Sterol-resistant transcription in CHO cells caused by gene rearrangement that truncates SREBP-2.
Sterol-resistant CHO cells (SRD-1 cells) fail to repress sterol synthesis and LDL receptor gene transcription when incubated with 25-hydroxycholesterol. Here we trace the defect to a rearrangement in the gene encoding SREBP-2, a membrane-bound transcription factor that regulates cholesterol homeostasis. SREBP-2 is an 1139-amino acid protein that is bound to extranuclear membranes via a carboxy-terminal attachment domain. In sterol-depleted cells a protease liberates the amino-terminal fragment (approximately 480 amino acids). This fragment, which contains the transcriptional activation and bHLH-Zip domains, translocates to the nucleus. 25-Hydroxycholesterol abolishes protease activity and halts transcription. SRD-1 cells produce a soluble, truncated form of SREBP-2 (amino acids 1-460) that lacks the membrane attachment domain and activates transcription directly, bypassing the sterol-regulated proteolytic step. Although SRD-1 cells produce full-length SREBP-2 from the wild-type allele and a related transcription factor, SREBP-1, they fail to cleave both of these precursors, indicating that the truncated form of SREBP-2 down-regulates the protease through a form of end-product feedback inhibition. The current data provide genetic evidence for the previously proposed model in which cholesterol homeostasis is controlled by sterol-regulated proteolysis of a membrane-bound bHLH-Zip transcription factor.
Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas 75235.
J
J
Yang
R
R
Sato
J L
JL
Goldstein
M S
MS
Brown
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015321:Gene Rearrangement; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D013261:Sterols / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C497387:SREBF1 protein, human; C497390:SREBF2 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D013261:Sterols; D014157:Transcription Factors