described as of by the was to that were were when All were at three are for 1 which was the of from more than one in of these activity of the 3.3 construct was induced by GnRH over A number of CAT 3 constructs containing of the csLHβ promoter were into LβT2 and of these cells were to 10 GnRH for The GnRH in an in the CAT activity for the promoter construct by more than although this was by a putative located between and bp, is not the longer of reporter activity was seen only by the 3.3-kbp promoter, the GnRH CAT over the 1 The 3.3-kbp into LβT2 to GnRH exposure with an in CAT activity of of the proximal Pitx-1 located at 232 bp, this of a more distal Pitx-1 located at 586 bp, of an and the GnRH induced an in promoter activity that was than in of both REs basal promoter activity to a of that of the 3.3-kbp of the Pitx-1 expression vector into COS1 cells over a spanning to activity of the csLHβ Moreover, the to CAT activity of of the Sf-1 expression vector CAT by as as of the Pitx-1 with of the Sf-1 expression vector more than the effect of the of Sf-1 alone 3 of Pitx-1 with ER synergistic effect 3 of all three expression vectors and ER at revealed that the of Pitx-1 expression vector promoter activity by as as 35-fold over the basal of promoter activity and over the synergistic effect of ER and Sf-1 to the two Pitx-1 REs located on the proximal csLHβ promoter, four putative Pitx-1 REs are located further upstream, between 1366 and 1506 these all a protein of similar assays were carried out using the putative RE and from LβT2 All of the including the consensus 232 bound a protein of similar The was not seen when the same was with the but was when a was used The of the protein binding these sequences was further by using DNA bound to a protein A protein bound the using a Pitx-1 RE or using the bp of the csLHβ Its molecular was or and as with the from the cDNA of that Pitx-1 contains a number of putative that the bound protein be Pitx-1 phosphorylated on three the from the This was by of the bound protein with for 1 which the of the bound protein to or of the of this protein as Pitx-1 was by of the bound which five and within and were a result of fragments of the of putative distal REs effect on the GnRH which at a in CAT activity of of the more proximal REs with RE basal of CAT activity to that of the although the by at of the two distal REs and 1506 basal of CAT activity to to a of in the controls, and the GnRH longer an of a RE basal further of for of the although for the construct with the two distal of the RE at 1366 or bp not the activity of all four REs CAT activity to the Pitx-1 to be in to the upstream promoter, mammalian two-hybrid assays were carried out to the of Pitx-1 to homodimerize. The cDNA of Pitx-1 was into both the pM and pVP16 vectors to two one comprised of the Pitx-1 protein and the Gal4 and the other the Pitx-1 protein and of both vectors revealed an of the reporter gene CAT activity to that of controls, of only one of the Pitx-1 containing with the or vector to CAT activity two-hybrid assays were carried out to the of Pitx-1 to with the CBP or expression vectors containing fragments of CBP or p300 cDNA were into the pVP16 vector and with the pM Pitx-1 all constructs the of CAT activity and not from assays were carried out using a containing a Pitx-1 RE at different within the DNA of the of the the a complex that was of similar when different constructs were used containing the at bp of the csLHβ promoter, which includes the three more distal Pitx-1 REs, the of the RE the of the when the REs were located in the of the DNA the was than when located at the studies that Pitx-1 is to the csLHβ gene through novel including interaction with and also through binding to multiple thus conformational changes in the DNA. We have also shown that Pitx-1 can homodimerize. indicate that Pitx-1 is phosphorylated on three residues on binding to its DNA of Pitx-1 with Sf-1 and ER expression vectors promoter activity by Although Pitx-1 has previously been shown to synergistically with Sf-1, indicate further synergistic action with the although interaction with ER alone was The of stimulation by of all three expression vectors is similar to that seen on the mammalian LHβ promoter when Pitx-1, Sf-1, and Egr-1 were (10Tremblay J.J. Drouin J. Mol. Cell. Biol. 1999; 19: 2567-2576Crossref PubMed Google and that the role of Egr-1 has been by ER in regulation of the LHβ gene (18Le Dréan Y. Liu D. Xiong F. Hew C.L. Mol. Cell. Endocrinol. 1997; 135: 31-40Crossref PubMed Scopus (18) Google Scholar). 2P. Melamed, J. Seah, and C. Hew, unpublished observations. of the Pitx-1 RE at 232 bp the csLHβ proximal promoter unresponsive to the of Pitx-1 in the GnRH of the synergistic action of Pitx-1 on the proximal promoter be through the direct activation of Sf-1, as it was previously shown that Pitx-1 can as a Sf-1 by the Sf-1 activation domain J.J. A. Gauthier Y. Drouin J. J. 1999; PubMed Scopus Google Scholar). the of stimulation by of Pitx-1 which is at with the for the mammalian homolog, that Pitx-1 is not a factor in stimulation of the promoter activity in these previously of (10Tremblay J.J. Drouin J. Mol. Cell. Biol. 1999; 19: 2567-2576Crossref PubMed Google and D. M. Dréan Y. H. Xiong F. Hew C.L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google that these three transcription factors to a number of Sf-1 has been shown to with P.A. G. Sadovsky Y. Mol. Endocrinol. 1997; PubMed Scopus Google and ER, interacts with and also with J. R. T. J. 1998; PubMed Scopus Google Scholar, 1999; 20: PubMed Scopus Google Scholar). Pitx-1 does not contain the motif for binding S. A. J. J. Rosenfeld M.G. Genes Dev. 1998; 12: PubMed Scopus Google and were not to demonstrate interaction between Pitx-1 and CBP or P300 in the The of interaction between Pitx-1 and with the recent that factors with CBP and P300 through the The result of this interaction is a of the acetyl transferase activity and also of the protein binding its DNA target K. C. Mol. Cell. Biol. 2001; PubMed Scopus Google an role for transcription factors on transcription of DNA. that the proteins were and using in The only of the interaction in was in The revealed that all Pitx-1 bound to its target are phosphorylated on three which indicate the of may be a of DNA The that the Pitx-1 not from the DNA as a result of does not this as the protein was bound to the DNA when the were The Pitx-1 contains a number of putative including three for of the and protein kinase both of which are by GnRH R. Trends Endocrinol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). It be that the not only binding of the Pitx-1 to but also interaction with which transcriptional other is on the bound by Pitx-1, but the appears to have two of one of which is by binding through which it the complex to the promoter and transcriptional activation F. R. 1995; PubMed Scopus Google the other mechanism is of these activation but has not yet been F. C. C. S. A. 1999; PubMed Scopus Google Scholar). The in csLHβ promoter activity seen here when of Pitx-1 expression vector were further that Pitx-1 does for to factors that basal transcription of this gene or for of the on the distal of the csLHβ promoter, four additional Pitx-1 REs 1366 and 1506 also mediate both basal and transcription of this The that of REs has effect on promoter activity that some of the REs are and indicates that Pitx-1 proteins with other to this of the distal two Pitx-1 REs and 1506 has a more effect on the basal and also GnRH from the two-hybrid assay study that Pitx-1 can homodimerize. it appears that binding to at one of the distal Pitx-1 REs or 1506 is crucial to of The between the proximal REs, is to interaction of binding these two The that Pitx-1 as a to the distal region of the csLHβ promoter and induces a conformational in the promoter, is in with The specific DNA by homeobox proteins is only bp and the to a of through with other specific proteins or as a (2Szeto D.P. Rodriguez-Esteban C. Ryan A.K. O'Connell S.M. Liu R. Kioussi C. Gleiberman A.S. Izpisua-Belmonte J.C. Rosenfeld M.G. Genes Dev. 1999; 13: 484-494Crossref PubMed Scopus (339) Google Scholar, 3Tremblay J.J. Lanctôt C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar, 7Poulin G. Lebel M. Chamberland M. Paradis F.W. Drouin J. Mol. Cell. Biol. 2000; 20: 4826-4837Crossref PubMed Scopus (133) Google Scholar, J.J. A. Gauthier Y. Drouin J. J. 1999; PubMed Scopus Google Scholar). are the or the and M.A. C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, B. C. J. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, T. C. 1995; PubMed Scopus Google both of which of the target DNA. by conformational changes in the target DNA also in other of DNA binding such as T. PubMed Scopus Google helix-loop-helix J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and Mol. Cell. Biol. 12: PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). The conformational in the promoter of csLHβ be further by from a located over upstream, at bp J. Biol. Chem. Full Text PDF PubMed Google Scholar). DNA can transcription by facilitating between distal and proximal binding factors J. G. 1996; PubMed Scopus Google Scholar, E. Anderson C. T. J. Mol. Biol. 1997; PubMed Scopus Google and distal REs on the csLHβ promoter are also involved in the mediating basal and the a distal ERE 2659 which was shown to with the proximal ERE 260 in a synergistic and also mediates the GnRH effect (20Liu D. Xiong F. Hew C.L. Endocrinology. 1995; 136: 3486-3493Crossref PubMed Scopus (37) Google Scholar). Melamed, J. Seah, and C. Hew, unpublished Moreover, studies on the promoter that the promoter which contains the four Pitx-1 REs but not upstream is not to the response to that the Pitx-1 binding these sites does not contain its transactivation have demonstrated novel through which Pitx-1 transcription of the csLHβ both synergistic with other protein and with additional Pitx-1 to form a to DNA The role of of Pitx-1 in its transactivation or interaction with has yet to be and may an additional mechanism regulating LHβ We J. K. Y. H. and H. Bernard for the expression vectors, and for the LβT2

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