[The study of cis-element HNF4 in the regulation of mfg12 prothrombinase/fibroleukin gene expression in response to nucleocapsid protein of MHV-3].
OBJECTIVE
To identify the transcription factor(s) that is essential for activation of mfgl2 prothrombinase/fibroleukin gene in response to nucleocapsid protein of murine hepatitis virus type 3 (MHV-3).


METHODS
Western blotting was performed to investigate whether HNF4 is expressed in macrophages of Ba1b/c mice where mfgl2 is expressed. Confocus microscope immunofluorescence was performed to show whether N protein of MHV enters into the nucleus of infected cells, which is a critical step for the N protein to facilitate its transactivation property. To facilitate the identification of three candidate factor(s) including hepatocyte nuclear factor 4 (HNF4)/liver factor A1 (LF-A1), cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element and granulocyte- macrophage colony stimulating factor (GM-CSF) in response to mfgl2 activation upon the stimulation of MHV-A59 N protein, gel mobility shift assay (GMSA), competition experiments and site directed mutagenesis were performed.


RESULTS
Western blotting displayed that HNF4 was constitutively expressed in macrophages and did not show significant change under the stimulation of different MHV. Confocus microscope immunofluorescence clearly showed that N protein of MHV entered into the nucleus of infected cells. GMSA and competition experiments demonstrated binding to both HNF4 and IE1.2 fragments could be competed with the cold specific oligonucleotides but not with the same amount of non-specific oligos nucleotides. A super shift band was observed when HNF4 antibody was pre-incubated with the nuclear extracts indicating the interaction between the HNF4 element and mfgl2 promoter. Site directed mutagenesis of cis-elements HNF4 (pfgl2HNF4mut) and HNF4/IE1.2 (pfgl2HNF4/IE1.2mut) mutations abolished over 75% of transcription from wild-type mfgl2 promoter. However the pfgl2IE1.2mut displayed almost wild-type promoter activity (75% approximately 80%).


CONCLUSIONS
The factor HNF4 binds to mfgl2 promoter and serves as an essential transcription factor for mfgl2/fibroleukin expression in response to MHV-3 N protein.
Ning
Qin
Q
Division of Clinical Immunology, Tongji Hospital, Institute of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. qning@tjh.tjmu.edu.cn
Luo
Xiao-ping
XP
Wang
Zhi-mo
ZM
Han
Mei-fang
MF
Yan
Wei-ming
WM
Liu
Ming-feng
MF
Levy
Gary
G
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005260:Female; D005340:Fibrinogen / Q000235:genetics*; D005786:Gene Expression Regulation; D051557:Hepatocyte Nuclear Factor 4; D051379:Mice; D008807:Mice, Inbred BALB C; D006517:Murine hepatitis virus / Q000472:pathogenicity*; D019590:Nucleocapsid Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D013925:Thromboplastin / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C490637:Fgl2 protein, mouse; D051557:Hepatocyte Nuclear Factor 4; D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D005340:Fibrinogen; D013925:Thromboplastin