CD11c Promoter Research Articles

Driving gene expression specifically in dendritic cells.

Lymphoid dendritic cells (DC) play an important role in the immune system. They are potent inducers of primary T cell responses and play a crucial part as MHC class II+ “interdigitating cells” in the thymus during thymocyte development. Most of our knowledge about DC was obtained using highly invasive and manipulatory experimental protocols. Since the functions of DC after these in vitro manipulations have been reported not to be identical to those of DC in vivo (1), we intended to establish a system that would allow us to investigate DC-function avoiding artificial interferences due to handling. Here we present a transgenic mouse system in which we targeted gene expression specifically to DC. Using the mouse CD11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types.

Sp1 Binds Two Sites in the CD11c Promoter In Vivo Specifically in Myeloid Cells and Cooperates with AP1 to Activate Transcription

The leukocyte integrin gene, CD11c, is transcriptionally regulated and is expressed predominantly on differentiated cells of the myelomonocytic lineage. In this study we have demonstrated that the regions -72 to -63 and -132 to -104 of the CD11c promoter contain elements responsible for phorbol ester-induced differentiation of the myeloid cell line HL60. DNase I footprinting analysis revealed that these regions can bind purified Sp1, and supershift analysis with Sp1 antibody confirmed that Sp1 in HL60 nuclear extracts could bind these regions. Transfection analysis of CD11c promoter-chloramphenicol acetyltransferase constructs containing deletions of these Sp1-binding sites revealed that these sites are essential for expression of the CD11c gene in HL60 cells but not in the T-cell line Molt4 or the cervical carcinoma cell line HeLa. Moreover, cotransfection of pPacSp1 along with these CD11c promoter-chloramphenicol acetyltransferase constructs into Sp1-deficient Drosophila Schneider 2 cells verified that these sites are essential for Sp1-dependent expression of the CD11c promoter. In vivo genomic footprinting revealed that Sp1 contacts the CD11c promoter within the regions -69 to -63 and -116 to -105 in phorbol 12-myristate 13-acetate-differentiated HL60 cells but not in undifferentiated HL60 cells or in Molt4 or HeLa cells. Cotransfection assays in HL60 cells revealed that Sp1 acts synergistically with Ap1 to activate CD11c. Further, both Sp1 sites are capable of cooperating with AP1. In vitro DNase I footprinting analysis with purified Sp1 and c-jun proteins showed that Sp1 binding could facilitate binding of c-jun. We propose that myeloid-specific expression of the CD11c promoter and is facilitated by cooperative interaction between the Sp1- and Ap1-binding sites.

AP-1 regulates the basal and developmentally induced transcription of the CD11c leukocyte integrin gene.

The p150,95 integrin (CD11c/CD18) mediates leukocyte/endothelium interactions during inflammatory reactions and certain CTL-target interactions, and is also a receptor for fibrinogen, LPS, and the complement component iC3b. CD11c/CD18 is expressed primarily on cells of the myeloid lineage and activated B lymphocytes, and is an important diagnostic marker for hairy cell leukemia. To identify the transcription factors and cis-acting elements involved in the regulated expression of CD11c/CD18 during myeloid cell differentiation and B lymphocyte activation, we have performed structural and functional analysis on the CD11c gene promoter. Electrophoretic mobility shift assays identified an AP-1 binding site (AP1-60) within the proximal promoter region and evidenced differences in the pattern of the Fos family members bound to the AP1-60 element in undifferentiated and differentiated myeloid cells, as well as between B lineage-derived cells. The involvement of the AP1-60 element in DNA-protein interactions was confirmed by means of in vivo footprinting experiments, and its functionality was demonstrated by trans activation of the CD11c promoter by c-Jun. Site-directed mutagenesis of AP1-60 greatly reduced the basal CD11c promoter activity in myeloid and B cells. Furthermore, mutations at AP1-60 inhibited the induction of the CD11c promoter activity during the PMA-triggered U937 cell differentiation, pointing out a key role for the AP-1 transcription factor complex in both the basal and the developmentally regulated expression of the p150,95 leukocyte integrin. The involvement of AP-1 in the transcription of the CD11c gene raises the possibility of altering leukocyte integrin expression by pharmacologic means and will greatly contribute to the characterization of the intracellular signals controlling the expression of leukocyte adhesion molecules.

Regulation of the leukocyte integrin gene CD11c is mediated by AP1 and Ets transcription factors

The leukocyte integrin gene, CD11c, encodes the α subunit of the p150,95 (CD11c·CD18) receptor. Expression of the CD11c gene is predominately seen in monocytes, but has also been detected in some B- and T-cell neoplasms and in some large-cell lymphomas of uncertain origin. To elucidate the molecular mechamisms that govern the expression of CD11c, we have cloned and characterized the promoter region of this gene. The DNase I footprint and mobility shift analyses revealed five sites within the −86 to +40 region that interact with nuclear proteins. The −62 to −44 region contains two consensus sequences for AP1 (referred to as AP1-1 and AP1-2) and were shown to bind purified c-jun protein. Co-transfection of c-fos and c-jun expression constructs with a CD11c promoter-CAT fusion into HL60 cells led to a 6.7-fold increase in CD11c promoter activity. We show that c-fos and c-jun mediate their effects through both AP1-1 and AP1-2 which function in an additive manner. Regions −42 to −34 and − 13 to −5 contain consensus sequences for Ets factors (referred to as Ets C and Ets A, respectively). Deletion of Ets C resulted in a significant reduction in phorbol ester-induced expression of CD11c, whereas deletion of Ets A led to only a modest loss in CD11c expression. We show that C Ets cooperates with the AP1 sites to regulate CD11c expression.

Identification of Sp1‐binding sites in the CD11c (p150,95α) and CD11a (LFA‐1α) integrin subunit promoters and their involvement in the tissuespecific expression of CD11c

The leukocyte integrins LFA-1 (CD11a/CD18) and p150,95 (CD11c/CD18) mediate cell-cell and cell-extracellular matrix interactions during inflammatory responses and signal transduction into the cytoplasm. While the CD11a integrin subunit is expressed on all leukocytes, CD11c is almost exclusively expressed on cells of the myeloid lineage and on activated B lymphocytes. Its expression is regulated during cell activation and differentiation by transcriptional mechanisms. We have previously demonstrated that the proximal region of the CD11c promoter directs tissue-restricted and developmentally-regulated expression of reporter genes. Structural studies by electrophoretic mobility shift assays have demonstrated the presence of two Sp1-binding sites at -70 (Sp1-70) and -120 (Sp1-120) which mediate the Sp1 transactivation of the CD11c promoter in Sp1-defective SL2 cells, and which are involved in cell lineage-specific DNA-protein interactions, as demonstrated by footprinting in vivo. More importantly, mutation of either Sp1 site inhibited the activity of the CD11c promoter both in myeloid U937 cells and the CD11c-expressing B lymphoblastoid JY cell line, while the opposite effect was observed in the CD11c-negative epithelial HeLa cell line, demonstrating the involvement of both Sp1-binding sites in the basal and the tissue-restricted expression of the CD11c integrin subunit gene. Interestingly, the analysis of the CD11a proximal promoter also revealed the existence of an Sp1-binding site at -70, indicating a common role for these cis-acting elements in the transcription of the leukocyte integrin alpha subunit genes. The binding of Sp1 to the regulatory regions of the leukocyte integrin genes raises the possibility that the retinoblastoma susceptibility gene product is implicated in integrin expression through its functional interaction with Sp1, thus establishing a link between integrin-dependent leukocyte adhesiveness and the state of cellular differentiation/proliferation.

Granulocyte-Macrophage Colony-Stimulating Factor, Phorbol Ester, and Sodium Butyrate Induce the CD11c Integrin Gene Promoter Activity During Myeloid Cell Differentiation

To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers. Unexpectedly, the inducibility of the CD11c gene promoter showed distinct kinetics and magnitude on the PMA-, SB-, GM-CSF-triggered differentiation. Moreover, SB synergized with either PMA or GM-CSF in enhancing both the CD11c promoter activity and the cell surface expression of p150,95 on differentiating U937 cells. Furthermore, we showed the existence of a c-Myb-binding site at -85, the importance of the -99/-61 region in the CD11c promoter inducibility during PMA- or SB-triggered differentiation, and the dependency of the GM-CSF and PMA responsiveness of the CD11c promoter on an intact AP-1-binding site located at -60. These results, together with the lack of functional effect of mutations disrupting the Sp1-and Myb-binding sites within the proximal region of the CD11c promoter, indicate that the myeloid differentiation pathways indicated by SB and phorbol esters (or GM-CSF) activate a distinct set of transcription factors and show that the myeloid differentiation-inducibility of the CD11c gene maps to the -99/-53 proximal region of the promoter.

Characterization of the p150,95 leukocyte integrin alpha subunit (CD11c) gene promoter. Identification of cis-acting elements.

The leukocyte integrin p150,95 (CD11c/CD18) is involved in a number of cell-cell and cell-extracellular matrix interactions and mediates signal transduction into the cytoplasm. p150,95 is expressed on cells of the myeloid lineage as well as on certain activated T and B lymphocytes, and its expression is regulated during cell activation and differentiation. Since CD18 is expressed on all leukocyte lineages, the restricted expression of p150,95 must be controlled at the level of CD11c gene transcription. To understand the mechanisms that direct the constitutive and regulated leukocyte expression of p150,95 we have structurally characterized the CD11c promoter region and initiated its functional dissection. The CD11c promoter lacks TATA- and CCAAT-boxes, directs the synthesis of transcripts with heterogeneous 5'-ends, and contains an initiator-like sequence at the major transcription initiation site. Several putative binding sequences for ubiquitous (Sp1, AP-1, AP-2, and NF-kB) and leukocyte-specific (PU.1) transcription factors have been identified in the proximal region of the CD11c promoter which may participate in the regulation of the expression of p150,95. Transient expression of CD11c-based reporter gene constructs indicates that the CD11c promoter dictates the tissue-specific expression of p150,95 and that sequences contained within 160 base pairs 5' from the major transcriptional start site are involved in the tissue-specific and regulated expression of p150,95. DNase I protection analysis on the promoter region spanning from -160 to +40 revealed four regions of DNA-protein interactions (FPI-FPIV), two of which (FPII and FPIV) correlate with the cell type-specific and regulated expression of the CD11c gene.