Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly. Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly. interferon class II transactivator polymerase chain reaction dimethylsulfate activation response element nuclear factor-1 major histocompatibility complex Major histocompatibility complex (MHC)1 class II molecules play a fundamental role in presenting exogenous antigenic peptides to CD4+ helper T lymphocytes (1Benacerraf B. Science. 1981; 212: 1229-1238Crossref PubMed Scopus (472) Google Scholar). These activated CD4+ T cells release stimulatory cytokines that augment the response of CD8+ cytotoxic T lymphocytes (2Frasca L. Piazza C. Piccolella E. Crit. Rev. Immunol. 1998; 18: 569-594Crossref PubMed Google Scholar). Constitutive expression of MHC class II molecules is restricted to professional antigen-presenting cells, such as B lymphocytes and dendritic cells, and can be induced by interferon-γ (IFN-γ) in macrophages, endothelial cells, and fibroblasts (3Glimcher L.H. Kara C.J. Annu. Rev. Immunol. 1992; 10: 13-49Crossref PubMed Scopus (504) Google Scholar, 4Ting J.P.-Y. Baldwin A.S. Curr. Opin. Immunol. 1993; 5: 8-16Crossref PubMed Scopus (202) Google Scholar). The MHC class II family of genes are coordinately regulated at the level of transcription through a conserved trimeric promoter motif (3Glimcher L.H. Kara C.J. Annu. Rev. Immunol. 1992; 10: 13-49Crossref PubMed Scopus (504) Google Scholar, 5Vilen B.J. Penta J.F. Ting J.P.-Y. J. Biol. Chem. 1992; 267: 23728-23734Abstract Full Text PDF PubMed Google Scholar). A major component of this transcription complex was cloned by genetic complementation of anin vitro mutagenized MHC class II negative B cell line, RJ2.2.5. This component was named the MHC class II transactivator (CIITA) and restored MHC class II cell surface expression in one subgroup of bare lymphocyte syndrome patient cells (6Steimle V. Otten L.A. Zufferey M. Mach B. Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (752) Google Scholar). CIITA has since been shown to be a key regulatory molecule in the expression of all the known genes involved in the MHC class II antigen-presenting pathway (6Steimle V. Otten L.A. Zufferey M. Mach B. Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 7Steimle V. Siegrist C.A. Mottet A. Lisowska-Grospierre B. Mach B. Science. 1994; 265: 106-109Crossref PubMed Scopus (679) Google Scholar, 8Chin K.-C. Mao C. Skinner C. Riley J.L. Wright K.L. Moreno C.S. Stark G.R. Boss J.M. Ting J.P.-Y. Immunity. 1994; 1: 679-689Abstract Full Text PDF Scopus (130) Google Scholar, 9Chang C.-H. Fontes J.D. Peterlin M. Flavell R.A. J. Exp. Med. 1994; 180: 1367-1374Crossref PubMed Scopus (297) Google Scholar). CIITA is required for constitutive expression of MHC class II on B-lymphocytes (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar) and with a few exceptions, such as in respiratory epithelial cells and dendritic cells (11Gao J. De B.P. Banerjee A.K. J. Virol. 1999; 73: 1411-1418Crossref PubMed Google Scholar, 12Williams G.S. Malin M. Vremec D. Chang C.H. Boyd R. Benoist C. Mathis D. Int. Immunol. 1998; 10: 1957-1967Crossref PubMed Scopus (81) Google Scholar), is required for cytokine-induced expression in other cell types. Mice in which functional CIITA protein was ablated clearly demonstrated an absolute requirement for CIITA in B cell expression of MHC class II (13Chang C.H. Guerder S. Hong S.C. van Ewijk W. Flavell R.A. Immunity. 1996; 4: 167-178Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). It has been shown that the CIITA gene has four distinct promoters each transcribing a unique first exon, which are located within approximately 13 kilobases of DNA (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). The promoters are utilized in a tissue-specific manner. CIITA expression in dendritic cells is primarily from promoter I. Promoter III is predominantly used in B-lymphocytes, while promoter IV is used predominantly in IFN-γ-induced expression. The first exon associated with the B cell and dendritic cell promoters encodes distinct amino-terminal residues of 24 and 101 amino acids, respectively, while mRNA from promoters II and IV initiates translation from within the common exon 2 (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). Promoter II has yet to be characterized. The IFN-γ-responsive control elements at CIITA promoter IV were recently characterized (14Piskurich J.F. Linhoff M.W. Wang Y. Ting J.P.Y. Mol. Cell. Biol. 1999; 19: 431-440Crossref PubMed Google Scholar, 15Muhlethaler-Mottet A. Di Berardino W. Otten L.A. Mach B. Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Both STAT-1 and IRF-1 are required at the proximal promoter for full response. Binding of the USF-1 transcription factor stabilizes STAT-1 binding (15Muhlethaler-Mottet A. Di Berardino W. Otten L.A. Mach B. Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Initial mapping of the CIITA promoter III revealed that sequences that lie between −545 and −113 base pairs relative to the transcription start site are required for constitutive expression in B-lymphocytes (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). While this region of the promoter is not responsive to IFN-γ, IFN-γ activation of CIITA promoter III can be observed when an additional 4000 base pairs of upstream DNA are present (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). However, nothing is known about the specific factors and promoter elements that control constitutive transcription from CIITA promoter III in B-lymphocytes. Tight of the expression of MHC class II genes has been observed B cell MHC class II gene transcription in the cell is B-lymphocytes M. S. S. S. J. Immunol. 1992; Google Scholar, D. Int. Immunol. 1993; 5: PubMed Scopus Google Scholar), and is when B-lymphocytes cells A. L. J. Immunol. Google Scholar, M. A. L. M. J.L. S. A. PubMed Scopus Google Scholar). CIITA expression is in the cell and Mottet A. Steimle V. W. Mach B. J. Exp. Med. 1994; 180: PubMed Google Scholar, A. Annu. Rev. Immunol. 1998; 16: PubMed Scopus Google Scholar). The of activation and are CIITA promoter III not sequences with to B cell a binding In this now the CIITA promoter III regulatory elements in B-lymphocytes and in vivo and in vitro five occupied two of the sites are required for CIITA transcription in B-lymphocytes. is a TEF-2-like while the other a binding site for a novel transcription activator. The CIITA promoter III and the CIITA promoter III sequences from to −545 to base respectively, relative to the transcription These two were in the and as and (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar), for this been the of the other and were and CIITA promoter III sequences from to the on the of the mutations as were in the were by the unique site 1992; PubMed Scopus Google Scholar). In the the sequence was to in In was to In the was to In the was to In the was to additional with mutations in the activation response sequence were in the The first is in the of the −545 base pair while the and are in the of the base pair The specific base changes are shown in I. mutations were by The expression was cloned by transcription and by sequence element and four activity is the activity of protein from was with the and the is the of activity of the by the activity of the and are in the of the pair promoter while and are in the of the pair sequence are as and in a activity is the activity of protein from was with the and the is the of activity of the by the activity of the and are in the of the pair promoter while and are in the of the pair sequence are as and is a cell that constitutively MHC class II B.J. Penta J.F. Ting J.P.-Y. J. Biol. Chem. 1992; 267: 23728-23734Abstract Full Text PDF PubMed Google Scholar). and are cell and were in 2 and and cells were with were by using the II were with at a of and cells were for cell were to protein with the protein In vivo of cells with dimethylsulfate and DNA were as 4: PubMed Scopus Google Scholar). In vivo footprinting was as by and S. 1997; PubMed Scopus Google Scholar). cells were by at and at in were on a and to light the cells were by the of an of The DNA was and as for the The DNA was at as using and of by A. of in vitro DNA were by first and the genomic DNA from cells, of DNA in of and the DNA as on and was as for the in vivo The polymerase chain in vivo genomic footprinting was as B. Science. PubMed Scopus Google Scholar) and by Wright and Ting K.L. Ting J.P. S. A. 1992; PubMed Scopus Google Scholar). The was revealed by using two and The sequence of the for are 2 and The sequence of the for are 2 and The was revealed by two and The sequence of the for are 2 and The sequence of the for are 2 and were to J.D. PubMed Scopus Google Scholar). analysis was as K.L. van J.L. G.S. 1992; PubMed Scopus Google Scholar) using and of the The from to base pairs of the promoter and is The the as in The from to of the promoter and is The the as in The site A from to base pairs of the promoter and is The site B from to of the promoter and is The and the as in and The site from to base pairs of the promoter relative to the transcription site and is The is van W. van EMBO J. 4: PubMed Scopus Google the is R. C. R.A. J. Exp. Med. 1994; PubMed Scopus Google the is Wang S. B. M. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar). The is M. E. A. Y. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). were used in a was from cells using the was transcribed using the and in of The of that a of for each was was in the of using and of the for each reaction was in the of each 2 of DNA polymerase and 2 of in a of The CIITA promoter III has been shown to be constitutively active in B-lymphocytes (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). the functional of the a of deletions of CIITA promoter III to the gene were and the B cell base pairs of the promoter the first base pairs of sequences of activity upstream sequences base pairs of promoter III not transcriptional activity (16Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). This that the first base pairs of CIITA promoter III are for transcriptional activation in B-lymphocytes. of the region from base pair to to a in transcriptional which suggests the of a in this pair of base pairs to not activity. However, of the region between base pairs and −113 to of that observed with the This suggests the of a element in this pair were by the B cell not In vivo genomic analysis of promoters has been in the of protein/DNA within the of the intact This was to occupied DNA elements present in the CIITA In B cells both of the promoter were from to base pairs using dimethylsulfate as the 2 in of transcription factors in vivo with DNA can of The of is and with the observed when the are to of the strong between base pairs and 2 2 and with These within the strong activation in between base pairs and of this element be as not residues on the reveal on the 2 and A region vivo protein/DNA is observed between base pairs and and is activation response A strong and a are on the while the strong and In to these two of in vivo sites of were A of five site A is between and to a sequence with to the transcription factor binding are observed on the and while the two and a and A of site is located at a site pairs to and is as a on the by a and on the a is observed at on the and and is site C. were not on upstream of base pair and two were observed at base pairs and of the sequence of CIITA promoter III and in vivo protein/DNA in B-lymphocytes. and in vivo protein/DNA observed of of of in vivo are by V. The of each in is by a and The transcription site is by at the CIITA promoter III correlates with CIITA and MHC class II expression in myeloma cell transcription demonstrates that not CIITA MHC class II while of B cell lines, and are shown as the are in the each of with of was to control for the and not in vivo analysis of CIITA promoter III in myeloma cell This is the as in 2 A are of the promoter in the cells, and are of the promoter in the A of is observed in the line, while the is completely of in vivo are as for 2 In vivo footprinting with is to analysis of which to In to the analysis of on CIITA promoter in vivo genomic footprinting using light as the in of Ultraviolet light the of that can be DNA on and promoters demonstrated of associated with known sites vivo protein/DNA R. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, S. J. Mol. Biol. PubMed Scopus Google Scholar). of CIITA promoter III demonstrates two strong on the DNA at the element 2 This is revealed by the in vivo at these two pairs with the pairs at of This the strong in vivo that were by on the and the of the A of strong is located between base pairs and to the on the at site C. This that site is a site of in vivo protein In the in vivo footprinting analyses five sites of protein/DNA within the first base pairs of the promoter of B-lymphocytes cells is by of MHC class II gene expression J. Wang R. J. Immunol. Google Scholar). In the cell to CIITA expression was and of CIITA MHC class II expression Mottet A. Steimle V. W. Mach B. J. Exp. Med. 1994; 180: PubMed Scopus Google Scholar). However, of other cell that MHC class II S. A. S. 1997; PubMed Google Scholar). This be to the of of the cell cell were to the level of CIITA expression and in vivo protein/DNA at the CIITA promoter were cell line, has MHC class II CIITA mRNA In the cell has of both with the B cell and by in vivo genomic CIITA promoter III was completely bare of all in vivo protein/DNA in the cell In in the cell were at the site and site B elements, which were from observed in the B cell site binding was in the cells when by in vivo footprinting not These findings that of CIITA transcription in cells is mediated through the of activity of a promoter However, transcriptional which in cell and was observed in cells is by of the CIITA promoter the functional of the in vivo binding site-specific mutations of the sequences protein/DNA were and these mutations for on transcriptional activity of the mutations were in the of the pair CIITA promoter the B cell of the region transcriptional activity to of activity The in activity the loss of activity observed when the pair region was these findings that the element is a critical transcriptional in B of the in vivo occupied element from base pair to transcriptional that is critical for transcription The of this was by the promoter in The of the in vivo binding at site site and site were by base pairs at the of each activity was not by of of the sites In to site site of mutations were and In each of transcription was in with the of the site binding site not activity with the of These findings that the elements for CIITA promoter III activity are and of the to the and elements was with a of in vitro protein/DNA binding the and DNA sequences known transcription factor binding of an the DNA element with nuclear from B-lymphocytes revealed two specific of each of the was by the of a of The two are not B since are in cell with a element an the site element not the binding and A between and the B Wang S. B. M. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar), as as the transcription factor A. C. R.A. Cell. Full Text PDF PubMed Scopus Google Scholar), was by with the transcription factor I. I. E. 1999; PubMed Scopus Google Scholar). A binding site from the promoter Wang S. B. M. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar) not for the two of the complex However, of a expression with CIITA promoter III a element not the activity of the CIITA while control promoter were responsive to This that is to be the factor at the element in A binding site not for of the a of with a region of the promoter that binding sites for and I. R. A. Cell. Full Text PDF PubMed Scopus Google Scholar, M. EMBO J. 8: PubMed Scopus Google Scholar). In addition, an with a binding sequence (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). the of these binding site a of site-specific mutations were in the CIITA promoter and for the loss of transcriptional activation in B cells of the of the and sites not transcriptional activity when with the However, mutations and that the central sequence of in a loss of activity to of the This sequence is a of to the the between the and in vitro binding were The are with a using the as a DNA binding site a strong specific complex was observed the of demonstrated that was as as the site in the This that binding is to the binding activity is a and the of strong in vivo in this region with the binding observed in that the of the complex on with other at the CIITA A of were to the binding A specific complex is when an the element is with B cell nuclear The complex is by the of a of an the sequence The of an that the element with the which transcriptional not for binding to this complex the element not to known transcription a of and DNA binding elements were used as to the of the complex of the for the binding The region from the CIITA promoter III of in the the sequence not for the complex The of is not to the B cell and can be in cell and not A and site B are located within the first base pairs upstream of the transcription start site and were on two of in vivo These sites were for specific factors binding in vitro. The site A sequence one complex when with a B cell nuclear This complex was by with a of site A not with the site A sequence the and A sequences with to the binding site for van W. van EMBO J. 4: PubMed Scopus Google Scholar) and a to the binding site R. Science. PubMed Scopus Google Scholar). the binding sequence the complex when used as a a sequence for binding not The a complex when is used as the the site in CIITA promoter III can for this complex This strong that is the factor at this in vivo site of protein/DNA B was to be an factor binding site on a of base pair to the sequence (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). However, of factor be observed at site B not In strong binding of both and factors was observed when the characterized MHC site was used as a in the This that site B has for factors and is with the in vivo and the of transcriptional activation from this In this characterized CIITA promoter which has been demonstrated to be the active promoter in B-lymphocytes (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar, J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar, C. J. M. R. C. 1997; PubMed Scopus Google Scholar). that the first base pairs of CIITA promoter III are for transcriptional activity in B now vivo genomic footprinting to the occupied promoter elements in intact B strong been in the first base pairs of the In addition, the region between and base pairs a in activity two and in were in this is located between to base pairs and a protein/DNA by both in vivo analysis and in The element has to transcription factor binding a factor that is in B cells and has a expression to was present in one of the of binding to in vitro. However, of the protein in B cells not CIITA promoter III activity. is to play an role at the site in CIITA to the binding site for the of the sequence not the conserved region in a loss of transcriptional activity. TEF-2-like binding activity was vitro at the with with the has been shown to be a transcriptional present in cell M. E. A. Y. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). to the family of transcription which the factor and to CIITA be activated by as yet family this sequence motif is conserved in the CIITA promoter III sequence at an relative to the transcription start site (10Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (429) Google Scholar). region critical for transcriptional activity is the region located from to base pairs relative to the transcription The element not to the binding site of known transcription of this site the binding of a transcriptional to this site is for CIITA the sequence of the CIITA promoter III is not conserved with the sequence in this the sequence not with the sequence for the binding This that in to a role for the binding factor be present at the CIITA promoter III. the factors at and are critical for CIITA transcription and MHC class II expression in B-lymphocytes. However, role in the B cell of the promoter is not Both of these factors are present in from all cell such as and However, this not from regulated in a B manner. It is that these factors to be activated by which be present of B cell to the of and M. Cell. Full Text PDF PubMed Scopus Google Scholar). The of B cell through binding of Y. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar) is to play a role in CIITA B cell The binding site in the promoter is present at site B and not transcription with located base pairs upstream of the transcription start has been demonstrated to the transcription factor is involved in both transcriptional activation and R.A. J. S. A. PubMed Scopus Google Scholar, B. Cell. Full Text PDF PubMed Scopus Google Scholar). It as a to the site of site is a pair with the sequence In vitro DNA binding as by demonstrated strong specific binding of to site A. of this site not transcriptional activity. this not a role for in CIITA transcription in has been demonstrated to the site of promoters such as the promoter and the promoter 1996; PubMed Scopus Google Scholar, Y. E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). play an role in transcription start site The CIITA promoter III a and yet predominantly initiates transcription from a site base pairs of the a report an element located at approximately base pairs upstream of the the site was not by the C. J. M. R. C. 1997; PubMed Scopus Google Scholar). with the transcriptional The functional of binding and the of start site at CIITA promoter III to be B-lymphocytes to cells, MHC class II expression is However, in cell lines, the level of MHC class II on the cell surface from to of two cell as that the level of MHC class II expression is with the level of CIITA expression. In footprinting that in cell the CIITA promoter III is while in cell full promoter is This suggests that of CIITA transcription in the of be mediated through of the activity of promoter In of the CIITA promoter and an of the of CIITA transcription in In characterized the CIITA promoter in B-lymphocytes and two activation to a TEF-2-like and a novel transcription both of which are to play a role in MHC class II expression. of these is key to at MHC class II expression such as in of expression in myeloma and other MHC class II negative cells in to for the of E.
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