Identification and characterization of a silencer regulatory element in the 3′-flanking region of the murine CD46 gene

Abstract

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3´-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein–silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

Mechanism of host cell protection from complement in murine cytomegalovirus (CMV) infection: identification of a CMV-responsive element in the CD46 promoter region

“…We identified a putative CMVRE in the 5'-flanking region of the mCD46 gene involved in up-regulation of CD46 gene expression after mCMV infection. This element consists of at least 19 bp, and shares significant homology with sequences in the promoter regions of CD46 gene [13,19], HCMV UL36 gene [15,20] and HHV-6 UL29 gene [16]. HCMV UL36 and HHV-6 UL29 genes were reported to be early inducible genes and have been speculated to be involved in viral replication [15,16].…”

“…Total RNA from NIH3T3 cells infected or non-infected with mCMV were isolated by the AGPC method [13]. First-strand cDNA synthesis was performed using SuperScript reverse transcriptase (GIBCO BRL).…”

“…g of poly(dI:dC) at room temperature for 5 min in a final volume of 25 ? l of binding buffer [7.5 mM Hepes-KOH (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM KCl, 10% glycerol] as described previously [13]. As a probe, the oligonucleotides: 5'-TTTGAC-ATTTGTCAGGATCCTGGGATGTTC-3' and 5'-GAACAT-CCCATTATCCTGACAAATGTCAAA-3' were synthesized, annealed and end-labeled with [ + -32 P]ATP by T4 polynucleotide kinase.…”

“…In humans, the levels of two SCR proteins, CD55 and CD46, were reported to be increased by several folds following infection with HCMV and protected HCMV-infected cells from the host immune response [12], although the mechanism responsible for this CD46 up-regulation remains elusive. In contrast, mouse CRRY was barely up-regulated by viral infection and thus was not involved in conferring resistance to complement on virus-infected cells [13]. The mechanism by which murine CMV (mCMV) circumvents host complement attack remains completely unknown.…”

Transcriptional control of complement receptor gene expression

“…While the human protein is ubiquitously expressed, in the mouse it is predominantly expressed in the testis [57]. Like the CR2/Cr2 genes, reporter gene constructs consisting of only murine 5 0 promoter sequences did not exhibit cell-specific regulation [58]. This repressor activity was not localized to the first intron, but rather was found between 600 and 800 bp downstream of the stop codon for the gene.…”