Effects Of The Transcription Factor SREBP2 On Cell Fate

SREBPs(sterol regulatory element binding proteins)is a family of transcription factors that regulate lipid metabolism by activating essential enzymes involved in endogenous cholesterol biosynthesis,fatty acids,triglycerides and phospholipids.Three closely related isoforms of SREBPs(SREBP1-a,SREBP1 c and SREBP2)exist in mammals,among which SREBP2 selectively regulates the genes encoding cholesterologenic enzymes.SREBP is an important transcription factor regulating cholesterol and lipid metabolism,and its precursor is a membrane protein located in the endoplasmic reticulum.The mature form of SREBP is cleaved on the golgi,then transported to the nucleus.It is not clear so far whether SREBP has other cytological functions.Our previous work found that there were no infected cells survived when we use p Lvx-nSREBP2-IRES-Zs Green to infect Hela cell.Then we constructed the Teton-Dox-nSREBP2-NIr D plasmid to infect Hela-Cas9 cell,finally we got a monoclone of Teton-nSREBP2-EGFP after we verified the expression of nSREBP2,Cas9 and EGFP and so on.Out of curiosity,we recorded the cell death process by confocal microscope.According to the dynamic response of cell death,we suspected that this phenomenon might be associated with GSDMD,which is a key regulator involved in pyroptosis.Next,we generated the GSDMD knock out TetonnSREBP2-EGFP cell line,and the result showed that GSDMD was not involved in the Dox-induced cell death.Furthermore,we found that the Dox-induced nSREBP2 overexpression upregulated the genes related to cholesterol synthesis pathway,which lead to the accumulation of sterols,then caused lipotoxic death.We infected cells with different knock out genes in cholesterol synthesis,respectively.However,the KO cells still all died.These results suggested that cholesterol was not the cause of cell death.To solve this problem,we used RNAseq technique to find 782 candidate genes that upregulated by nSREBP2.We further optimized the concentration and time curve of Dox-induced cell death,and conducted a large-scale screening.Finally,we obtained343 candidate genes.Analysed with the RNAseq results,we got 44 candidate genes.In summary,firstly we found the special way of cell death caused by nSREBP2 overexpression through generating the Tet-on-Dox-nSREBP2 system.Secondly,we confirmed that the key gene of pyroptosis,GSDMD,and related genes in the cholesterol synthesis pathway were not involved in the cell death caused by the overexpression of nSREBP2.Finally,44 potential candidate genes were selected for validation after the analysis with the large-scale screening and RNAseq results,which laid a foundation for further revealing the mechanism of effects of nSREBP2 on cell fate.Cell death has always been one of most important issue in cell biology,and is closely related to many human diseases.This work will not only broaden our understanding of cell death,but also provide theoretical basis for the diagnosis and treatment of many metabolic diseases.