Abstract
To study the mechanism of gene regulation for coagulation factor XIII A subunit (FXIIIA), we characterized its 5'-flanking region using a monocytoid (U937), a megakaryocytoid (MEG-01), and other cells. Our results confirmed that U937 and MEG-01 contained FXIIIA mRNA. A tentative transcription start site was determined to be 76 bases upstream from the first exon/intron boundary. Reporter gene assays revealed that a 5'-fragment (-2331 to +75) was sufficient to support basal expression in U937 and MEG-01 but not in the other cells. Deletion analysis confined a minimal promoter sequence from -114 to +75. DNase footprinting, electrophoretic mobility shift, and reporter gene assays demonstrated that promoter elements for a myeloid-enriched transcription factor (MZF-1-like protein) and two ubiquitous transcription factors (NF-1 and SP-1) in this region were important for the basal FXIII expression. It was also revealed that an upstream region (-806 to -290) had enhancer activity in MEG-01 but silencer activity in U937. DNA sequences for binding of myeloid-enriched factors (GATA-1 and Ets-1) were recognized in this region, and the GATA-1 element was found to be responsible for the enhancer activity. These transcription factors play a major role in the cell type-specific expression of FXIIIA, which differs from other transglutaminases.
Highlights
Human coagulation factor XIII (FXIII,1 fibrin-stabilizing factor) is a plasma transglutaminase (TGase) and circulates in blood as a heterotetramer consisting of two catalytic A (FXIIIA) and two noncatalytic B (FXIIIB) subunits (A2B2) [1]
Semi-quantitation of the bands by an image analyzer revealed that the factor XIII A subunit (FXIIIA) mRNA level in U937 cells was 3-fold that in the MEG-01 cells, when normalized to -actin mRNA levels
Electrophoretic Mobility Shift Assay (EMSA) for the putative Ets-1b site showed the same results as those for Ets-1a (Fig. 8, right). These results suggested that GATA-1 and Ets-1 might contribute to the expression of FXIIIA in MEG-01 cells
Summary
Human Cell Culture—The megakaryoblastic leukemia cell lines MEG-01 and MEG-J and the histiocyte lymphoma cell line U937 were maintained in RPMI 1640 medium with 10% fetal bovine serum and. The cDNA purified by a GlassMax spin cartridge (Life Technologies, Inc.) was used in terminal dideoxynucleotide transferase (TdT) tailing, and PCR was performed with an internal antisense primer (5Ј-ACTGCTCTTCTGCCTCCAAA-3Ј designed for exon II of FXIIIA) and a 5Ј-sense primer termed Abridged Anchor Primer. A probe for S1 nuclease mapping was produced by the single-strand PCR method using Taq DNA polymerase, an antisense primer designed from the sequence in exon II (GSP2, 5Ј-GGTCCTGGAAGTTTCTGACAT-3Ј), and the chimeric DNA plasmid as a template. The samples were electrophoresed on a 6% polyacrylamide gel containing 8 M urea, together with a sequencing reaction mixture produced by employing the same antisense primer and template chimeric DNA as used in the generation of the probe.
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