Nuclear Protein Interactions with the Human KDR/flk-1 Promoter in Vivo

The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs −110 and −25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1. The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs −110 and −25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1. INTRODUCTIONKDR/flk-1 is a membrane-bound receptor of the tyrosine kinase family with expression restricted predominantly to endothelial cells (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar). KDR/flk-1 and a similar tyrosine kinase, flt-1, are receptors for the specific endothelial cell mitogen and angiogenic peptide vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorEMSAelectrophoretic mobility shift assayDMSdimethylsulfateLM-PCRligation-mediated polymerase chain reactionHUVEChuman umbilical vein endothelial cellbpbase pairBSAbovine serum albumin. (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar, 2de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1879) Google Scholar, 3Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (670) Google Scholar). Both receptors are expressed early in murine development, with KDR/flk-1 appearing a full day earlier (day 7.0-7.5 of the developing mouse embryo) than flt-1 (4Dumont D.J. Fong G.H. Puri M.C. Gradwohl G. Alitalo K. Breitman M.L. Dev. Dyn. 1995; 203: 80-92Crossref PubMed Scopus (444) Google Scholar, 5Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development (Camb.). 1993; 118: 489-498Crossref PubMed Google Scholar). Of the two, KDR/flk-1 has a wider pattern of expression among endothelial cell populations than does flt-1 (6Kaipainen A. Korhonen J. Pajusola K. Aprelikova O. Persico M.G. Terman B.I. Alitalo K. J. Exp. Med. 1993; 178: 2077-2088Crossref PubMed Scopus (215) Google Scholar). In addition, only KDR/flk-1 has been shown to autophosphorylate in the presence of VEGF (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar), and signal transduction mechanisms downstream of the two receptors differ (7Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). These differences in expression and signaling suggest that the two receptors mediate different physiologic functions of VEGF in endothelial cell growth and angiogenesis.Recent studies in which the genes for the two VEGF receptors were deleted in mice by homologous recombination have provided critical data about KDR/flk-1 function (8Shalaby F. Rossant J. Yamaguchi T. Gertsenstein M. Wu X.-F. Breitman M. Schuh A. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3332) Google Scholar, 9Fong G. Rossant J. Gertsenstein M. Breitman M. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2201) Google Scholar). In mice with homozygous deletions of flt-1, endothelial cells develop normally, but vessel formation is impaired, and lethality occurs at the midsomitic stages of development. In contrast, homozygous deletion of KDR/flk-1 also results in early embryo death (at day 8.5-9.5), but histologic examination reveals that embryonic endothelial cells are completely absent. Thus, KDR/flk-1 appears to be upstream of flt-1 in the cascade of vascular development; moreover, the presence of KDR/flk-1 is absolutely required for the development of endothelial cells from hemangioblastic precursors.In addition to its important developmental role, KDR/flk-1 is implicated in the pathogenesis of a number of diseases with significant angiogenic components. For example, expression of both KDR/flk-1 and its ligand VEGF are up-regulated in neoplastic processes (10Plate K.H. Breier G. Millauer B. Ullrich A. Risau W. Cancer Res. 1993; 53: 5822-5827PubMed Google Scholar, 11Takahashi Y. Kitadai Y. Bucana C. Cleary K. Ellis L. Cancer Res. 1995; 55: 3964-3968PubMed Google Scholar), and administration of a dominant-negative form of KDR/flk-1 inhibits angiogenesis and experimental tumor growth (12Millauer B. Shawver L.K. Plate K.H. Risau W. Ullrich A. Nature. 1994; 367: 576-579Crossref PubMed Scopus (1158) Google Scholar). Likewise, a critical role for KDR/flk-1 has been established in angiogenesis associated with proliferative retinopathies (13Aiello L. Pierce E. Foley E. Takagi H. Chen H. Riddle L. Ferrara N. King G. Smith L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10457-10461Crossref PubMed Scopus (1155) Google Scholar).In view of the importance of KDR/flk-1 in endothelial cell differentiation and angiogenesis, and to address the mechanisms of endothelial cell type-specific gene regulation, we have cloned and begun to analyze the regulatory elements of the human KDR/flk-1 gene (14Patterson C. Perrella M. Hsieh C.-M. Yoshizumi M. Lee M.-E. Haber E. J. Biol. Chem. 1995; 270: 23111-23118Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). We have previously demonstrated in transient transfection assays that maximal promoter activity of the 5′-flanking region resides within a fragment from −225 to +127 relative to the transcription initiation site, and that deletions from −95 to −37 result in complete loss of promoter activity, defining this segment as the core promoter for human KDR/flk-1.Because putative binding sites for AP-2, NFκB, and Sp1 are found within the KDR/flk-1 core promoter, we have now examined which factors interact with the proximal promoter region in vitro and in vivo and how nonspecifically expressed trans-acting factors might function to regulate an endothelial cell type-specific gene. We demonstrate by electrophoretic mobility shift assays (EMSAs) and in vitro DNase I footprinting experiments that a large nucleoprotein complex forms over the human KDR/flk-1 core promoter, and that purified Sp1 alone is sufficient to recapitulate this binding pattern. Nuclear extracts from endothelial and nonendothelial cells produce the same binding pattern over the core promoter, as would be expected, since Sp1 is expressed ubiquitously in mammalian cells (15Briggs M. Kadonanga J. Bell S. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1055) Google Scholar). In marked contrast, we show by in vivo dimethylsulfate (DMS) and DNase I footprinting experiments using ligation-mediated polymerase chain reaction (LM-PCR) that Sp1 or Sp1-like proteins bind to the human KDR/flk-1 core promoter in endothelial cells but not in fibroblasts or HeLa cells in vivo, despite the presence of Sp1 in fibroblasts and HeLa cells. Moreover, we provide evidence that the exclusion of Sp1 from the KDR/flk-1 core promoter in nonendothelial cells is associated with changes in chromatin structure. Our data support a model whereby proximal promoter elements are regulated by chromatin structure to provide access to or to exclude ubiquitous trans-acting factors for the regulation of cell type-specific gene expression.DISCUSSIONUsing complementary methods of EMSA, in vitro DNase I footprinting, and in vivo DMS and DNase I footprinting, we have defined the DNA-protein interactions within the proximal promoter of human KDR/flk-1. We have demonstrated that the important Sp1 sites are bound in vivo in endothelial cells but not in nonendothelial cells. Furthermore, our data suggest that the lack of Sp1 binding to this promoter in nonendothelial cells is associated with changes in chromatin structure.The results of in vivo DMS and DNase I footprinting in HUVECs demonstrate that four Sp1 motifs (Sp1 I-IV) within the KDR/flk-1 promoter are occupied in vivo, and that contacts are made predominantly with the G-rich strand of each motif. Our in vivo results correspond closely to in vitro binding of purified Sp1 to the SV40 promoter (26Gidoni D. Dynan W. Tjian R. Nature. 1984; 312: 410-413Crossref Scopus (334) Google Scholar), in which DMS protection is also restricted to the G-rich strand of Sp1 elements. In addition, although the crystal structure of Sp1 binding to DNA has not yet been solved, the coordinates for binding of Egr-1, a related zinc finger protein, to DNA have been resolved and are also restricted to G residues on one strand (29Pavletich N. Pabo C. Science. 1991; 252: 809-817Crossref PubMed Scopus (1723) Google Scholar), suggesting that binding to the G-rich strand is the mechanism of DNA interaction for three-zinc finger nuclear proteins. Our results are therefore entirely consistent with Sp1 binding to the KDR/flk-1 promoter in endothelial cells in vivo.On the basis of our results, the human KDR/flk-1 promoter bears strong similarities to the SV40 promoter (26Gidoni D. Dynan W. Tjian R. Nature. 1984; 312: 410-413Crossref Scopus (334) Google Scholar). The SV40 promoter contains repeated Sp1 elements, and Sp1 binds cooperatively to these elements to induce bending toward the minor groove of the DNA helix; this bending may bring distant regulatory elements in proximity to the core transcriptional apparatus (30Sun D. Hurley L. Biochemistry. 1994; 33: 9578-9587Crossref PubMed Scopus (33) Google Scholar). It is thought that cooperative binding of Sp1 on the SV40 promoter involves alignment of protected residues along a single face of the DNA helix (26Gidoni D. Dynan W. Tjian R. Nature. 1984; 312: 410-413Crossref Scopus (334) Google Scholar). The periodicity between the axes of protected residues in the Sp1 I-III sites in the KDR/flk-1 promoter is approximately 14 bp (Fig. 9), suggesting that if similar cooperative alignment occurs in this promoter, the helix may be slightly unwound, perhaps due to steric or sequence-related considerations. Alignment may also explain why the Sp1 IV site has a greater periodicity between the axis of its protected residues and those of Sp1 III; since Sp1 IV is G-rich and therefore protected on the opposite strand from the other three Sp1 sites, an additional half turn of the helix would be necessary to bring it into alignment. In the SV40 promoter, all Sp1 sites are located on the same strand; whether the orientation of Sp1 IV on the opposite strand from the other Sp1 sites in the KDR/flk-1 promoter has any functional significance remains to be determined.Although Sp1 has primarily been considered a transcription factor for housekeeping genes, convincing evidence suggests that Sp1 is functionally regulated (31Saffer J. Jackson S. Thurston S. Genes Dev. 1990; 4: 659-666Crossref PubMed Scopus (99) Google Scholar, 32Persengiev S. Raval P. Rabinovitch S. Millette C. Kilpatrick D. Endocrinology. 1996; 137: 638-646Crossref PubMed Scopus (58) Google Scholar) and participates in cell type-specific gene expression (33Baker D. Dave V. Reed T. Periasamy M. J. Biol. Chem. 1996; 271: 5921-5928Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34D'Angelo D. Oliver B. Davis M. McCluskey T. Dorn G. J. Biol. Chem. 1996; 271: 19696-19704Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 35Noti J. Reinemann B. Petrus M. Mol. Cell. Biol. 1996; 16: 2940-2950Crossref PubMed Scopus (94) Google Scholar). Moreover, Sp1 is regulated during development (36Saffer J. Jackson S. Annarella M. Mol. Cell. Biol. 1991; 11: 2189-2199Crossref PubMed Scopus (483) Google Scholar), is important for the expression of ρ-globin in differentiating erythroid cells (37Minie M. Kimura T. Felsenfeld G. Development (Camb.). 1992; 115: 1149-1164PubMed Google Scholar), and is highly expressed in areas of vasculogenesis such as the developing hearts of mouse embryos (36Saffer J. Jackson S. Annarella M. Mol. Cell. Biol. 1991; 11: 2189-2199Crossref PubMed Scopus (483) Google Scholar). It is plausible, then, that Sp1 may participate not only in constitutive expression of KDR/flk-1 in human endothelial cells, but also in the developmental expression of this gene as endothelial cells differentiate from hemangioblastic precursors. If this is the case, it is likely to interact functionally with other, more cell type-specific transcription factors, as is true for developmentally regulated erythroid genes (38Merika M. S. Mol. Cell. Biol. 1995; PubMed Scopus Google the of areas of protection, the pattern of in vivo DNase I of the human KDR/flk-1 5′-flanking sequence in endothelial cells is to that of DNA to DNase I in suggesting that the promoter is not bound by T. 1993; PubMed Scopus Google Scholar). In contrast, the pattern of in vivo DNase in with the lack of protection from DMS in vivo that the promoter is likely to be associated with in cells. periodicity by in vivo DNase I footprinting is a defining of in the 5′-flanking of a number of genes G. A. Genes Dev. 1991; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). studies be necessary to the positioning of the KDR/flk-1 5′-flanking sequence in nonendothelial cells and how such nucleosomal positioning is of nucleosomal positioning on Sp1 binding and remains other nuclear factors may Sp1 for nucleosomal and transcription initiation M. P. K. J. Kadonanga J. K. Genes Dev. 1996; PubMed Scopus Google Scholar), it is that Sp1 is for nucleosomal since Sp1 is ubiquitous in mammalian cells (15Briggs M. Kadonanga J. Bell S. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1055) Google Scholar), and Sp1 alone does not in vitro B. C. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). It also has not been demonstrated that Sp1 interact with binding sites that are bound by in Sp1 with nucleosomal DNA in but its for nucleosomal DNA is by greater than of with its for DNA B. C. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and transcription initiation by Sp1 from is or P. C. K. M. M. J. K. Genes Dev. 1995; PubMed Scopus Google Scholar). of nucleosomal has been shown to Sp1 binding in vitro H. B. J. J. 1994; PubMed Scopus Google data that Sp1 is from binding the KDR/flk-1 promoter in nonendothelial cells that not this and we have evidence that this exclusion is associated with changes in chromatin structure. A between the two is to in vivo, In this results of experiments in mice using a KDR/flk-1 gene are this to in all cell examined endothelial cells, although KDR/flk-1 is in endothelial cells of these would suggest that the proximal of 5′-flanking sequence although in transient assays of gene expression (14Patterson C. Perrella M. Hsieh C.-M. Yoshizumi M. Lee M.-E. Haber E. J. Biol. Chem. 1995; 270: 23111-23118Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), is in a and that this region are required for expression of KDR/flk-1. A is that distant regulate by chromatin structure to Sp1 to bind the core promoter, or by Sp1 to bind chromatin structure is In an structure for the gene is in endothelial but not in nonendothelial a number of genes are expressed with of specificity in endothelial cells, we of no transcription factors that function in endothelial cells. We that factors, or a of more factors, KDR/flk-1 expression in early in development in the of to the endothelial cell this of cells to the and of In our changes in chromatin structure within the core promoter, regulated by elements at a from the transcription initiation site, are associated with cell type-specific regulation of this gene. A similar on regulation a is used to explain gene expression within the in the developmentally related erythroid D. S. M. Dev. 1996; PubMed Scopus Google Scholar, J. K. A. D. N. S. F. J. 1996; PubMed Scopus Google Scholar). as our results KDR/flk-1 is indeed regulated by a mechanism similar to that of the methods used to regulatory elements within the such as DNase I assays and and homologous promoter may be in the mechanisms KDR/flk-1 as In addition, the results of our experiments that one or more functional elements regulate chromatin structure such that the KDR/flk-1 gene is and expressed in endothelial cells. of elements is of importance to the mechanisms of expression of the human KDR/flk-1 gene. INTRODUCTIONKDR/flk-1 is a membrane-bound receptor of the tyrosine kinase family with expression restricted predominantly to endothelial cells (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar). KDR/flk-1 and a similar tyrosine kinase, flt-1, are receptors for the specific endothelial cell mitogen and angiogenic peptide vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorEMSAelectrophoretic mobility shift assayDMSdimethylsulfateLM-PCRligation-mediated polymerase chain reactionHUVEChuman umbilical vein endothelial cellbpbase pairBSAbovine serum albumin. (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar, 2de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1879) Google Scholar, 3Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (670) Google Scholar). Both receptors are expressed early in murine development, with KDR/flk-1 appearing a full day earlier (day 7.0-7.5 of the developing mouse embryo) than flt-1 (4Dumont D.J. Fong G.H. Puri M.C. Gradwohl G. Alitalo K. Breitman M.L. Dev. Dyn. 1995; 203: 80-92Crossref PubMed Scopus (444) Google Scholar, 5Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development (Camb.). 1993; 118: 489-498Crossref PubMed Google Scholar). Of the two, KDR/flk-1 has a wider pattern of expression among endothelial cell populations than does flt-1 (6Kaipainen A. Korhonen J. Pajusola K. Aprelikova O. Persico M.G. Terman B.I. Alitalo K. J. Exp. Med. 1993; 178: 2077-2088Crossref PubMed Scopus (215) Google Scholar). In addition, only KDR/flk-1 has been shown to autophosphorylate in the presence of VEGF (1Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar), and signal transduction mechanisms downstream of the two receptors differ (7Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). These differences in expression and signaling suggest that the two receptors mediate different physiologic functions of VEGF in endothelial cell growth and angiogenesis.Recent studies in which the genes for the two VEGF receptors were deleted in mice by homologous recombination have provided critical data about KDR/flk-1 function (8Shalaby F. Rossant J. Yamaguchi T. Gertsenstein M. Wu X.-F. Breitman M. Schuh A. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3332) Google Scholar, 9Fong G. Rossant J. Gertsenstein M. Breitman M. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2201) Google Scholar). In mice with homozygous deletions of flt-1, endothelial cells develop normally, but vessel formation is impaired, and lethality occurs at the midsomitic stages of development. In contrast, homozygous deletion of KDR/flk-1 also results in early embryo death (at day 8.5-9.5), but histologic examination reveals that embryonic endothelial cells are completely absent. Thus, KDR/flk-1 appears to be upstream of flt-1 in the cascade of vascular development; moreover, the presence of KDR/flk-1 is absolutely required for the development of endothelial cells from hemangioblastic precursors.In addition to its important developmental role, KDR/flk-1 is implicated in the pathogenesis of a number of diseases with significant angiogenic components. For example, expression of both KDR/flk-1 and its ligand VEGF are up-regulated in neoplastic processes (10Plate K.H. Breier G. Millauer B. Ullrich A. Risau W. Cancer Res. 1993; 53: 5822-5827PubMed Google Scholar, 11Takahashi Y. Kitadai Y. Bucana C. Cleary K. Ellis L. Cancer Res. 1995; 55: 3964-3968PubMed Google Scholar), and administration of a dominant-negative form of KDR/flk-1 inhibits angiogenesis and experimental tumor growth (12Millauer B. Shawver L.K. Plate K.H. Risau W. Ullrich A. Nature. 1994; 367: 576-579Crossref PubMed Scopus (1158) Google Scholar). Likewise, a critical role for KDR/flk-1 has been established in angiogenesis associated with proliferative retinopathies (13Aiello L. Pierce E. Foley E. Takagi H. Chen H. Riddle L. Ferrara N. King G. Smith L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10457-10461Crossref PubMed Scopus (1155) Google Scholar).In view of the importance of KDR/flk-1 in endothelial cell differentiation and angiogenesis, and to address the mechanisms of endothelial cell type-specific gene regulation, we have cloned and begun to analyze the regulatory elements of the human KDR/flk-1 gene (14Patterson C. Perrella M. Hsieh C.-M. Yoshizumi M. Lee M.-E. Haber E. J. Biol. Chem. 1995; 270: 23111-23118Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). We have previously demonstrated in transient transfection assays that maximal promoter activity of the 5′-flanking region resides within a fragment from −225 to +127 relative to the transcription initiation site, and that deletions from −95 to −37 result in complete loss of promoter activity, defining this segment as the core promoter for human KDR/flk-1.Because putative binding sites for AP-2, NFκB, and Sp1 are found within the KDR/flk-1 core promoter, we have now examined which factors interact with the proximal promoter region in vitro and in vivo and how nonspecifically expressed trans-acting factors might function to regulate an endothelial cell type-specific gene. We demonstrate by electrophoretic mobility shift assays (EMSAs) and in vitro DNase I footprinting experiments that a large nucleoprotein complex forms over the human KDR/flk-1 core promoter, and that purified Sp1 alone is sufficient to recapitulate this binding pattern. Nuclear extracts from endothelial and nonendothelial cells produce the same binding pattern over the core promoter, as would be expected, since Sp1 is expressed ubiquitously in mammalian cells (15Briggs M. Kadonanga J. Bell S. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1055) Google Scholar). In marked contrast, we show by in vivo dimethylsulfate (DMS) and DNase I footprinting experiments using ligation-mediated polymerase chain reaction (LM-PCR) that Sp1 or Sp1-like proteins bind to the human KDR/flk-1 core promoter in endothelial cells but not in fibroblasts or HeLa cells in vivo, despite the presence of Sp1 in fibroblasts and HeLa cells. Moreover, we provide evidence that the exclusion of Sp1 from the KDR/flk-1 core promoter in nonendothelial cells is associated with changes in chromatin structure. Our data support a model whereby proximal promoter elements are regulated by chromatin structure to provide access to or to exclude ubiquitous trans-acting factors for the regulation of cell type-specific gene