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Sterol-resistant transcription in CHO cells caused by gene rearrangement that truncates SREBP-2
J Yang1, R Sato, J L Goldstein
1Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas 75235.
Abstract:
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.
Insights
Sterol-resistant cells reveal a defect in SREBP-2 gene regulation, impacting cholesterol homeostasis. A truncated SREBP-2 form bypasses normal sterol control, disrupting lipid metabolism.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Cholesterol homeostasis is tightly regulated by gene transcription.
- Sterol regulatory element-binding proteins (SREBPs) are key transcription factors controlling lipid metabolism.
- SREBP-2 is a membrane-bound protein activated by proteolytic cleavage in sterol-depleted cells.
Purpose of the Study:
- To identify the genetic defect in sterol-resistant CHO cells (SRD-1) that resist cholesterol synthesis repression.
- To elucidate the mechanism by which 25-hydroxycholesterol fails to inhibit sterol synthesis and LDL receptor gene transcription in SRD-1 cells.
- To investigate the role of SREBP-2 in cholesterol homeostasis and its regulation by sterol levels.
Main Methods:
- Genetic analysis of sterol-resistant CHO cells (SRD-1).
- Characterization of the SREBP-2 gene and protein in wild-type and mutant cells.
- Analysis of SREBP-2 processing and transcriptional activity under different sterol conditions.
Main Results:
- SRD-1 cells possess a rearranged SREBP-2 gene, leading to a truncated, soluble SREBP-2 form (amino acids 1-460).
- This truncated SREBP-2 lacks the membrane attachment domain and constitutively activates transcription, bypassing sterol-regulated proteolysis.
- SRD-1 cells exhibit impaired cleavage of both full-length SREBP-2 and SREBP-1, suggesting feedback inhibition of the processing protease by the truncated SREBP-2.
Conclusions:
- The study provides genetic evidence for a model where cholesterol homeostasis is controlled by sterol-regulated proteolysis of membrane-bound transcription factors like SREBP-2.
- A defect in SREBP-2 processing can lead to constitutive activation of cholesterol synthesis genes, contributing to sterol resistance.
- The findings highlight the critical role of SREBP-2 processing in maintaining cellular lipid balance and suggest a feedback mechanism involving the protease responsible for SREBP activation.