Multiple Ets Factors and Interferon Regulatory Factor-4 Modulate CD68 Expression in a Cell Type-specific Manner

CD68 is a transmembrane glycoprotein expressed in all cells of the mononuclear phagocyte lineage including monocytes and tissue resident macrophages. Deletion analysis of the 5′-flanking sequences of the gene demonstrated that the proximal –150-bp sequence of the CD68 promoter exhibits high level promoter activity in macrophages. Mutations that abolish Ets factor binding at positions –106 and –89 reduce promoter activity in macrophages to 12 and 30%, respectively. Band shift experiments show that PU.1 associates with the –89 site whereas, Elf-1 preferentially binds the –106 Ets binding site and enhances CD68 activity in vitro. Furthermore, chromatin immunoprecipitation experiments confirm that Elf-1 and PU.1 associate with the CD68 proximal promoter in vivo in THP-1 cells. PU.1 does not bind to the CD68 promoter alone but instead forms heterocomplexes with members of the interferon regulatory factor family (IRF) including IRF-4 and IRF-8. IRF-4 and IRF-8 typically mediate transcriptional activation when associated with PU.1 on composite elements. However, our data show that PU.1/IRF-4 and IRF-8 heterocomplexes down-regulate CD68 promoter activity in macrophages and repression is dependent on the integrity of both the IRF and PU.1 half-sites of this composite element. Chromatin immunoprecipitation data reveal that neither IRF-4 nor IRF-8 associate with the CD68 proximal promoter in macrophages in vivo but IRF-4 is associated with the promoter in B lymphocytes. We propose that expression of CD68 in myeloid cells requires the Ets transcription factors Elf-1 and PU.1 and CD68 expression is down-regulated in lymphoid cells by combinatorial interactions between PU.1 and IRF-4. CD68 is a transmembrane glycoprotein expressed in all cells of the mononuclear phagocyte lineage including monocytes and tissue resident macrophages. Deletion analysis of the 5′-flanking sequences of the gene demonstrated that the proximal –150-bp sequence of the CD68 promoter exhibits high level promoter activity in macrophages. Mutations that abolish Ets factor binding at positions –106 and –89 reduce promoter activity in macrophages to 12 and 30%, respectively. Band shift experiments show that PU.1 associates with the –89 site whereas, Elf-1 preferentially binds the –106 Ets binding site and enhances CD68 activity in vitro. Furthermore, chromatin immunoprecipitation experiments confirm that Elf-1 and PU.1 associate with the CD68 proximal promoter in vivo in THP-1 cells. PU.1 does not bind to the CD68 promoter alone but instead forms heterocomplexes with members of the interferon regulatory factor family (IRF) including IRF-4 and IRF-8. IRF-4 and IRF-8 typically mediate transcriptional activation when associated with PU.1 on composite elements. However, our data show that PU.1/IRF-4 and IRF-8 heterocomplexes down-regulate CD68 promoter activity in macrophages and repression is dependent on the integrity of both the IRF and PU.1 half-sites of this composite element. Chromatin immunoprecipitation data reveal that neither IRF-4 nor IRF-8 associate with the CD68 proximal promoter in macrophages in vivo but IRF-4 is associated with the promoter in B lymphocytes. We propose that expression of CD68 in myeloid cells requires the Ets transcription factors Elf-1 and PU.1 and CD68 expression is down-regulated in lymphoid cells by combinatorial interactions between PU.1 and IRF-4. Monoclonal antibodies of the CD68 cluster show strong staining of all monocytes and macrophages in normal human tissues and CD68 is widely regarded as a pan macrophage marker in immunohistochemistry studies (1Parwaresch M.R. Radzun H.J. Kreipe H. Hansmann M.L. Barth J. Am. J. Pathol. 1986; 125: 141-151PubMed Google Scholar, 2Pulford K.A. Sipos A. Cordell J.L. Stross W.P. Mason D.Y. Int. Immunol. 1990; 2: 973-980Crossref PubMed Scopus (158) Google Scholar). The protein recognized by CD68 antibodies is a member of the lysosome-associated membrane protein family (3Holness C.L. Simmons D.L. Blood. 1993; 81: 1607-1613Crossref PubMed Google Scholar). Human CD68 shows 72% identity with the murine macrosialin protein and displays the same overall organization with an N-terminal 117-amino acid mucin domain separated from a 165-amino acid lysosome-associated membrane protein domain by a 13-amino acid proline-rich spacer region (4Holness C.L. da Silva R.P. Fawcett J. Gordon S. Simmons D.L. J. Biol. Chem. 1993; 268: 9661-9666Abstract Full Text PDF PubMed Google Scholar, 5Rabinowitz S. Horstmann H. Gordon S. Griffiths G. J. Cell Biol. 1992; 116: 95-112Crossref PubMed Scopus (229) Google Scholar, 6Rabinowitz S.S. Gordon S. J. Exp. Med. 1991; 174: 827-836Crossref PubMed Scopus (188) Google Scholar). Its function, as yet, has not been established but CD68 and macrosialin have been shown to bind and uptake oxidized low density lipoproteins, which are accumulated in macrophage-derived foam cells within atherosclerotic plaques (7Ramprasad M.P. Fischer W. Witztum J.L. Sambrano G.R. Quehenberger O. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9580-9584Crossref PubMed Scopus (296) Google Scholar, 8Ramprasad M.P. Terpstra V. Kondratenko N. Quehenberger O. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14833-14838Crossref PubMed Scopus (313) Google Scholar, 9Van Velzen A.G. Da Silva R.P. Gordon S. Van Berkel T.J. Biochem. J. 1997; 322: 411-415Crossref PubMed Scopus (49) Google Scholar). CD68 is one of 9 genes in a 140-kb region of the human chromosome 17p13 (10Jones E. Quinn C.M. See C.G. Montgomery D.S. Ford M.J. Kolble K. Gordon S. Greaves D.R. Genomics. 1998; 53: 248-250Crossref PubMed Scopus (13) Google Scholar). It lies 669 bp 3′ to the human eukaryotic initiation factor 4AI gene and 5′ to a MPDU1 gene, which encodes a repressor of the Lec 15 and Lec 35 glycosylation mutants of Chinese hamster ovary cells. All of the genes clustered around CD68 have very different expression profiles, being expressed in different tissues with very different developmental specificities. Its close proximity, however, to a housekeeping gene, EIF-4A1, suggests that the CD68 gene is likely to be in a permanently open chromatin configuration (11Quinn C.M. Wiles A.P. El-Shanawany T. Catchpole I. Alnadaf T. Ford M.J. Gordon S. Greaves D.R. Genomics. 1999; 62: 468-476Crossref PubMed Scopus (15) Google Scholar). We have shown that the first intron of the EIF-4A1 gene contains a methylation free island, which as a in a of D.R. Quinn C.M. Gordon S. Genomics. 1998; PubMed Scopus Google Scholar). the CD68 gene not a chromatin region but which to expression to macrophages. The to regulatory to expression to macrophages to and the CD68 studies show that sequences between and of the human CD68 gene promoter activity in macrophage and that Ets factors bind this promoter in and in of the promoter is a composite site that binds PU.1 and interferon regulatory factor members IRF-4 interferon of myeloid shift interferon of myeloid shift and IRF-8. both factors are to CD68 promoter activity in a in in THP-1 IRF-4 is associated with the promoter in vivo in B lymphocytes. We propose that CD68 expression is in different by and of transcription including Ets family members and IRF-4. from human macrophages by of 5′ a 5′ transcription of of human macrophage the first as a with a 5′ and the 3′ and with and from (10Jones E. Quinn C.M. See C.G. Montgomery D.S. Ford M.J. Kolble K. Gordon S. Greaves D.R. Genomics. 1998; 53: 248-250Crossref PubMed Scopus (13) Google Scholar). The by with and the site of to The 3′ site contains the CD68 initiation which by the activity of bp of of the CD68 the to the with to the All CD68 promoter in by as a and that a site and a 3′ that the site of The to CD68 promoter and between the and of the to the 5′ of the CD68 promoter in and that a 5′ by from to the 5′ between and of 5′ in the Ets binding site and the composite site the and the as to the composite element. The contains a within the IRF binding site and the contains a at the PU.1 binding The Ets sequences at –106 and the and are All promoter by and from by and by in by and J. E. T. Scholar). expression murine PU.1 and human Elf-1 of The a of and a murine a of expression by All Ets the expression to Ets factors expressed in the IRF-4 a from and murine IRF-8 in a of genes Cell and murine macrophage and the murine B the human the human and and the human B and from cells in with and cells in with and and and cells in with and and cells to and in and in cells cells. of cells with of of and of to a and in a at of that been to between and with and cells. 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PubMed Scopus Google Scholar). by a of with in and activity with with and by at density in in and at a of of at the and in by and the at in The B cells from the cells by the of at a of of at with and The B cells a in and from the to the and B cells in and as Chromatin immunoprecipitation to the of T. Biol. PubMed Scopus Google Scholar). immunoprecipitation and cells with a of to the at by the of at in and in and on at low and in B and to bp by the on at 15 at and with cells 15 at S. cells with and at with and and of a from S. to the chromatin and at of a to the the at and to and in in and as J. J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). by the of of and at at and at with and at with in of and by chromatin from cells in of and at in of of of and of in a of at 15 by of and and a at separated by a and by The sequences of the as CD68 proximal promoter and CD68 and human and and and and of the CD68 at with genes the CD68 promoter contains neither a binding nor The transcription initiation of the human CD68 gene by and 5′ analysis of human macrophage cluster of 5′ and in the site of CD68 initiation by sequence analysis of CD68 5′ The positions of CD68 transcription are by the CD68 gene sequence CD68 in human macrophages a region of bp of the initiation of the CD68 of clustered transcription has been in the macrosialin gene, which encodes the murine of CD68 J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. Genomics. 1998; PubMed Scopus Google Scholar). of the in CD68 transcription the of the initiation of the CD68 of the CD68 in and Cell the of the CD68 promoter gene expression to cells of the mononuclear lineage the murine macrophage and bp of sequence 5′ of the CD68 initiation the to the shown to high of gene expression in macrophages the of sequence in the CD68 5′-flanking which contains of the 3′ of the EIF-4A1 gene, a of 5′ of the CD68 5′-flanking sequence in the The level of expression in cells with a bp of sequence 5′ of the CD68 initiation The level of expression with this of the CD68 promoter is that with the in the same gene Deletion of sequences between and to a in CD68 promoter of sequences between and abolish promoter the CD68 of CD68 promoter activity in the activity by in and and as as in cells as with with macrophage cells. experiments that sequences between and expression in macrophages. The of the CD68 analysis of the proximal promoter region a of on at positions and with the sequence sequence is recognized by transcription factors of the Ets family including a factor to be expression of a of genes Biol. PubMed Scopus Google Scholar, S. Biol. 1995; PubMed Google Scholar, K. H. Biol. PubMed Scopus Google Scholar, T. 1995; Google Scholar). binding the CD68 proximal to of the gene in and a of and to the Ets on the experiments in THP-1 cells and by with a the sequence at by with an site a has the on CD68 promoter of expression and the –89 site promoter activity by to and has and data that the Ets binding at positions –106 and –89 are promoter activity in macrophages. 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All Ets factor the same expression to The with and PU.1 expression shown in Elf-1 CD68 promoter activity and expression We that the Ets factors and PU.1 not the CD68 We chromatin immunoprecipitation analysis to Ets factors are to the CD68 proximal promoter in macrophages in THP-1 cells as and with antibodies to and an as with the proximal promoter region of the CD68 a to the CD68 shows a of the CD68 gene and the by the CD68 promoter and alone and to The data of show that and Elf-1 are all associated with the proximal promoter of the CD68 gene in THP-1 cells. The this region a of from the and the and the of the CD68 gene an of the from the of this region that are Ets binding However, is that associates with the at a binding site that binds to the by with a factor to a different sequence element. to a in the same not data show that Ets factors the CD68 proximal promoter in and bind the CD68 promoter in of the PU.1 to the –89 CD68 the factor to the –89 CD68 the sequence and a binding site members of the IRF 5′ of the of this binding with composite strong sequence are IRF family members to with IRF-4 and IRF-8 to lymphoid and myeloid cells M.J. H. J. 1999; PubMed Scopus Google Scholar, T. K. A. N. Immunol. PubMed Scopus Google Scholar, S. M.J. J. PubMed Scopus Google Scholar, S. A. D. J. PubMed Scopus Google Scholar). between PU.1 and IRF members have been E. U. H. 1996; PubMed Scopus Google Scholar, H. J. 1999; PubMed Scopus Google Scholar, M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, D. H. J. Immunol. 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PU.1 and PU.1/IRF-4 that neither IRF-4 nor IRF-8 bind to the in the of of the composite from including human with the composite 3′ in a show that the on the –89 CD68 on the binding of IRF-4 IRF-8 to the sequence 5′ of the PU.1 the IRF binding site the PU.1 site of the IRF binding site binding but does not the binding of Elf-1 PU.1 alone have a composite binding factors on the CD68 promoter and shown that binding of IRF-4 IRF-8 requires with IRF-4 and IRF-8 CD68 in the composite the PU.1 and IRF binding within the of the to CD68 experiments and CD68 in THP-1 cells in the of IRF-4 IRF-8. the of IRF of the PU.1 binding site promoter activity by of as of the IRF site to on transcription It that the CD68 promoter does not the IRF binding site activity in macrophages. however, when IRF-4 promoter activity of the is by and of the IRF binding site this suggests that IRF-4 the this transcriptional repression to IRF-4 is to promoter by of when the PU.1 binding site is We from data that IRF-4 associate with the in the of PU.1 However, IRF-4 be to associate with the and activity in the of PU.1 when this factor bind to the at this Elf-1 preferentially associates with the –106 site PU.1 bind this site and is that IRF-4 repression on the CD68 promoter interactions with PU.1 to the –106 site when the –89 site is data that IRF-8 likely to associate with the composite with of IRF-8 in THP-1 cells CD68 activity by of of the IRF site the PU.1 site this data that IRF-4 and IRF-8 the transcriptional repression to and that both factors with PU.1 to this of IRF-4 and IRF-8 in is expressed in cells of the mononuclear both IRF-4 and IRF-8 to CD68 promoter activity to of factors are expressed in that not CD68 experiments on from including and B and and a murine All protein by and as with the B and shown in IRF-4 and IRF-8 to be expressed in the different IRF-8 is expressed in and cells IRF-4 to be to the B and cells very and cells to the and cells. We to this expression of IRF-4 and IRF-8 human B cells and human from B which from human from different and one IRF-4 is expressed in the human B cells with of IRF-8 expression level is very to in human and murine B Human on the show a expression to and cells and low of IRF-4 expression is the expression of IRF-4 and IRF-8 in human B and human monocytes to the of expression in the human in IRF-4 with the CD68 in B to IRF-4 binds the CD68 promoter in vivo in macrophages B cells. THP-1 and cells as and with an alone and as and respectively. It has been that IRF-4 with PU.1 at a composite within an of the human gene S. M.J. J. Immunol. PubMed Scopus Google and a site within the proximal promoter of the human gene in in experiments myeloid A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We to both of promoter as IRF-4 binding and to the human gene as a We not binding of the IRF-4 factor to the gene in of the and However, IRF-4 binding to of the gene and in the but to show of IRF-4 with the CD68 proximal promoter in macrophages IRF-4 is associated with the CD68 promoter in B and cells and with an a IRF-4 binding the composite within the proximal promoter of the gene, which shown to bind IRF-4 in in B A. A. Blood. 1997; PubMed Google Scholar). We to the gene as and to the gene as a IRF-4 associates with the promoter in both B and to bind the IRF-4 binds the human gene in both monocytes and B and However, in to the IRF-4 with the CD68 proximal promoter in B lymphocytes. IRF-4 as a of CD68 activity in THP-1 cells propose that IRF-4 associates with the CD68 promoter in vivo in B to down-regulate CD68 expression in cells. this have the transcriptional of CD68 in human macrophages and the proximal promoter that activity in cells. We have that are CD68 activity in macrophages that bind members of the Ets family of transcription factors including and of is a composite site that binds PU.1 and interferon regulatory factor IRF-4 and IRF-8. PU.1/IRF-4 and heterocomplexes transcriptional activation activity in cells. our that PU.1/IRF-4 and CD68 promoter activity in human cells. Furthermore, chromatin immunoprecipitation experiments reveal that IRF-4 associates with the CD68 promoter in vivo in B and not show that the Ets family transcription factors and bind the CD68 proximal promoter in and in are 35 members of the Ets transcription factor family to be in a of from and PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar). a with the and a sequence with sequences the binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PU.1 is the member and is the of myeloid and lymphoid J. H. PubMed Scopus Google Scholar, H. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that Elf-1 preferentially binds the –106 site and enhances CD68 promoter activity by in Mutations that the sequence at –106 abolish promoter activity in macrophages of Furthermore, chromatin immunoprecipitation analysis that Elf-1 is to the proximal promoter in vivo in THP-1 cells analysis of cells including and including and Chinese hamster ovary show expression of Elf-1 protein not We to that not Elf-1 to promoter activity and propose that the of activity that with the CD68 promoter in is to the of the that when Elf-1 in is an of the of this Ets factor on the CD68 Chromatin immunoprecipitation to in vivo transcription factor that binds the CD68 proximal promoter with as with the region are Ets binding within this region and as is to that binds to an sequence by with a factor to the It however, to associate with the –106 site within the proximal promoter in and the CD68 promoter in We have shown that Elf-1 preferentially binds this site and as is to that activity by with Elf-1 the –106 this is the this an to that CD68 expression is in all macrophage in with the CD68 promoter a of THP-1 cells in a of the a activation J. 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IRF-4 and IRF-8 typically as transcription when heterocomplexes with However, our data that IRF-4 and IRF-8 CD68 activity in a in THP-1 cells B and repression requires as that abolish IRF as by this experiments with that neither IRF-4 nor IRF-8 bind the composite in the of with PU.1 IRF-8 to CD68 promoter activity when the PU.1 IRF-4 to activity by as as when the PU.1 site in binding between IRF-4 and IRF-8 the in the of PU.1 is that repression is interactions with Elf-1 at the –106 analysis that PU.1 bind the –106 site and and as is likely that in the of the –89 PU.1 PU.1 with Elf-1 the –106 site and IRF-4. with the mutants and on CD68 promoter activity that of the IRF to have on promoter activity in THP-1 of the PU.1 binding site activity by as as that IRF factors are not CD68 promoter activity in myeloid cells but that Ets factor binding is data and chromatin immunoprecipitation analysis to both the the CD68 promoter activity in THP-1 cells and the of the PU.1/IRF-4 and repression of CD68 activity in cells. data show that IRF-8 is expressed in myeloid including cells from IRF-4 is expressed in cells but expressed in B including murine and B and B cells not which the that the of IRF-4 IRF-8 be to CD68 activity in Chromatin immunoprecipitation data our and that IRF-4 to the CD68 promoter in vivo in the B and and not to associate with this promoter in THP-1 cells and It demonstrated that IRF-4 in a lineage and A. A. Blood. 1997; PubMed Google Scholar). expressed in cells and as cells to cells. dependent on interactions with IRF-4 and PU.1 at the composite PU.1 the experiments are in to this of IRF-4 to the CD68 promoter in B cells is an that an IRF-4 is to lymphoid cells be a factor as cells myeloid which CD68 expression in on data propose that IRF-4 with PU.1 and binds the composite within the CD68 promoter in vivo in B to down-regulate CD68 expression in cells. It is that the in our chromatin immunoprecipitation experiments and show the first that IRF-4 is associated in vivo with human and human We propose that IRF-4 an in CD68 expression in B lymphocytes. studies have a regulatory within the CD68 promoter and of the that expression of the human CD68