1. Characterization of the Human Platelet/Endothelial Cell Adhesion Molecule-1 Promoter: Identification of a GATA-2 Binding Element Required for Optimal Transcriptional Activity
by Richard J. Gumina, Nancy E. Kirschbaum, Kim Piotrowski, and Peter J. Newman
Blood
Volume 89(4):
February 15, 1997
©1997 by American Society of Hematology

2. Characterization of PECAM-1 expression on leukocyte tumor cell lines.
Characterization of PECAM-1 expression on leukocyte tumor cell lines. The human leukocyte cell lines HEL, Dami, and Raji were examined for PECAM-1 expression. (A) Flow cytometric analysis of PECAM-1 expression. Cells were stained with either normal rabbit IgG or the human PECAM-1–specific polyclonal rabbit IgG, SEW16. Detection was accomplished by staining with a dichlorotriazinyl amino fluorescein-conjugated goat antirabbit IgG. As shown, HEL and Dami cells, express PECAM-1 on their surface, whereas Raji cells are negative. (B) Western blot analysis of PECAM-1 expression in HEL, Dami, and Raji cells was performed using SEW16. Detection was accomplished using alkaline phosphatase-conjugated goat antirabbit secondary antibody. Note that Raji cells do not contain even an intracellular pool of detectable PECAM-1. (C) RT-PCR analysis of PECAM-1 mRNA levels. mRNA was isolated from HEL, Dami, and Raji cells and subjected to RT-PCR using primers spanning a 400-bp region of PECAM-1 that include exons 2 and 3. These primers would yield a PCR product of greater than 12-kb product if amplification were to occur from genomic DNA. As shown, both HEL and Dami cells express PECAM-1 mRNA, whereas Raji cells are negative.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

3. Characterization of a genomic clone containing the 5′-flanking region of the PECAM-1 gene.
Characterization of a genomic clone containing the 5′-flanking region of the PECAM-1 gene. The map of Clone 7.2 (5.2 kb) indicates the relative position of selected introns and exons, as well as the restriction sites for several enzymes. As shown, digestion with Pst I, Sph I, or Sma I, alone or in combination with HindIII, yielded the following fragments: Pst I to Pst I (4.7 kb), Sph I to HindIII (2.8 kb), and Sma I to HindIII (1.5 kb). These fragments were subcloned into the vector pGEM7 for sequence analysis.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

4. Sequence and structural features of the 5′-flanking region of the PECAM-1 gene.
Sequence and structural features of the 5′-flanking region of the PECAM-1 gene. The 1,588-bp sequence shown contains 1,252 bases of the 5′-flanking region of the PECAM-1 gene, as well as the 5′ untranslated region, exon 1 and the beginning of intron 1. All numbering is relative to the Transcription Initiation Site (arrow at +1). The translated protein sequence corresponding to exon 1 is shown as single letter amino acids. Putative regulatory elements are boxed and labeled, and selected restriction enzymes sites used in subcloning this fragment, either in part or in its entirety, are also shown. GATA elements potentially involved in transcriptional regulation of the PECAM-1 promoter are shown in white against a blue background. An Alu sequence spanning nucleotides −859 to −557 is backshadowed in yellow, and contains one of three putative SSREs (shown in red). The ATG translation start site at +205 is shown in bold.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

5. Identification of multiple transcription initiation sites for the PECAM-1 gene.
Identification of multiple transcription initiation sites for the PECAM-1 gene. (A) 5′-RACE analysis. Total RNA was isolated from HUVEC, HEL, and Dami cells, all which express PECAM-1, as well as from the PECAM-1-negative Raji cell line. 5′-RACE analysis was conducted using a forward primer complementary to the anchor together with one of two different antisense primers, beginning either 63 bp upstream from the major TIS (lanes A) or 101 bp upstream from the TIS (lanes B), as described in Materials and Methods. The resulting PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (A) and (B) represent the two separate primer sets used (see Materials and Methods). As shown, HUVECs and HEL and Dami cells yielded comparably sized products, indicating that all three cell lines use the same region for transcription initiation. Note that the bands on the gel are somewhat broad, suggesting that a number of closely spaced PCR products might exist within each band. Raji cells showed no specific amplification products. (B) Sequence analysis of the 5′-RACE products. The 5′-RACE products shown in (A) were excised, subcloned, and sequenced. A summary of the 5′-termini derived from sequencing 15 individual clones is shown. The number under a specific nucleotide indicates the number of clones whose 5′-end corresponded to that particular base. The TIS, as previously determined by primer extension analysis,28 is shown above the sequence. Based on the 5′-RACE data, it appears that transcription of the PECAM-1 gene initiates at several closely spaced sites within this region.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

6. Analysis of transcriptional activity of the 5′-flanking region of the PECAM-1 gene.
Analysis of transcriptional activity of the 5′-flanking region of the PECAM-1 gene. HEL, Dami, and Raji cells were transfected with PECAM-1 promoter/luciferase reporter constructs (shown schematically on the left) containing selected segments of the 5′-flanking region of the PECAM-1 gene. Luciferase assays were conducted as described in Materials and Methods to assess transcriptional activity, which was normalized to β-galactosidase activity derived from equal transfection of a separate reporter plasmid encoding lacZ. The boundaries of the constructs are numbered relative to the transcription initiation site, and the relative 5′ → 3′ orientation of each construct is indicated by the direction of the arrow. Normalized luciferase activity present in each transfected cell line is indicated on the right. The data shown represent the mean ± SEM of three independent experiments, each performed in duplicate. (A) Orientation-specific transcriptional activity of the 5′-flanking region of the PECAM-1 gene. Note that both the longer 4,500-bp construct, as well as the shorter 1,100-bp construct are capable of driving orientation- and lineage-specific transcription of luciferase activity. (B) Localization of transcriptional activity to a core region of the PECAM-1 promoter. 5′-serially-truncated PECAM-1 promoter/luciferase constructs were assayed for luciferase activity to assess PECAM-1 promoter activity in HEL, Dami, and Raji cells as described in the text. Note that the region encompassing nucleotides −163 to +204 retains transcriptional activity in the PECAM-1–positive HEL and Dami cells and that all constructs failed to drive transcription in Raji cells.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

7. The GATA element at position −24 contributes to the transcriptional activity of the PECAM-1 promoter.
The GATA element at position −24 contributes to the transcriptional activity of the PECAM-1 promoter. HEL and Dami cells were transfected with the reporter constructs shown and cellular lysates assayed for luciferase activity to access transcriptional activity. Note that substitution of the sequence 5′-CTTT-3′ for the wild-type 5′-GATA-3′ resulted in a 70% reduction in PECAM-1 promoter activity in both megakaryocytic cell lines. PECAM-1-negative Raji cells showed no transcriptional activity when transfected with either reporter construct (data not shown). Data shown represent the mean ± SEM of three independent experiments, each performed in duplicate.
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology

8. Binding of the transcription factor, GATA-2, to the −24 GATA element of the PECAM-1 promoter.
Binding of the transcription factor, GATA-2, to the −24 GATA element of the PECAM-1 promoter. Nuclear extracts derived from DAMI cells were incubated with a 32P-labeled double-stranded oligonucleotide containing either a GATA consensus binding site (lanes 2 to 5) or a mutated CTTT sequence in its place (lanes 6 and 7). Competition reactions were performed using a 100-fold molar excess of unlabeled double-stranded oligonucleotide containing either the wild-type (lane 3) or mutant CTTT sequence (lane 7). Immunodepletion studies (lanes 4 and 5) were conducted by preincubating the nuclear extracts with 1 μg of a monoclonal antibody specific for either GATA-1 (lane 4) or GATA-2 (lane 5). Note that a GATA-2-specific DNA-protein complex (arrow) forms only when the GATA sequence is present. Comparable results were obtained using nuclear extracts derived from HEL cells (data not shown).
Richard J. Gumina et al. Blood 1997;89:
©1997 by American Society of Hematology