Chromatin-dependent Cooperativity between Site-specific Transcription Factors in Vivo

Abstract

Accessing binding sites in DNA wrapped around histones in condensed chromatin is an obstacle that transcription factors must overcome to regulate gene expression. Here we demonstrate cooperativity between two transcription factors, the glucocorticoid receptor (GR) and nuclear factor 1 (NF1) to bind the mouse mammary tumor virus promoter organized as regular chromatin in vivo. This cooperativity is not observed when the promoter is introduced transiently into cells. Using RNA interference to deplete NF1 protein levels in the cells, we confirmed that NF1 promotes binding of GR to the promoter. Furthermore, we observed a similar synergism between GR and NF1 binding on the endogenous 11β-hydroxysteroid dehydrogenase promoter, also regulated by GR and NF1. Our results suggest that the chromatin architecture of the promoters does not permit strong association of GR in the absence of NF1. Therefore we propose that cooperativity among DNA binding factors in binding to their cognate recognition sites in chromatin may be an important feature in the regulation of gene expression. Accessing binding sites in DNA wrapped around histones in condensed chromatin is an obstacle that transcription factors must overcome to regulate gene expression. Here we demonstrate cooperativity between two transcription factors, the glucocorticoid receptor (GR) and nuclear factor 1 (NF1) to bind the mouse mammary tumor virus promoter organized as regular chromatin in vivo. This cooperativity is not observed when the promoter is introduced transiently into cells. Using RNA interference to deplete NF1 protein levels in the cells, we confirmed that NF1 promotes binding of GR to the promoter. Furthermore, we observed a similar synergism between GR and NF1 binding on the endogenous 11β-hydroxysteroid dehydrogenase promoter, also regulated by GR and NF1. Our results suggest that the chromatin architecture of the promoters does not permit strong association of GR in the absence of NF1. Therefore we propose that cooperativity among DNA binding factors in binding to their cognate recognition sites in chromatin may be an important feature in the regulation of gene expression. Eukaryotic DNA is condensed and highly compacted into chromatin, which allows multiple levels of transcriptional regulation (1Wolffe A.P. Guschin D. J. Struct. Biol. 2000; 129: 102-122Crossref PubMed Scopus (293) Google Scholar). Chromatin architecture can inhibit transcription by preventing the access of transacting factors to their binding sites on the DNA (2Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar). The mechanism by which transcription factors access their cognate binding sites buried in the nucleosome is not clear. Chromatin modifying enzymes such as the ATP-dependent chromatin remodeling factors are known to aid unwrapping or translocation of DNA on the nucleosomes such that binding sites for transcription factors are exposed (3Langst G. Becker P.B. Biochim. Biophys. Acta. 2004; 1677: 58-63Crossref PubMed Scopus (125) Google Scholar). Recent evidence suggests that remodeling complexes may be recruited to the target promoters by site-specific DNA binding proteins, which initially engage their target sites (4Peterson C.L. Workman J.L. Curr. Opin. Genet. Dev. 2000; 10: 187-192Crossref PubMed Scopus (383) Google Scholar). One such class of transcription factors is the steroid hormone receptors that can interact with the repressive chromatin structure and remodel the chromatin by recruiting chromatin-remodeling complexes to eventually allow other transcription factors to bind (5Hager G.L. Fletcher T.M. Xiao N. Baumann C.T. Muller W.G. McNally J.G. Biochem. Soc. Trans. 2000; 28: 405-410Crossref PubMed Scopus (25) Google Scholar). The hormone-inducible mouse mammary tumor virus (MMTV) 2The abbreviations used are: MMTV, mouse mammary tumor virus; GR, glucocorticoid receptor; ChIP, chromatin immunoprecipitation; HRE, hormone response element; NF1, nuclear factor 1; BRG1, brahma-related gene 1; 11βHSD2, 11 β-hydroxysteroid dehydrogenase 2; sgk, serum/glucocorticoid inducible kinase; OTF, octamer transcription factor; dex, dexamethasone; WT, wild-type; mNF1, mutation in NF1 binding site; siRNA, small interfering RNA; PEPCK, phosphoenolpyruvate carboxykinase. promoter has been used extensively as a model to study the relationship between chromatin structure and transcriptional activation by the steroid hormone receptors (6Hebbar P.B. Archer T.K. Chromosoma. 2003; 111: 495-504Crossref PubMed Scopus (85) Google Scholar). When stably integrated into mammalian cells, MMTV promoter assumes a phased array of six nucleosomes (A to F). The minimal promoter is made up of nucleosomes A and B, which encompasses binding sites for glucocorticoid receptor (GR) hormone response elements (HREs), nuclear factor 1 (NF1), octamer transcription factor (OTF), and the transcription factor IID complex. In the absence of hormone, the chromatin architecture of the promoter excludes the binding of NF1 and OTFs. Upon hormone administration, GR recruits ATP-dependent BRG1 remodeling complex and multiple coactivator proteins to the promoter to remodel the chromatin (7Trotter K.W. Archer T.K. Mol. Cell. Biol. 2004; 24: 3347-3358Crossref PubMed Scopus (87) Google Scholar). This remodeling or “opening” of the promoter permits binding of NF1 and OTFs, which eventually leads to the assembly of a transcription initiation complex and the subsequent induction of mRNA (6Hebbar P.B. Archer T.K. Chromosoma. 2003; 111: 495-504Crossref PubMed Scopus (85) Google Scholar). Previously we established that NF1 protein is required for both transcription and chromatin remodeling of the MMTV promoter (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). Furthermore, we demonstrated that NF1 promotes the binding of GR with the stably integrated promoter. This synergism in binding by transcription factors may be a common occurrence on regulatory regions in the genome. Indeed, most eukaryotic promoter regions contain binding sites for several DNA-binding proteins, which probably allows multiple levels and combinatorial control of gene expression (9Miller J.A. Widom J. Mol. Cell. Biol. 2003; 23: 1623-1632Crossref PubMed Scopus (135) Google Scholar). Therefore we wanted to investigate the role of cooperative binding by transcription factors to their cognate binding sites within an in vivo chromatin context. In this study, we report that cooperative binding by GR and NF1 is observed only in the context of chromatin because the synergism in binding is absent on a transiently transfected promoter. We confirmed these results by using RNA interference to deplete NF1 protein levels in the cell followed by assessment of GR binding to the promoter. Furthermore we show that GR and NF1 cooperativity is not unique to the MMTV promoter but is also observed on the GR-regulated 11 β-hydroxysteroid dehydrogenase (11βHSD2) promoter. Plasmids—pLS-WT (wild-type MMTV promoter) and pmNF1 (mNF1, MMTV promoter with mutation in NF1 binding site) plasmids have the promoter fused to thymidine kinase gene as reporter (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). Cell Culture and Transfections—WT (stably integrated pLS-WT construct) and mNF1 (stably integrated pmNF1 construct) cell lines were derived from C127 mouse mammary carcinoma cells, which were grown at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 μg/ml penicillin/streptomycin, 10 mm HEPES, and 600 μg of G418/ml (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). MCF7M cells were derived from human breast cancer cells MCF7 (10Kinyamu H.K. Archer T.K. Mol. Cell. Biol. 2003; 23: 5867-5881Crossref PubMed Scopus (134) Google Scholar). These cells contained stably integrated copies of MMTV promoter fused to luciferase gene in the genome and overexpressed rat GR. MCF7M cells were grown at 37 °C with 5% CO2 in modified Eagle’s medium supplemented with 2 mm glutamine, 100 μg of penicillin-streptomycin/ml, 10 mm HEPES, 10% fetal bovine serum, and 300 μg of G418/ml. Cells were treated with a synthetic glucocorticoid dexamethasone (dex) (10-7 M). Transient transfections were performed by the Lipofectamine PLUS method (Invitrogen) as described by the manufacturer. For knockdown of NF1 mRNA, cells were transfected with NF1-C siRNA (Santa Cruz Biotechnology, Santa Cruz, CA), control lamin A/C-specific siRNA, or control non-targeting siRNA (Dharmacon, Lafayette, CO) for 48 h using Lipofectamine 2000 (Invitrogen). Chromatin Immunoprecipitation (ChIP) and ReChIP Assays—Cells were fixed with 1% formaldehyde at 37 °C for 20 min. ChIP analysis was carried out as described previously (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). Antibodies used for ChIP assay were: GR antibody (M20) (Santa Cruz Biotechnology), human GR (N499) (Keith Yamamoto, University of California, San Francisco, CA), NF1 (H300) (Santa Cruz Biotechnology), NF1-C-specific (8199) (Naoko Tanese, New York University School of Medicine, NY), and normal serum immunoglobulin G (IgG) (Santa Cruz Biotechnology). After immunoprecipitation, the eluted samples were reverse cross-linked for 12 h at 65 °C, treated with proteinase K (Invitrogen), and purified using PCR purification kit (Qiagen, Valencia, CA). ReChIP assay was performed following the protocol described (11Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar). For reChIP assays, to perform the first ChIP, nuclei were isolated after formaldehyde cross-linking, and the complexes were eluted with 50 μl of 10 mm dithiothreitol at 37 °C for 30 min. The eluted complex was diluted 20 times with the reChIP buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.0), and the ChIP procedure was repeated again. Real-time PCR was performed on ChIP DNA samples using real-time PCR primers. For MMTV promoter (nucleosome B region), primer pair 5′-GGT TAC AAA CTG TTC AAA and was serum/glucocorticoid inducible kinase promoter was with and primer and human promoter was with TTC and primer the cells were two times with were by For cells were with buffer mm mm NaCl, 1% 1 mm EDTA, and and nuclear were as described previously H.K. Archer T.K. J. Biol. 2000; Full Text Full Text PDF PubMed Google Scholar). nuclei were in buffer and by with at The was by at for 10 on a 50 μg of protein was by and to a RNA and Cells were treated or for h with (10-7 and RNA was isolated using (Invitrogen) as described by the manufacturer. For the was as described previously Archer T.K. J. Biol. Full Text Full Text PDF PubMed Scopus Google and real-time PCR was performed using with the following of primers. For mRNA, and were NF1 primer for mRNA, and CTG for mRNA, and for NF1-C mRNA, and and for mRNA, and were dehydrogenase mRNA was with the primer pair and and human lamin mRNA was with primer and CTG For mRNA, primer pair CTG and and for mRNA, and were In were isolated as described previously T.K. PubMed Scopus Google and with DNA was purified and to with The samples were by primer and the were on The were exposed to for GR an NF1 to to an MMTV but a with the stably integrated MMTV promoter have that binding of NF1 is (6Hebbar P.B. Archer T.K. Chromosoma. 2003; 111: 495-504Crossref PubMed Scopus (85) Google Scholar). We demonstrated previously that binding of GR to the stably integrated promoter is on NF1 binding (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). to investigate this of GR and NF1 for binding their recognition we first the of promoter chromatin We the binding of GR to the transiently transfected MMTV promoter in the absence of NF1 has been that in to the stably integrated when MMTV promoter DNA is introduced transiently into cells, the DNA to the phased array of nucleosomes T.K. G.L. PubMed Scopus Google Scholar). The MMTV promoter permits binding of NF1 but the of the the promoter We transiently transfected the MMTV promoter and a with mutation in NF1 binding into MCF7M cells (10Kinyamu H.K. Archer T.K. Mol. Cell. Biol. 2003; 23: 5867-5881Crossref PubMed Scopus (134) Google Scholar). in the the MMTV promoter within nucleosome B of the phased The NF1 binding also within nucleosome B to the with nucleosome A We performed a of ChIP using an antibody and the nucleosome B of the MMTV promoter For we performed ChIP assay to GR binding on integrated copies of MMTV promoter in and mNF1 cell on the the binding of GR is on NF1 binding (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). In to we on the the binding of GR to the transiently transfected promoter is not by the mutation in NF1 binding Therefore the for a NF1 bind to allow GR binding is observed only on the stably integrated promoter. with this in the absence of organized chromatin, the GR is to bind the promoter of NF1. These suggest that NF1 binding is required for GR to overcome chromatin architecture and to bind cognate within the promoter. of NF1 by siRNA of GR from the promoter in the absence of NF1 binding is the that in in the absence of NF1, GR is to bind to on the promoter and remodel the chromatin with complex T.M. Baumann C.T. G. G.L. Mol. Cell. Biol. 2000; PubMed Scopus Google H. Mol. Cell. Biol. PubMed Scopus Google Scholar). we have previously that the in the mNF1 does not to nucleosome (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google the results be by of the following be in the that may the binding of GR in the absence of NF1 when stably by the NF1 binding for transcription factor may be in this or on the stably integrated promoter on the these we RNA interference to deplete NF1 protein levels in the cells followed by chromatin this the synergism in binding of these two transcription factors can be to the promoter. This also allows to the of this on other GR-regulated The NF1 gene highly and 2000; PubMed Scopus Google Scholar). of the expression of these and their is not which is required for with GR J. F. J. Biol. 2003; PubMed Scopus Google Scholar). NF1-C has been to be most in the mouse mammary R. D. F. Biol. 2000; PubMed Google we the role of NF1-C in gene regulation in MCF7M cells. Using ChIP assay and NF1-C-specific we that NF1-C to the MMTV promoter in a A and we used siRNA to NF1-C to deplete NF1 protein levels required for activation of the promoter. MCF7M cells were transfected with siRNA lamin and NF1-C with NF1-C siRNA in in NF1-C mRNA levels is knockdown of other NF1 as In with the mRNA we also in NF1 protein levels when were with antibody that This suggests that by siRNA to NF1-C mRNA, we levels of NF1 proteins in these cells. of NF1 on MMTV and GR to the this in vivo model for NF1 we the of NF1 on MMTV NF1 protein levels in a in the of MMTV transcription after hormone with control This in transcriptional activation is not with in GR levels in the cell the of NF1 on chromatin remodeling of the MMTV promoter, assay was with (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google we observed in of in samples with the control samples 1 and 2 with and This that NF1 is required for chromatin remodeling of the MMTV promoter. we performed ChIP with GR and NF1 after NF1 In control siRNA GR and NF1 to the promoter in a NF1 binding to the MMTV promoter was NF1 knockdown in both control and that is NF1 binding in the absence of GR as by ChIP with we observed in GR binding to the promoter of NF1 proteins that GR required NF1 to association with binding on the MMTV promoter organized as GR and NF1 the GR and NF1 to bind to the MMTV promoter, we these proteins to the promoter. demonstrate binding of GR and NF1 on the promoter, we performed a We performed the first ChIP with GR antibody and as was of GR binding to the promoter in the of Using the with GR we with or NF1 NF1 antibody the complexes with GR antibody in the first This suggests that GR and NF1 on the MMTV promoter. of NF1 on of GR-regulated the of NF1 and GR in binding the MMTV promoter, we wanted to the of this NF1 is known to as an transcription factor for regulated by a of by other steroid and GR and NF1 have been to a of promoters 2000; PubMed Scopus Google Scholar). both GR and have similar with in and are a of by these two proteins J.A. G. J. Biochem. Mol. Biol. PubMed Scopus Google G. G. Mol. Cell. Biol. PubMed Scopus Google Scholar). Therefore to the between GR and NF1 on other GR-regulated we two GR-regulated and We the and promoters with the for GR and NF1 binding sites A and promoter is a glucocorticoid gene G.L. Mol. Cell. Biol. PubMed Scopus Google and has NF1 binding sites but their role in activation has not been When we transcription from promoter of NF1 we to on in mRNA levels This suggests that the of binding NF1 may not a role in of the promoter. This also that of NF1 does not of the GR to gene has on and mRNA MCF7M cells were transfected with siRNA to lamin and NF1-C-specific siRNA for 48 h and treated with for RNA was and real-time PCR from RNA were performed using and In to the promoter, NF1 was as a strong factor for promoter and binding at was to be important for this activation R. F. J. 2004; PubMed Scopus Google are NF1 binding and within the promoter. has been to expression of in a of cell lines (7Trotter K.W. Archer T.K. Mol. Cell. Biol. 2004; 24: 3347-3358Crossref PubMed Scopus (87) Google H. J. 2004; PubMed Scopus Google Mol. 2002; PubMed Scopus Google Scholar). when we mRNA levels NF1 is a in response that NF1 is required for activation of this promoter. GR and NF1 on GR-regulated in vivo model to cooperativity between GR and NF1 proteins on other endogenous we the binding of GR to promoter and to promoter. We performed ChIP with GR antibody and for these two The promoter has at between and and we used real-time PCR to this with the mRNA levels GR binding to the promoter in the of hormone was not NF1 knockdown Therefore NF1 to have on mRNA levels or on GR binding to the promoter. with the in mRNA we observed a of the GR at the in the promoter. In to was with the promoter, when we the on promoter with a HRE, the binding of GR in the of hormone was NF1 knockdown and This that binding of GR to promoter is also This is similar to we on the MMTV promoter, which suggests that the binding of GR to promoters that is regulated by both GR and NF1 may be on NF1 These between the and MMTV promoters may a of GR-regulated with a role for NF1 with to GR binding cognate in vivo. transcription multiple the of which are to promoters by transcription We have used the GR and NF1 transcription factors within the context of the MMTV promoter to the of chromatin to combinatorial in vivo. We previously demonstrated that NF1 a as a transcription factor and the other as an protein for chromatin remodeling of the promoter (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). Furthermore we that NF1 binding is required for of GR with the promoter (8Hebbar P.B. Archer T.K. Mol. Cell. Biol. 2003; 23: 887-898Crossref PubMed Scopus (49) Google Scholar). In this study, we that binding of GR on transiently transfected MMTV promoter was not on NF1 the GR NF1 to bind to only in the context of organized chromatin We also demonstrate that both these transcription factors the promoter, which is a for in demonstrated that GR but not NF1 can bind nucleosomes J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). we propose that not only is GR to permit NF1 binding but also that NF1 in or GR association with the promoter in vivo. is a and synergism between GR and NF1 on MMTV promoter, which we propose may be for the of GR with the promoter J.G. Muller W.G. D. R. G.L. 2000; PubMed Scopus Google Scholar). this does not to between GR and NF1 as we to these proteins not This of GR and NF1 binding to the promoter both regulate into transcription factors to bind to their recognition sites on DNA organized as The of combinatorial binding sites for multiple site-specific transcription factors on promoters may be an of the of between with a similar cooperativity has been for phosphoenolpyruvate promoter GR binding has been to be by factors J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). in breast cancer cells, the with regions in with response elements to the of receptor with chromatin J. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Our with GR and NF1 on the MMTV promoter are because this is the first study in which two transcription factors have been to to overcome the chromatin We a similar may on the promoter. Indeed, as with the MMTV promoter, promoter is to BRG1 remodeling complex for activation (7Trotter K.W. Archer T.K. Mol. Cell. Biol. 2004; 24: 3347-3358Crossref PubMed Scopus (87) Google Scholar). the in the of these two promoters with to binding sites for GR and NF1, the chromatin architecture may be similar such that between GR and NF1 may be to permit association of the activation complex with the promoter. the MMTV and the absence of GR and NF1 cooperativity on the promoter is One for such a may be that the chromatin architecture of the promoter permits association of GR in the absence of cooperative GR may other transcription to with the the chromatin architecture and other transcription factor for activation of promoter is are binding sites for transcriptional and transcription factors on the promoter on be to of these factors have similar cooperative binding as NF1. The of cooperativity between NF1 and GR on the and the promoters may factor to the cell and of the The of the HRE, of and the of other regulatory are known to a response to hormone 2004; PubMed Scopus Google Scholar). Recent suggest that of and by GR also transcription Mol. PubMed Scopus Google Scholar). Furthermore, show that can the of GR to bind and in in the of regulatory complexes recruited to the promoter N. Dev. PubMed Scopus Google Scholar). a of factors to cell and control by GR, and we propose that the cooperative binding of site-specific DNA binding proteins can be factor to this We two to the cooperativity between GR and NF1 on promoters both regulate In the first cooperativity between transcription factors can be to be the first in target recognition in which both GR and NF1 bind to their sites to overcome the chromatin This cooperative factor binding may by as by a using DNA-binding proteins, and and protein in (9Miller J.A. Widom J. Mol. Cell. Biol. 2003; 23: 1623-1632Crossref PubMed Scopus (135) Google Scholar). This is followed by the of remodeling which the of the of other regulatory on this cooperativity in the absence of chromatin Therefore in the absence of NF1 we of GR binding and chromatin In this be important to the DNA binding of NF1 are to GR binding to the MMTV promoter. In the we that NF1 a role in a subsequent to factor In this GR initially to the promoter and recruits BRG1 remodeling which the promoter. with this GR is to bind to the promoter in the absence of BRG1 remodeling complex (7Trotter K.W. Archer T.K. Mol. Cell. Biol. 2004; 24: 3347-3358Crossref PubMed Scopus (87) Google Scholar). We propose that NF1 may an role in the remodeling or may be to the of the promoter because in the absence of NF1 chromatin remodeling is Therefore on this cooperativity between GR and NF1 at the remodeling The of the association of the GR with the promoter in the of J.G. Muller W.G. D. R. G.L. 2000; PubMed Scopus Google the of a factor such as NF1, which can the is that in the absence of NF1 binding to the promoter, the of GR with the promoter is This in in the observed of both chromatin remodeling and GR binding on the promoter as in the ChIP the role of BRG1 remodeling complex in cooperative binding of GR and NF1 can aid in a mechanism for this with the that NF1 has a role in GR is evidence that NF1 with and is to chromatin of target promoters and M. N. Dev. PubMed Scopus Google Scholar). Furthermore, of NF1 with is to the of chromatin structure Muller G. N. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This of NF1 is to the protein of factor DNA-binding protein on the N. F. M. M. G. J. 2000; PubMed Scopus (135) Google Scholar). these suggest that NF1 has an role in transcriptional and such a may cooperativity between GR and NF1 in chromatin These results suggest a in of the of these two transcription factors (5Hager G.L. Fletcher T.M. Xiao N. Baumann C.T. Muller W.G. McNally J.G. Biochem. Soc. Trans. 2000; 28: 405-410Crossref PubMed Scopus (25) Google Scholar). Previously the GR was as the that recruited the remodeling complex to the chromatin structure (6Hebbar P.B. Archer T.K. Chromosoma. 2003; 111: 495-504Crossref PubMed Scopus (85) Google Scholar). The binding of NF1 was to be subsequent to this chromatin remodeling Our suggest that NF1 has a role such that the of the GR and remodeling complex both the NF1 protein and a NF1 binding This a in previously of the GR, NF1, and the BRG1 remodeling complex on this promoter (6Hebbar P.B. Archer T.K. Chromosoma. 2003; 111: 495-504Crossref PubMed Scopus (85) Google Scholar). Our suggest a mechanism to cooperative binding of of transcription factors that is to gene We N. for the antibody and for GR (N499) We also M. and for the