Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique.
Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.
Department of Immunology, and Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Anhui 233030, People's Republic of China.
Shu-Jun
SJ
Guo
Jian-Guo
JG
Hu
Bao-Ming
BM
Zhao
Lin
L
Shen
Rui
R
Wang
Jian-Sheng
JS
Zhou
He-Zuo
HZ
Lü
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D005453:Fluorescence; D005786:Gene Expression Regulation; D006801:Humans; D051796:Inhibitor of Differentiation Proteins / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D051796:Inhibitor of Differentiation Proteins; D009419:Nerve Tissue Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011994:Recombinant Proteins; C490126:Id4 protein, rat; C498633:Olig1 protein, rat