Molecular regulation of KIR3DL1 gene expression by transcription factor E2F1 / 中华血液学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To study the role of transcription factor E2F1 in the transcription of KIR3DL1 promoter, and to identify its molecular mechanism of regulation of KIR3DL1 gene expression.</p><p><b>METHODS</b>The mutant promoter fragment of KIR3DL1 gene was amplified from genomic DNA of K562 cells by PCR. The PCR product was cloned into pGL3-basic reporter vector to construct KIR3DL1 promoter-luciferase reporter plasmid (PLRP). The binding of E2F1 to KIR3DL1 promoter was detected by chromatin immunoprecipitation (CHIP) assays. The KIR3DL1 PLRP construction was transfected into K562 cells using cationic liposome SuperFect. The binding of E2F1 to the construction was detected by CHIP assays and reporter activity was quantitated by the dual-luciferase reporter assay system. The mammalian expression vector containing E2F1 cDNA was co-transfected into K562 cells with wild-type KIR3DL1 PLR construction and reporter activity was quantitated.</p><p><b>RESULTS</b>The mutant KIR3DL1 PLR recombinant was constructed successfully and a naturally point mutation (TTTGGCGC-->TTCGGCGC) within a putative E2F1 binding site in the KIR3DL1 promoter region was authenticated by DNA sequencing. E2F1 absolutely could not bind to the mutant KIR3DL1 promoter in K562 cells, but could bind to the wild-type one in NK-92MI cells. The binding of E2F1 to the mutant KIR3DL1 PLR construction was partially reserved, however, its relative luciferase activity was decreased by 50% than that of wild-type. On the other hand, when co-transfected with E2F1 mammalian expression vector, the relative luciferase activity of wild-type construction was increased, over 2-fold higher than that of control group.</p><p><b>CONCLUSION</b>E2F1 participates in the regulation of the transcriptional activation of KIR3DL1 gene. The number of CpG dinucleotide and methylation pattern within the E2F1 binding site probably influence the binding of E2F1 to target sequence.</p>